British Pharmacopea - 02 PDF

24-1
Liquorice
Liquorice complies with the requirements of the 3rd edition of the European Pharmacopoeia for Liquorice Root
[0277]. These requirements are reproduced after the heading Definition below.
Action and use Flavour.
Preparations
Liquorice Liquid Extract
When Powdered Liquorice is prescribed or demanded, material complying with the appropriate
requirements below shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Liquorice root consists of the dried unpeeled or peeled, whole or cut root and stolons of Glycyrrhiza
glabra L. It contains not less than 4.0 per cent glycyrrhizic acid (C42H62O16, Mr 823), calculated with
reference to the dried drug.
CHARACTERS
It has the macroscopic and microscopic characters described under Identification tests A and B.
IDENTIFICATION
A.The root has few branches. Its bark is brownish-grey to brown with longitudinal striations and
bears traces of lateral roots. The cylindrical stolons are 1 cm to 2 cm in diameter; their external
appearance is similar to that of the root but there are occasional small buds. The fracture of the root
and the stolon is granular and fibrous. The cork layer is thin; the secondary phloem region is thick
and light yellow with radial striations. The yellow xylem cylinder is compact, with a radiate structure.
The stolon has a central pith, which is absent from the root. The external part of the bark is absent
from the peeled root.
B.Reduce to a powder (355). The powder is light yellow to faintly greyish. Examine under a
microscope using chloral hydrate solution R. The powder shows fragments of yellow thick-walled
fibres, 700 m to 1200 m long and 10 m to 20 m wide with a punctiform lumen, often
accompanied by crystal sheaths containing prisms of calcium oxalate 10 m to 35 m long and 2 m
to 5 m wide. The walls of the large vessels are yellow, 5 m to 10 m thick lignified and have
numerous bordered pits with a slit-shaped aperture; fragments of cork consisting of thin-walled cells
and isolated prisms of calcium oxalate occur as well as fragments of parenchymatous tissue. Fragments of cork are absent from the peeled root. Examine under a microscope using a mixture of equal
volumes of glycerol R and water R. The powder shows simple, round or oval starch granules, 2 m to
20 m in diameter.
C.Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable silica gel
with a fluorescent indicator having an optimal intensity at 254 nm.
Test solution. To 0.50 g of the powdered drug (180) in a 50 ml round-bottomed flask add 16.0 ml of
water R and 4.0 ml of hydrochloric acid R1 and heat on a water-bath under a reflux condenser for
30 min. Cool and filter. Dry the filter and the round-bottomed flask at 105C for 60 min. Place the
filter in the round-bottomed flask, add 20.0 ml of ether R and heat in a water-bath at 40C under a
reflux condenser for 5 min. Cool and filter. Evaporate the filtrate to dryness. Dissolve the residue in
5.0 ml of ether R.
Reference solution. Dissolve 5.0 mg of glycyrrhetic acid R and 5.0 mg of thymol R in 5.0 ml of ether R.
Apply separately to the plate as bands 10 l of each solution. Develop over a path of 15 cm using a
mixture of 1 volume of concentrated ammonia R, 9 volumes of water R, 25 volumes of alcohol R and 65
volumes of ethyl acetate R. Allow the plate to dry in air for 5 min and examine in ultraviolet light at
254 nm. The chromatograms obtained with the test solution and with the reference solution show in
the lower half a quenching zone due to glycyrrhetic acid. Spray the plate with anisaldehyde solution R,
and heat at 100C to 105C for 5 min to 10 min. Examine in daylight. The chromatogram obtained
with the reference solution shows in the lower half the violet zone of glycyrrhetic acid and in the
upper third the red zone of thymol. The chromatogram obtained with the test solution shows in the
lower half of violet zone corresponding to the zone of glycyrrhetic acid in the chromatogram obtained
with the reference solution and a yellow zone (isoliquiridigenine) in the upper third under the zone of
thymol in the chromatogram obtained with the reference solution. Further zones may be present.
TESTS
Loss on drying (2.2.32). Not more than 10.0 per cent, determined on 1.000 g of powdered drug
(355) by drying in an oven at 100C to 105C for 2 h.
Total ash (2.4.16). Not more than 10.0 per cent for the unpeeled drug and not more than 6.0 per
cent for the peeled drug.
24-2
Ash insoluble in hydrochloric acid (2.8.1). Not more than 2.0 per cent for the unpeeled drug and
not more than 0.5 per cent for the peeled drug.
ASSAY
Examine by liquid chromatography (2.2.29).
Test solution. Place 1.000 g of the powdered drug (180) in a 150 ml ground glass conical flask. Add
100.0 ml of an 8 g/l solution of ammonia R and treat in a ultrasonic bath for 30 min. Centrifuge a
part of the supernatant layer and dilute 1.0 ml to 5.0 ml with the same solvent. Filter the solution
through a filter (0.45 m) and use the filtrate as the test solution.
Stock solution. Dissolve 0.130 g of monoammonium glycyrrhizate CRS in an 8 g/l solution of ammonia R
and dilute to 100.0 ml with the same solvent.
Reference solution (a). Dilute 5.0 ml of the stock solution to 100.0 ml with an 8 g/l solution of
ammonia R.
Reference solution (b). Dilute 10.0 ml of the stock solution to 100.0 ml with an 8 g/l solution of
ammonia R.
Reference solution (c). Dilute 15.0 ml of the stock solution to 100.0 ml with an 8 g/l solution of
ammonia R.
The chromatographic procedure may be carried out using:
a stainless steel column 0.10 m long and 4 mm in internal diameter packed with octadecylsilyl
silica gel for chromatography R (5 m),
as mobile phase at a flow rate of 1.5 ml/min a mixture of 6 volumes of acetic acid R, 30 volumes
of acetonitrile R and 64 volumes of water R,
as detector a spectrophotometer set at 254 nm,
a 10 l loop injector.
Inject reference solution (c). Adjust the sensitivity of the system so that the height of the peaks are
at least 50 per cent of the full scale of the recorder. Inject each reference solution and determine the
peak areas.
Establish a calibration curve with the concentration of the reference solutions (g/100 ml) as the
abscissa and the corresponding areas as the ordinate.
Inject the test solution. Using the retention time and the peak area determined from the chromatograms obtained with the reference solutions, locate and integrate the peak due to glycyrrhizic acid in
the chromatogram obtained with the test solution.
Calculate the percentage content of glycyrrhizic acid from the expression:
5
822
B
840
m
A = concentration of monoammonium glycyrrhizate in the test solution determined from the
calibration curve in g/100 ml,
B = declared percentage content of monoammonium glycyrrhizate CRS,
m = mass of the drug in grams,
822 = molecular weight of glycyrrhizic acid,
840 = molecular weight of the monoammonium glycyrrhizate (without any water of crystallisation).
A
STORAGE
Store in a well-closed container, protected from light.
LABELLING
The label states whether the drug is peeled or unpeeled.
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24-3
Lisinopril Dihydrate
1/01
H 2N
HOOC
H
N
N
H
O
C21H31N3O5,2H2O
COOH
441.5
83915-83-7
Lisinopril Dihydrate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1120].
These requirements are reproduced after the heading Definition below.
Action and use Angiotensin-converting enzyme inhibitor.
Preparation
Lisinopril Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Lisinopril dihydrate contains not less than 98.5 per cent and not more than the equivalent of
101.5 per cent of N-[N-[(1S)-1-carboxy-3-phenylpropyl]-L-lysyl]-L-proline, calculated with reference
to the anhydrous substance.
CHARACTERS
A white, crystalline powder, soluble in water, sparingly soluble in methanol, practically insoluble in
acetone and in ethanol.
IDENTIFICATION
Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained
with lisinopril dihydrate CRS. Examine the substances prepared as discs.
TESTS
Specific optical rotation (2.2.7). Dissolve 0.5 g in zinc acetate solution R and dilute to 50.0 ml with
the same solvent. The specific optical rotation is 43 to 47, calculated with reference to the
anhydrous substance.
Related substances Examine by liquid chromatography (2.2.29).
Test solution. Dissolve 20.0 mg of the substance to be examined in mobile phase A and dilute to
10.0 ml with the same mobile phase.
Reference solution (a). Dissolve 20.0 mg of lisinopril dihydrate for performance test CRS in mobile phase
A and dilute to 10.0 ml with the same mobile phase.
Reference solution (b). Dilute 0.5 ml of the test solution to 50.0 ml with mobile phase A.
a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with octylsilyl silica
gel for chromatography R,
as mobile phase at a flow rate of 1.8 ml/min:
Mobile phase A. Prepare a mixture of 30 volumes of acetonitrile R and 970 volumes of a 3.12 g/l
sodium dihydrogen phosphate R solution adjusted to pH 5.0 with a 50 g/l solution of sodium
hydroxide R,
Mobile phase B. Prepare a mixture of 200 ml of acetonitrile R and 800 ml of a 3.12 g/l sodium
dihydrogen phosphate R solution adjusted to pH 5.0 with a 50 g/l solution of sodium hydroxide R,
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
0 35
35 45
45 50
100 70
70
70 100
0 30
30
30 0
50 = 0
100
Comment
Linear gradient
Isocratic
Switch to initial
eluent composition
Restart gradient
24-4
maintaining the temperature of the column at 50C.
Equilibrate the column with mobile phase A for at least 30 min. Adjust the sensitivity of the
system so that the height of the principal peak in the chromatogram obtained with 20 l of reference solution (b) is at least 50 per cent of the full scale of the recorder.
Inject 20 l of reference solution (a). The resulting chromatogram resembles that of the
specimen chromatogram supplied with lisinopril dihydrate for performance test CRS in that the peaks
due to impurity A and impurity E fall on either side of the peak due to lisinopril. Measure the
heights A1 and A2 above the baseline of the peaks due to impurity A and impurity E and the heights
B1 and B2 above the baseline of the lowest points of the curve separating these peaks from the peak
due to lisinopril. The test is not valid unless A1 is greater than nine times B1 and A2 is greater than
nine times B2.
If necessary, adjust the pH of the mobile phase to 4.5 with phosphoric acid R and repeat the
chromatography. A further adjustment to pH 4.0 may be necessary with some columns before
satisfactory separation of impurity A, lisinopril and impurity E is obtained. If, after adjustment, the
retention time of the peak due to impurities C and D becomes extended to the point where integration becomes difficult, increase the content of mobile phase B from 30 per cent to 40 per cent over
the interval from 35 min to 45 min from the start of the chromatogram. Maintain this concentration for a further 10 min. Return the concentration of mobile phase A to 100 per cent over the
next 10 min prior to the next injection.
Inject 20 l of the test solution and 20 l of reference solution (b). In the chromatogram
obtained with the test solution: the area of any peak due to impurity E is not greater than 0.3 times
the area of the principal peak in the chromatogram obtained with reference solution (b) (0.3 per
cent); the area of any peak, apart from the principal peak and any peak due to impurity E, is not
greater than 0.3 times the area of the principal peak in the chromatogram obtained with reference
solution (b) (0.3 per cent) and the sum of the areas of all such peaks is not greater than half the
area of the principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent).
Disregard any peak due to the solvent, any peak occurring in the first 3 minutes and any peak with
an area less than 0.05 times the area of the principal peak in the chromatogram obtained with
reference solution (b).
Water (2.5.12). 8.0 to 9.5 per cent, determined on 0.200 g by the semi-micro determination of
water.
Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g.
ASSAY
Dissolve 0.350 g in 50 ml of distilled water R. Titrate with 0.1M sodium hydroxide, determining the
end-point potentiometrically (2.2.20).
1 ml of 0.1M sodium hydroxide is equivalent to 40.55 mg of C21H31N3O5.
IMPURITIES
HOOC H
NH2 and enantiomer
A. (RS)-2-amino-4-phenylbutanoic acid,
SO3H
Me
B. toluene-4-sulphonic acid,
HOOC H
N
O
NH2
O
N
H
C. (S)-2-[(3S,8aS)-3-(4-aminobutyl)-1,4-dioxo-1,2,3,4,6,7,8,8a-octahydropyrrolo[1,2a]piperazin-2-yl]-4-phenylbutanoic acid ((S,S,S)-diketopiperazine),

HOOC H
N
O
NH2
O
N
H
D. (S)-2-[(3S,8aR)-3-(4-aminobutyl)-1,4-dioxo-1,2,3,4,6,7,8,8a-octahydropyrrolo[1,2a]piperazin-2-yl]-4-phenylbutanoic acid ((R,S,S)-diketopiperazine),
24-5
H2N
HOOC H
N
H
N
H
COOH
E. N-[N-[(R)-1-carboxy-3-phenylpropyl)-L-lysyl]-L-proline (lisinopril (R,S,S)-isomer),

H2N
HOOC H
N
H
H
O
N
H
COOH
F. N-[N-[(S)-1-carboxy-3-cyclohexylpropyl)-L-lysyl]-L-proline (cyclohexyl analogue).

__________________________________________________________________________________________________________ Ph Eur
24-6
Lithium Carbonate
Li2CO3
73.9
554-13-2
Lithium Carbonate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0228].
Action and use Prophylaxis of affective disorders.
Preparations
Lithium Carbonate Tablets
Slow Lithium Carbonate Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Lithium carbonate contains not less than 98.5 per cent and not more than the equivalent of
100.5 per cent of Li2CO3.
CHARACTERS
A white powder, slightly soluble in water, practically insoluble in alcohol.
IDENTIFICATION
A. When moistened with hydrochloric acid R, it gives a red colour to a non-luminous flame.
B. Dissolve 0.2 g in 1 ml of hydrochloric acid R. Evaporate to dryness on a water-bath. The residue
dissolves in 3 ml of alcohol R.
C. It gives the reaction of carbonates (2.3.1).
TESTS
Solution S Suspend 10.0 g in 30 ml of distilled water R and dissolve by the addition of 22 ml of nitric
acid R. Add dilute sodium hydroxide solution R until the solution is neutral and dilute to 100 ml with
distilled water R.
Appearance of solution Solution S is clear (2.2.1) and colourless (Method II, 2.2.2).
Chlorides (2.4.4). 2.5 ml of solution S diluted to 15 ml with water R complies with the limit test for
chlorides (200 ppm).
Sulphates (2.4.13). Disperse 1.25 g in 5 ml of distilled water R and dissolve by adding 5 ml of hydrochloric acid R1. Boil for 2 min. Cool and add dilute sodium hydroxide solution R until neutral. Dilute to
25 ml with distilled water R. The solution complies with the limit test for sulphates (200 ppm).
Arsenic (2.4.2). 0.5 g complies with limit test A for arsenic (2 ppm).
Calcium (2.4.3). 5 ml of solution S diluted to 15 ml with distilled water R complies with the limit test
for calcium (200 ppm).
Heavy metals (2.4.8). 12 ml of solution S complies with limit test A for heavy metals (20 ppm).
Prepare the standard using lead standard solution (2 ppm Pb) R.
Iron (2.4.9). 5 ml of solution S diluted to 10 ml with water R complies with the limit test for iron
(20 ppm).
Magnesium (2.4.6). Dilute 1 ml of solution S to 10 ml with water R. 6.7 ml of this solution diluted
to 10 ml with water R complies with the limit test for magnesium (150 ppm).
Potassium Not more than 300 ppm of K, determined by atomic emission spectrometry (Method I,
2.2.22).
Test solution. Dissolve 1.0 g of the substance to be examined in 10 ml of hydrochloric acid R1 and
dilute to 50.0 ml with water R.
Reference solutions. Prepare the reference solutions using a solution of potassium chloride R containing
500 g of K per millilitre, diluted as required.
Measure the emission intensity at 766.5 nm.
Sodium Not more than 300 ppm of Na, determined by atomic emission spectrometry (Method I,
2.2.22).
Test solution. Dissolve 1.0 g of the substance to be examined in 10 ml of hydrochloric acid R1 and
Reference solutions. Prepare the reference solutions using a solution of sodium chloride R containing
500 g of Na per millilitre, diluted as required.
Measure the emission intensity at 589 nm.
24-7
ASSAY
Dissolve 0.500 g in 25.0 ml of 1M hydrochloric acid. Titrate with 1M sodium hydroxide, using methyl
orange solution R as indicator.
1 ml of 1M hydrochloric acid is equivalent to 36.95 mg of Li2CO3.
__________________________________________________________________________________________________________ Ph Eur
24-8
Lithium Citrate
CH2 COOLi
HO C
COOLi
CH2 COOLi
C6H5Li3O7,4H2O
282.0
6080-58-6
Lithium Citrate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0621].
Action and use Antidepressant.
Preparation
Lithium Citrate Oral Solution
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Lithium citrate contains not less than 98.0 per cent and not more than the equivalent of 102.0 per
cent of trilithium 2-hydroxypropane-1,2,3-tricarboxylate, calculated with reference to the anhydrous
substance.
CHARACTERS
A white or almost white, fine crystalline powder, freely soluble in water, slightly soluble in alcohol.
IDENTIFICATION
A. When moistened with hydrochloric acid R, it gives a red colour to a non-luminous flame.
B. Dilute 3 ml of solution S (see Tests) to 10 ml with water R. Add 3 ml of potassium ferriperiodate
solution R. A white or yellowish-white precipitate is formed.
C. To 1 ml of solution S add 4 ml of water R. The solution gives the reaction of citrates (2.3.1).
TESTS
Solution S Dissolve 10.0 g in carbon dioxide-free water R prepared from distilled water R and dilute to
100 ml with the same solvent.
Acidity or alkalinity To 10 ml of solution S add 0.1 ml of phenolphthalein solution R. Not more than
0.2 ml of 0.1M hydrochloric acid or 0.1M sodium hydroxide is required to change the colour of the
indicator.
Readily carbonisable substances To 0.20 g of the powdered substance to be examined add 10 ml
of sulphuric acid R and heat in a water-bath at 90 1C for 60 min. Cool rapidly. The solution is not
more intensely coloured than reference solution Y2 or GY2 (Method II, 2.2.2).
Chlorides (2.4.4). Dilute 5 ml of solution S to 15 ml with water R. The solution complies with the
limit test for chlorides (100 ppm).
Oxalates Dissolve 0.50 g in 4 ml of water R, add 3 ml of hydrochloric acid R and 1 g of granulated
zinc R and heat on a water-bath for 1 min. Allow to stand for 2 min, decant the liquid into a test-tube
containing 0.25 ml of a 10 g/l solution of phenylhydrazine hydrochloride R and heat to boiling. Cool
rapidly, transfer to a graduated cylinder and add an equal volume of hydrochloric acid R and 0.25 ml
of potassium ferricyanide solution R. Shake and allow to stand for 30 min. Any pink colour in the
solution is not more intense than that in a standard prepared at the same time and in the same
manner using 4 ml of a 0.05 g/l solution of oxalic acid R (300 ppm, calculated as anhydrous oxalate
ion).
Sulphates (2.4.13). To 3 ml of solution S add 2 ml of hydrochloric acid R1 and dilute to 17 ml with
distilled water R. The solution complies with the limit test for sulphates (500 ppm). Prepare the
standard using 15 ml of a mixture of 2 ml of hydrochloric acid R1 and 15 ml of sulphate standard
solution (10 ppm SO4) R and compare the opalescence after 15 min.
Water (2.5.12). 24.0 per cent to 27.0 per cent, determined on 0.100 g by the semi-micro determination of water. After adding the substance to be examined, stir for 15 min before titrating. Carry out a
blank titration.
24-9
ASSAY
Dissolve 80.0 mg in 50 ml of anhydrous acetic acid R, heating to about 50C. Allow to cool. Titrate
with 0.1M perchloric acid, using 0.25 ml of naphtholbenzein solution R as indicator, until the colour
changes from yellow to green.
1 ml of 0.1M perchloric acid is equivalent to 7.00 mg of C6H5Li3O7.
STORAGE
Store in an airtight container.
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24-10
Lofepramine Hydrochloride
Cl
N
,HCl
N
Me
C26H27ClN2O,HCl
455.4
O
26786-32-3
Definition Lofepramine Hydrochloride is 5-(3-[N-(4-chlorophenacyl)-N-methylamino]propyl)10,11-dihydro-5H-dibenz[b,f]azepine hydrochloride. It contains not less than 98.5% and not more
than 101.0% of C26H27ClN2O,HCl, calculated with reference to the dried substance.
Characteristics A fine, yellowish white to greenish yellow powder with a faint characteristic odour.
Very soluble in ethanol (96%) and in methanol; slightly soluble in acetone; very slightly soluble in
water.
It exhibits polymorphism.
Identification
A. The infrared absorption spectrum, Appendix II A, is concordant with the reference spectrum of
lofepramine hydrochloride (form A) (RS 399A).
B. 2 ml of a 1% w/v solution in ethanol (96%) complies with the test for chlorides, Appendix VI.
Related substances Carry out the method for liquid chromatography, Appendix III D, using the
following solutions. Solution (1) contains 0.1% w/v of the substance being examined in the mobile
phase. For solution (2) dilute 1 volume of solution (1) to 200 volumes with the mobile phase.
Solution (3) contains 0.0002% w/v each of desipramine hydrochloride EPCRS and imipramine hydrochloride EPCRS in the mobile phase.
Inject 20 l of solutions (1) and (2). Inject 20 l of solution (3) and allow the chromatography to
continue for 4 times the retention time of the principal peak.
The chromatographic procedure may be carried out using (a) a stainless steel column (25 cm
4.6 mm) packed with base-deactivated end-capped octylsilyl silica gel for chromatography (5 m)
(Lichrospher 60 RP-select B is suitable) maintained at 50, (b) as the mobile phase with a flow rate
of 1.5 ml per minute a 0.9% w/v solution of sodium dodecyl sulphate in a mixture of 550 volumes of
acetonitrile, 325 volumes of water and 125 volumes of a buffer solution of pH 1.0 containing 0.015%
w/v of glycine, 0.018% w/v of sodium chloride and 0.44% w/v of hydrochloric acid and (c) a detection
wavelength of 254 nm.
The test is not valid unless, in the chromatogram obtained with solution (3), the resolution factor
between the two principal peaks is at least 0.9.
In the chromatogram obtained with solution (1) the area of any secondary peak is not greater than
the area of the peak in the chromatogram obtained with solution (2) (0.5%) and the sum of the areas
of any secondary peaks is not greater than twice the area of the peak in the chromatogram obtained
with solution (2) (1%).
Loss on drying When dried at a temperature of 100 at a pressure not exceeding 0.2 kPa, loses not
more than 0.5% of its weight. Use 1 g.
Assay Carry out the method for liquid chromatography, Appendix III D, using the following solutions.
Solution (1) contains 0.02% w/v of the substance being examined in the mobile phase. Solution (2)
contains 0.02% w/v of lofepramine hydrochloride BPCRS in the mobile phase.
The chromatographic procedure described under the test for Related substances may be used.
Inject 20 l of each solution.
Calculate the content of C26H27ClN2O,HCl from the chromatograms obtained and using the
declared content of C26H27ClN2O,HCl in lofepramine hydrochloride BPCRS.
Storage Lofepramine Hydrochloride should be kept in an airtight container and protected from
light.
Preparation
Lofepramine Tablets
24-11
IMPURITIES
Cl
eHN
O
A. 1-methylamino-4-chloroacetophenone,
Cl
HOOC
B. 4-chlorobenzoic acid,
N
NHMe
C. desipramine,
N
H
D. iminodibenzyl,
N
N
CHO
Me
E. N-formyldesipramine,
Cl
N
Me
O
N
O
Br
Cl
F. N,N-bis(4-chlorophenacyl)desipramine bromide,
N
NMe2
G. imipramine,
Cl
r
O
H. 2-bromo-4-chloroacetophenone.
24-12
Lomustine
NO
H
N
N
Cl
O
C9H16ClN3O2
233.7
13010-47-4
Lomustine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0928]. These
requirements are reproduced after the heading Definition below.
Action and use Cytotoxic.
Preparation
Lomustine Capsules
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Lomustine contains not less than 98.5 per cent and not more than the equivalent of 100.5 per cent of
1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, calculated with reference to the dried substance.
CHARACTERS
A yellow, crystalline powder, practically insoluble in water, freely soluble in acetone and in methylene
chloride, soluble in alcohol.
Carry out the tests protected from light and prepare all the solutions immediately before use.
IDENTIFICATION
First identification: C.
Second identification: A, B, D, E.
A. Melting point (2.2.14): 89C to 91C.
B. Dissolve 50.0 mg in alcohol R and dilute to 50.0 ml with the same solvent. Dilute 2.0 ml to
100.0 ml with alcohol R. Examined between 220 nm and 350 nm (2.2.25), the solution shows an
absorption maximum at 230 nm. The specific absorbance at the maximum is 250 to 270.
C. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum
obtained with lomustine CRS. Examine the substances prepared as discs.
D. Examine the chromatograms obtained in the test for related substances. The principal spot in the
chromatogram obtained with test solution (b) is similar in position, colour and size to the principal
spot in the chromatogram obtained with reference solution (a).
E. Dissolve about 25 mg in 1 ml of methanol R, add 0.1 ml of dilute sodium hydroxide solution R and
2 ml of water R. Acidify by adding dropwise dilute nitric acid R. Filter. The filtrate gives reaction (a) of
chlorides (2.3.1).
TESTS
Related substances
A. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance.
Test solution (a). Dissolve 0.25 g of the substance to be examined in methanol R and dilute to 10 ml
with the same solvent.
Test solution (b). Dilute 1 ml of test solution (a) to 25 ml with methanol R.
Reference solution (a). Dissolve 10 mg of lomustine CRS in methanol R and dilute to 10 ml with the
same solvent.
Reference solution (b). Dilute 1 ml of test solution (b) to 10 ml with methanol R.
Reference solution (c). Dilute 1 ml of test solution (b) to 20 ml with methanol R.
Reference solution (d). Dissolve 10 mg of lomustine CRS and 10 mg of dicyclohexylurea R in methanol R
and dilute to 10 ml with the same solvent.
Apply separately to the plate 5 l of each solution. Develop over a path of 15 cm using a mixture of
20 volumes of glacial acetic acid R and 80 volumes of toluene R. Dry the plate at 110C for 1 h. At the
bottom of a chromatography tank, place an evaporating dish containing a mixture of 1 volume of
hydrochloric acid R1, 1 volume of water R and 2 volumes of a 15 g/l solution of potassium
permanganate R, close the tank and allow to stand for 15 min. Place the dried plate in the tank and
24-13
close the tank. Leave the plate in contact with the chlorine vapour for 5 min. Withdraw the plate
and place it in a current of cold air until the excess of chlorine is removed and an area of coating
below the points of application does not give a blue colour with a drop of potassium iodide and starch
solution R. Spray with potassium iodide and starch solution R. Any spot in the chromatogram obtained
with test solution (a), apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (b) (0.4 per cent) and at most one such spot is more
intense than the spot in the chromatogram obtained with reference solution (c) (0.2 per cent). The
test is not valid unless the chromatogram obtained with reference solution (d) shows two clearly
separated principal spots.
B. Examine by liquid chromatography (2.2.29).
Test solution. Dissolve 0.25 g of the substance to be examined in methanol R and dilute to 10.0 ml the
same solvent.
Reference solution. Dilute 1.0 ml of the test solution to 100.0 ml with methanol R.
silica gel for chromatography R (5 m to 10 m),
as mobile phase at a flow rate of 2 ml per minute a mixture of 50 volumes of methanol R and 50
volumes of water R,
a loop injector.
Inject 20 l of the reference solution. Adjust the sensitivity of the system so that the height of the
principal peak in the chromatogram obtained with the reference solution is at least 50 per cent of the
full scale of the recorder. Inject separately 20 l of each solution. In the chromatogram obtained with
the test solution, the sum of the areas of any peaks apart from the principal peak is not greater than
the area of the principal peak in the chromatogram obtained with the reference solution (1 per cent).
Disregard any peak due to the solvent and any peak with an area less than 0.05 times that of the
principal peak in the chromatogram obtained with the reference solution.
Chlorides (2.4.4). Dissolve 0.24 g in 4 ml of methanol R and add 20 ml of water R. Allow to stand
for 20 min and filter. To 10 ml of the filtrate, add 5 ml of methanol R. The solution complies with the
limit test for chlorides (500 ppm). When preparing the standard, replace the 5 ml of water R with
5 ml of methanol R.
Loss on drying (2.2.32). Not more than 1.0 per cent, determined on 1.000 g by drying in a
desiccator over diphosphorus pentoxide R at a pressure not exceeding 0.7 kPa for 24 h.
ASSAY
Dissolve 0.200 g in about 3 ml of alcohol R and add 20 ml of a 200 g/l solution of potassium
hydroxide R and boil under a reflux condenser for 2 h. Add 75 ml of water R and 4 ml of nitric acid R.
Cool and titrate with 0.1M silver nitrate, determining the end-point potentiometrically (2.2.20). Carry
out a blank titration.
1 ml of 0.1M silver nitrate is equivalent to 23.37 mg of C9H16ClN3O2.
STORAGE
IMPURITIES
H
N
Cl
H
N
Cl
A. 1,3-bis(2-chloroethyl)urea,
H
N
H
N
Cl
B. 1-(2-chloroethyl)-3-cyclohexylurea,
H
N
H
N
O
C. 1,3-dicyclohexylurea.
__________________________________________________________________________________________________________ Ph Eur
24-14
Loosestrife
1/01
Loosestrife complies with the requirements of the 3rd edition of the European Pharmacopoeia [1537]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Loosestrife consists of the dried flowering tops, whole or cut, of Lythrum salicaria L. It contains not
less than 5.0 per cent of tannins, expressed as pyrogallol (C6H6O3; Mr 126.1) and calculated with
CHARACTERS
It has the macroscopic and microscopic characters described under identification tests A and B.
IDENTIFICATION
A. The stems are rigid, four-angled, branching at the top, brownish-green, longitudinally wrinkled
and pubescent. The leaves are opposite, decussate, rarely verticillate in threes and sometimes
alternate at the inflorescence which forms a long terminal spike. The leaves are sessile, lanceolate and
cordate at the base, 5 cm to 15 cm long and 1 cm to 2.5 cm wide, pubescent on the lower surface;
the subsidiary veins form arcs that anastomose near the leaf margin. The flowers have a pubescent,
tubular, persistent gamosepalous calyx, 4 mm to 8 mm long, consisting of 6 sepals bearing 6 small,
triangular teeth alternating with 6 large acute teeth at least half as long as the tube; a polypetalous
corolla consisting of 6 violet-pink petals, each expanded at the top with a wavy outline and narrowing
at the base. The androecium consists of 2 verticils of 6 stamens (one verticil with short, barely
emerging stamens, the other with long stamens extending well out of the corolla). The fruit, if
formed, is a small capsule included in the persistent calyx.
B. Reduce to a powder (355). The powder is greenish-yellow. Examine under a microscope using
chloral hydrate solution R. The powder shows unicellular or bicellular, uniseriate, thick-walled, finely
pitted covering trichomes from the lower epidermis of the stem and leaf; numerous uniseriate,
unicellular or bicellular, thin-walled, finely pitted covering trichomes from the calyx; transparent
violet-pink fragments from the petals; numerous cluster crystals of calcium oxalate; pollen grains with
3 pores and a thin and slightly granular exine; fragments of the upper epidermis with large polygonal
cells and sinuous walls; fragments of the lower epidermis with smaller polygonal cells and anomocytic
stomata (2.8.3).
C. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel plate R.
Test solution. To 1.0 g of the powdered drug (355) add 10 ml of methanol R and heat in a water-bath
at 65C for 5 min with frequent shaking. Cool and filter. Dilute the filtrate to 10 ml with methanol R.
Reference solution. Dissolve 0.5 mg of chlorogenic acid R, 1 mg of hyperoside R, 1 mg of rutin R and
1 mg of vitexin R in 10 ml of methanol R.
Apply to the plate as bands 10 l of each solution. Develop over a path of 15 cm using a mixture of
7.5 volumes of anhydrous acetic acid R, 7.5 volumes of anhydrous formic acid R, 18 volumes of water R
and 67 volumes of ethyl acetate R. Dry the plate at 100C to 105C and spray it while still warm with
a 10 g/l solution of diphenylboric acid aminoethyl ester R in methanol R. Subsequently spray the plate
with a 50 g/l solution of macrogol 400 R in methanol R. Allow the plate to dry in air for 30 min and
examine in ultraviolet light at 365 nm. The chromatogram obtained with the reference solution
shows in the lower third a yellowish-brown fluorescent zone (rutin) and in the middle third a light
blue fluorescent zone (chlorogenic acid), above it a yellowish-brown fluorescent zone (hyperoside)
and a green fluorescent zone (vitexin). The chromatogram obtained with the test solution shows a
bright green fluorescent zone slightly above the rutin zone in the chromatogram obtained with the
reference solution, a yellow fluorescent zone similar in position to the chlorogenic acid zone in the
chromatogram obtained with the reference solution, a yellow fluorescent zone similar in position to
the hyperoside zone in the chromatogram obtained with the reference solution and a bright green
fluorescent zone corresponding to the vitexin zone in the chromatogram obtained with the reference
solution.
TESTS
Foreign matter (2.8.2). It complies with the test for foreign matter.
Loss on drying (2.2.32). Not more than 12.0 per cent, determined on 1.000 g of the powdered drug
(355) by drying in an oven at 100C to 105C.
Total ash (2.4.16). Not more than 7.0 per cent.
24-15
ASSAY
Carry out the determination of tannins in herbal drugs (2.8.14). Use 0.750 g of the powdered drug
(180).
STORAGE
Store protected from light.
__________________________________________________________________________________________________________ Ph Eur
24-16
Loperamide Hydrochloride
Cl
OH
,HCl
O
N
NMe2
Ph
C29H33ClN2O2,HCl
513.5
Ph
34552-83-5
Loperamide Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Antidiarrhoeal.
Preparation
Loperamide Capsules
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Loperamide hydrochloride contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of 4-[4-(4-chlorophenyl)-4-hydroxypiperidino]-N,N-dimethyl-2,2-diphenylbutyramide hydrochloride, calculated with reference to the dried substance.
CHARACTERS
A white or almost white powder, slightly soluble in water, freely soluble in methanol, soluble in
alcohol.
It melts at about 225C, with decomposition.
IDENTIFICATION
First identification: A, C.
Second identification: B, C.
A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum
obtained with loperamide hydrochloride CRS. Examine the substances prepared as discs.
B. Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable octadecylsilyl silica gel.
Test solution. Dissolve 30 mg of the substance to be examined in the mobile phase and dilute to 5 ml
with the mobile phase.
Reference solution (a). Dissolve 30 mg of loperamide hydrochloride CRS in the mobile phase and dilute
to 5 ml with the mobile phase.
Reference solution (b). Dissolve 30 mg of loperamide hydrochloride CRS and 30 mg of ketoconazole CRS
in the mobile phase and dilute to 5 ml with the mobile phase.
20 volumes of ammonium acetate solution R, 40 volumes of dioxan R and 40 volumes of methanol R.
Dry the plate in a current of air for 15 min and expose it to iodine vapour until the spots appear.
Examine in daylight. The principal spot in the chromatogram obtained with the test solution is
similar in position, colour and size to the principal spot in the chromatogram obtained with reference
solution (a). The test is not valid unless the chromatogram obtained with reference solution (b)
shows two clearly separated spots.
C. Dissolve 50 mg in a mixture of 0.4 ml of ammonia R and 2 ml of water R. Mix, allow to stand for
5 min and filter. Acidify the filtrate with dilute nitric acid R. It gives reaction (a) of chlorides (2.3.1).
TESTS
Appearance of solution Dissolve 1.0 g in methanol R and dilute to 10 ml with the same solvent.
The solution is clear (2.2.1) and not more intensely coloured than reference solution BY7 (Method II,
2.2.2).
Test solution. Dissolve 0.10 g of the substance to be examined in methanol R and dilute to 10.0 ml
Reference solution (a). Dissolve 2.5 mg of loperamide hydrochloride CRS and 2.5 mg of haloperidol CRS
in methanol R and dilute to 100.0 ml with the same solvent.
24-17
Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with methanol R. Dilute 5.0 ml of
this solution to 20.0 ml with methanol R.
a stainless steel column 0.10 m long and 4.6 mm in internal diameter packed with octadecylsilyl
as mobile phase at a flow rate of 2 ml per minute a mixture of 1 volume of acetonitrile R and 9
volumes of a 17 g/l solution of tetrabutylammonium hydrogen sulphate R, changing by linear
gradient elution over 10 min to a mixture of 7 volumes of acetonitrile R and 3 volumes of a 17 g/l
solution of tetrabutylammonium hydrogen sulphate R; elute for a further 5 min with the final eluent
mixture,
as detector a spectrophotometer set at 220 nm.
Equilibrate the column for at least 30 min with acetonitrile R and then equilibrate at the initial
eluent composition for at least 5 min.
Adjust the sensitivity of the detector so that the height of the principal peak in the chromatogram
obtained with reference solution (b) is 70 per cent to 90 per cent of the full scale of the recorder.
Inject 10 l of reference solution (a). When the chromatogram is recorded in the prescribed conditions, the retention times are: haloperidol about 3 min; loperamide hydrochloride about 4.5 min. The
test is not valid unless the resolution between the peaks corresponding to haloperidol and loperamide
hydrochloride is at least 8.0. If necessary, adjust the final concentration of acetonitrile in the mobile
phase or adjust the conditions of the linear gradient.
Inject separately 10 l of methanol R as a blank, 10 l of the test solution and 10 l of reference
solution (b). In the chromatogram obtained with the test solution: the area of any peak, apart from
the principal peak, is not greater than the area of the principal peak in the chromatogram obtained
with reference solution (b) (0.25 per cent); the sum of the areas of all the peaks, apart from the
principal peak, is not greater than twice the area of the principal peak in the chromatogram obtained
with reference solution (b) (0.5 per cent). Disregard any peak obtained with methanol and any peak
with an area less than 0.2 times the area of the principal peak in the chromatogram obtained with
Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at
100C to 105C.
ASSAY
Dissolve 0.400 g in 50 ml of alcohol R and add 5.0 ml of 0.01M hydrochloric acid. Carry out a potentiometric titration (2.2.20), using 0.1M sodium hydroxide. Read the volume added between the two
points of inflexion.
1 ml of 0.1M sodium hydroxide is equivalent to 51.35 mg of C29H34Cl2N2O2.
STORAGE
IMPURITIES
Cl
OH
N
CONMe2
Ph Ph
A. 4-[4-(4-chlorobiphenyl-4-yl)-4-hydroxypiperidino]-N,N-dimethyl-2,2-diphenylbutyramide,
Cl
OH
+
N
Br
CONMe2
Ph Ph
B. 4-(4-chlorophenyl)-1,1-bis[4-(dimethylamino)-4-oxo-3,3-diphenylbutyl]-4hydroxypiperidinium bromide,
24-18
Cl
OH
NH
C. 4-(4-chlorophenyl)piperidin-4-ol,
OH
N
CONMe2
Ph Ph
D. 4-(4-hydroxy-4-phenylpiperidino)-N,N-dimethyl-2,2-diphenylbutyramide,
Cl
OH
Cl
OH
N
N
O
Ph Ph
E. 4-(4-chlorophenyl)-1-[4-[4-(4-chlorophenyl)-4-hydroxy-piperidino]-2,2diphenylbutyryl]piperidin-4-ol.
__________________________________________________________________________________________________________ Ph Eur
24-19
Loprazolam Mesilate
O
H
N
O2N
NMe
,CH3SO3H
Cl
C23H21ClN6O3,CH4O3S,H2O
579.1
70111-54-5
Definition Loprazolam Mesilate is (Z)-6-(2-chlorophenyl)-2,4-dihydro-2-(4-methylpiperazin-1ylmethylene)-8-nitroimidazo[1,2-a][1,4]benzodiazepin-1-one methanesulphonate monohydrate. It

contains not less than 98.5% and not more than 101.0% of C23H21ClN6O3,CH4O3S, calculated
with reference to the dried substance.
Characteristics A yellow, crystalline powder.
Slightly soluble in water, in chloroform and in ethanol (96%); very slightly soluble in ether.
Identification
loprazolam mesilate (RS 205).
B. The light absorption, Appendix II B, in the range 210 to 370 nm of a 0.001% w/v solution in
ethanol (96%) exhibits a maximum at 330 nm and a shoulder at 240 nm. The absorbance at the
maximum is about 0.7.
N-Methylpiperazine Carry out the method for thin-layer chromatography, Appendix III A, using a
silica gel precoated plate (Merck silica gel 60 plates are suitable) and a mixture of 80 volumes of
chloroform, 20 volumes of methanol and 2 volumes of 13.5M ammonia as the mobile phase. Apply
separately to the plate 10 l of each of two solutions in a mixture of equal volumes of chloroform and
methanol containing (1) 2.0% w/v of the substance being examined and (2) 0.0050% w/v of
N-methylpiperazine. After removal of the plate, allow it to dry in air, heat at 110 for 15 minutes and
spray with a mixture of 50 volumes of a 2% w/v solution of potassium iodide and 1 volume of a 10%
w/v solution of chloroplatinic(IV) acid. Any spot corresponding to N-methylpiperazine in the chromatogram obtained with solution (1) is not more intense than the spot in the chromatogram obtained
with solution (2).
Related substances Carry out the method for thin-layer chromatography, Appendix III A, using a
chloroform and 20 volumes of methanol as the mobile phase. Before use, stand the plate in methanol,
allowing the solvent front to ascend 17 cm, allow to dry in air, heat the plate at 100 to 105 for 1
hour and use with the flow of mobile phase in the same direction as that used for the pretreatment.
Apply separately to the plate 10 l of each of three solutions in a mixture of 46 volumes of chloroform,
46 volumes of methanol and 8 volumes of water containing (1) 2.0% w/v of the substance being
examined, (2) 0.0020% w/v of the substance being examined and (3) 0.010% w/v of 6-(2chlorophenyl)-2,4-dihydro-2-[(dimethylamino)methylene]-8-nitroimidazo[1,2-a][1,4]benzodiazepin-1-one
BPCRS (dimethylamino analogue). After removal of the plate, allow it to dry in air, spray the plate
with a 5% w/v solution of titanium(III) chloride in a solution of hydrochloric acid containing 10% w/v
of HCl and then spray with a solution containing 0.4 g of 4-dimethylaminocinnamaldehyde in a mixture
of 20 ml of 6M hydrochloric acid and 100 ml of ethanol (96%). Heat at 100 until spots appear (about
10 minutes). Any spot corresponding to the dimethylamino analogue in the chromatogram obtained
with solution (1) is not more intense than the spot in the chromatogram obtained with solution (3)
and any other secondary spot is not more intense than the spot in the chromatogram obtained with
solution (2).
Loss on drying When dried at 100 to 105 for 3 hours, loses 2.5 to 4.5% of its weight. Use 1 g.
Sulphated ash Not more than 0.1%, Appendix IX A.
Assay Dissolve 0.25 g in 60 ml of a 50% v/v solution of propan-2-ol and titrate with 0.05M sodium
hydroxide VS determining the end point potentiometrically. Each ml of 0.05M sodium hydroxide VS is
equivalent to 28.05 mg of C23H21ClN6O3,CH4O3S.
Storage Loprazolam Mesilate should be kept in a well-closed container.
Action and use Hypnotic.
24-20
Preparation
Loprazolam Tablets
When loprazolam mesylate is prescribed or demanded, Loprazolam Mesilate shall be dispensed or
supplied.
IMPURITIES
O
O2N
NMe2
N
Cl
A. 6-(2-chlorophenyl)-2,4-dihydro-2-[(dimethylamino)methylene]-8-nitroimidazo[1,2-a][1,4]benzodiazepin-1-one,
HN
NMe
B. N-methylpiperazine.
24-21
Lorazepam
H
OH
H
N
Cl
Cl
and enantiomer
C15H10Cl2N2O2
321.2
846-49-1
Lorazepam complies with the requirements of the 3rd edition of the European Pharmacopoeia [1121]. These
Action and use Anxiolytic.
Preparations
Lorazepam Injection
Lorazepam Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Lorazepam contains not less than 98.5 per cent and not more than the equivalent of 102.0 per cent
of (RS)-7-chloro-5-(2-chlorophenyl)-3-hydroxy-1,3-dihydro-2H-1,4-benzodiazepin-2-one, calculated
CHARACTERS
A white or almost white, crystalline powder, practically insoluble in water, sparingly soluble in
alcohol, sparingly soluble or slightly soluble in methylene chloride.
It shows polymorphism.
IDENTIFICATION
First identification: B.
Second identification: A, C.
A. Dissolve 10.0 mg in alcohol R and dilute to 100.0 ml with the same solvent. Dilute 10.0 ml of this
solution to 100.0 ml with alcohol R. Examined between 210 nm and 280 nm (2.2.25), the solution
shows an absorption maximum at 230 nm. The specific absorbance at the maximum is 1070 to
1170.
B. Examine by infrared absorption spectrophotometry between 600 cm-1 and 2000 cm-1 (2.2.24),
comparing with the spectrum obtained with lorazepam CRS. Examine the substances as discs
prepared using potassium bromide R.
C. Examine the chromatograms obtained in the test for related substances. The principal spot in the
chromatogram obtained with test solution (b) is similar in position and size to the spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained
with reference solution (d) shows two clearly separated spots.
TESTS
Related substances Examine by thin-layer chromatography (2.2.27), using a TLC silica gel F254
plate R. Place the plate in a chromatographic tank containing methanol R and allow the solvent front
to migrate over a path of 17 cm. Allow the plate to dry in air and heat it at 100C to 105C for 1 h.
Test solution (a). Dissolve 0.200 g of the substance to be examined in acetone R and dilute to 10 ml
Test solution (b). Dilute 2 ml of test solution (a) to 50 ml with acetone R.
Reference solution (a). Dissolve 20 mg of lorazepam CRS in acetone R and dilute to 25 ml with the
same solvent.
Reference solution (b). Dilute 1 ml of test solution (b) to 20 ml with acetone R.
Reference solution (c). Dilute 5 ml of reference solution (b) to 10 ml with acetone R.
Reference solution (d). Dissolve 4 mg of nitrazepam CRS in acetone R, add 5 ml of reference solution
(a) and dilute to 20 ml with acetone R.
24-22
Apply to the plate 20 l of each solution and develop in the direction used for the migration of
methanol over a path of 12 cm with a mixture of 10 volumes of methanol R and 100 volumes of
methylene chloride R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Any spot
in the chromatogram obtained with test solution (a), apart from the principal spot, is not more
intense than the spot in the chromatogram obtained with reference solution (b) (0.2 per cent) and at
most one such spot is more intense than the spot in the chromatogram obtained with reference
solution (c) (0.1 per cent).
Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g under high vacuum at
100C to 105C.
ASSAY
Dissolve 0.250 g in 30 ml of dimethylformamide R. Titrate with 0.1M tetrabutylammonium hydroxide,
determining the end-point potentiometrically (2.2.20). Protect the solution from atmospheric carbon
dioxide throughout the titration.
1 ml of 0.1M tetrabutylammonium hydroxide is equivalent to 32.12 mg of C15H10Cl2N2O2.
STORAGE
Store in an airtight container, protected from light.
IMPURITIES
NH2
O
Cl
Cl
A. 2-amino-2,5-dichlorobenzophenone,
H
N
O
OAc
Cl
and enantiomer
Cl
B. (RS)-3-(acetyloxy)-7-chloro-5-(2-chlorophenyl)-1,3-dihydro-2H-1,4benzodiazepin-2-one.
__________________________________________________________________________________________________________ Ph Eur
24-23
Lormetazepam
Me
O
OH
H
Cl
Cl
and enantiomer
C16H12Cl2N2O2
335.2
848-75-9
Definition Lormetazepam is (RS)-7-chloro-5-(2-chlorophenyl)-1,3-dihydro-3-hydroxy-1-methyl1,4-benzodiazepin-2-one. It contains not less than 99.0% and not more than 101.0% of
C16H12Cl2N2O2, calculated with reference to the dried substance.
Characteristics A white, crystalline powder.
Practically insoluble in water; soluble in ethanol (96%) and in methanol.
Identification A. The infrared absorption spectrum, Appendix II A, is concordant with the reference
spectrum of lormetazepam (RS 207).
B. In the test for Related substances, the chromatogram obtained with solution (2) shows a peak with
the same retention time as the principal peak in the chromatogram obtained with solution (4).
Related substances Carry out the method for liquid chromatography, Appendix III D, using five
solutions in methanol containing (1) 1.0% w/v of the substance being examined, (2) 0.0020% w/v of
the substance being examined, (3) 0.0010% w/v of the substance being examined, (4) 0.0020% w/v
of lormetazepam BPCRS, (5) 0.0010% w/v each of lormetazepam BPCRS and lorazepam BPCRS.
The chromatographic procedure may be carried out using (a) a column (20 cm 4.6 mm) packed
with stationary phase C (5 m) (Hypersil ODS is suitable), (b) as the mobile phase with a flow rate of
2 ml per minute a mixture of 48 volumes of methanol and 52 volumes of a phosphate buffer prepared
by dissolving 4.91 g of sodium dihydrogen orthophosphate and 0.633 g of disodium hydrogen orthophosphate in sufficient water to produce 1000 ml and (c) a detection wavelength of 230 nm.
The test is not valid unless the resolution factor between the two principal peaks in the chromatogram obtained with solution (5) is at least 4.
that of the principal peak in the chromatogram obtained with solution (2) (0.2%) and not more than
two such peaks have an area greater than the area of the principal peak in the chromatogram obtained
with solution (3) (0.1%). The sum of the areas of all such peaks is not greater than 2.5 times the area
of the principal peak obtained with solution (2) (0.5%).
Loss on drying When dried to constant weight at 105 for 3 hours, loses not more 1.0% of its
weight. Use 1 g.
Assay Dissolve 0.5 g in 50 ml of nitroethane and carry out Method I for non-aqueous titration,
Appendix VIII A, determining the end point potentiometrically. Each ml of 0.1M perchloric acid VS is
equivalent to 33.52 mg of C16H12Cl2N2O2.
Storage Lormetazepam should be kept in a well-closed container and protected from light.
Action and use Hypnotic; anxiolytic.
Preparation
Lormetazepam Tablets
IMPURITIES
NHMe
Cl
O
Cl
A. 2-methylamino-2,5-dichlorobenzophenone
24-24
Me
OAc
H
Cl
Cl
B. O3-acetyl-lormetazepam
Me
O
Cl
NH
Cl
C. 7-chloro-1-methyl-5-(2-chlorophenyl)-4,5-dihydro-2H-1,4-benzodiazepin-2,3(1H)-dione
24-25
Lovage Root
Lovage Root complies with the requirements of the 3rd edition of the European Pharmacopoeia [1233]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Lovage root consists of the whole or cut, dried rhizome and root of Levisticum officinale Koch. The
whole drug contains not less than 4.0 ml/kg of essential oil and the cut drug not less than 3.0 ml/kg of
essential oil, calculated with reference to the anhydrous drug.
CHARACTERS
IDENTIFICATION
A. The rhizome and the large roots are often split longitudinally. The rhizome is short, up to 5 cm in
diameter, light greyish-brown or yellowish-brown, simple or with several protuberances; the roots,
showing little ramification, are the same colour as the rhizome; they are usually up to 1.5 cm thick
and up to about 25 cm long; the fracture is usually smooth and shows a very wide yellowish-white
bark and a narrow brownish-yellow wood.
B. Reduce to a powder (355). The powder is brownish-yellow. Examine under a microscope using
chloral hydrate solution R. The powder shows: cork cells polygonal or rounded in surface view brown
contents; abundant parenchyma, mostly thin-walled and rounded but some with thicker walls; groups
of small, reticulately thickened vessels embedded in small-celled, unlignified parenchyma; fragments
of larger vessels with reticulate thickening, up to 125 m in diameter; fragments of secretory canals
up to 180 m wide. Examine under a microscope using a 50 per cent V/V solution of glycerol R. The
powder shows starch granules, simple, rounded to ovoid, up to about 12 m, and numerous larger,
compound granules, many with several components.
C. Examine the chromatogram obtained with the reference solution in the test for angelicae radix in
ultraviolet light at 254 nm and locate the quenching zone (eugenol). Examine in ultraviolet light at
365 nm. The chromatogram obtained with the test solution shows a principal zone with intense pale
blue to greenish-blue fluorescence. This zone is slightly below that of eugenol in the chromatogram
obtained with the reference solution. There are one or two smaller zones with the same fluorescence
immediately below the principal zone. Other zones with less intense fluorescence are visible in the
lower part of the chromatogram obtained with the test solution.
TESTS
Angelicae radix Examine by thin-layer chromatography (2.2.27) using as the coating substance a
suitable silica gel with a fluorescent indicator having an optimal intensity at 254 nm.
Test solution. Shake 2 g of the freshly powdered drug (500) with 10 ml of a mixture of equal volumes
of methanol R and of methylene chloride R for 10 min and filter.
Reference solution. Dissolve 50 mg of eugenol R in a mixture of equal volumes of methanol R and of
methylene chloride R. Dilute to 10.0 ml with the same mixture of solvents.
mixture of equal volumes of methylene chloride R and of toluene R. Allow the plate to dry in air and
develop again over a path of 10 cm using the same mobile phase. Allow the plate to dry in air and
examine in ultraviolet light at 365 nm. The chromatogram obtained with the test solution shows no
intense blue or bluish-violet fluorescent zone in the lower third.
Foreign matter (2.8.2). Not more than 3 per cent, determined on 50 g.
Ash insoluble in hydrochloric acid (2.8.1). Not more than 2.0 per cent.
Water (2.2.13). Not more than 12.0 per cent, determined on 25.00 g by distillation.
ASSAY
Carry out the determination of essential oils in vegetable drugs (2.8.12). Use a 2 litre flask, ten drops
of liquid paraffin R, 500 ml of water R as the distillation liquid and 0.50 ml of xylene R in the
graduated tube. Reduce the drug to a powder (500) and immediately use 40.0 g for the determination. Distil at a rate of 2 ml/min to 3 ml/min for 4 h.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
24-26
Lovastatin
1/01
HO
H
O
O
H3C
H
O
H
H
H
Me
Me
H
Me
C24H36O5
404.5
75330-75-5
Lovastatin complies with the requirements of the 3rd edition of the European Pharmacopoeia [1538]. These
Action and use Hypolipidaemic.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Lovastatin contains not less than 97.0 per cent and not more than the equivalent of 102.0 per cent of
(1S,3R,7S,8S,8aR)-8-[2-[(2R,4R)-4-hydroxy-6-oxotetrahydro-2H-pyran-2-yl]ethyl]-3,7-dimethyl1,2,3,7,8,8a-hexahydronaphthalen-1-yl (2S)-2-methylbutanoate, calculated with reference to the
dried substance.
PRODUCTION
Where applicable, it complies with the requirements of the monograph on Products of fermentation
(1468).
CHARACTERS
A white or almost white crystalline powder, practically insoluble in water, soluble in acetone, sparingly soluble in ethanol.
IDENTIFICATION
A. It complies with the test for specific optical rotation (see Tests).
B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum
obtained with lovastatin CRS. Examine the substances prepared as discs.
TESTS
Appearance of solution Dissolve 0.200 g in acetonitrile R and dilute to 20.0 ml with the same
solvent. The solution is clear (2.2.1) and not more intensely coloured than reference solution B6 or
BY6 (Method II, 2.2.2).
Specific optical rotation (2.2.7). Dissolve 0.125 g in acetonitrile R and dilute to 25.0 ml with the
same solvent. The specific optical rotation is +325 to +340, calculated with reference to the dried
substance.
Related substances Examine by liquid chromatography (2.2.29) as described under Assay.
Inject 10 l of reference solution (b). Adjust the sensitivity of the system so that the height of the
principal peak is at least 20 per cent of the full scale of the recorder. Inject 10 l of test solution (a).
When the chromatograms are recorded under the prescribed conditions the relative retentions are:
impurity A about 0.8, impurity B about 0.6, impurity C about 1.2 and impurity D about 2.3 (retention time of lovastatin: about 7 min). In the chromatogram obtained with test solution (a): the area of
any peak, apart from the principal peak, is not greater than 0.6 times the area of the principal peak in
the chromatogram obtained with reference solution (b) (0.3 per cent); the sum of the areas of all
peaks, apart from the principal peak, is not greater than twice the area of the principal peak in the
chromatogram obtained with reference solution (b) (1 per cent). Disregard any peak with an area less
than 0.1 times the area of the principal peak in the chromatogram obtained with reference solution
(b) (0.05 per cent).
Heavy metals (2.4.8).1.0 g complies with limit test C for heavy metals (20 ppm). Prepare the
standard using 2 ml of lead standard solution (10 ppm Pb) R.
24-27
desiccator under high vacuum at 60C for 3 h.
ASSAY
Test solution (a). Dissolve 20.0 mg of the substance to be examined in acetonitrile R and dilute to
50.0 ml with the same solvent.
Test solution (b). Dilute 10.0 ml of test solution (a) to 20.0 ml with acetonitrile R.
Reference solution (a). Dissolve 10.0 mg of lovastatin CRS in acetonitrile R and dilute to 50.0 ml with
the same solvent.
Reference solution (b). Dilute 0.5 ml of test solution (a) to 100.0 ml with acetonitrile R.
Reference solution (c). To 5.0 ml of reference solution (a) add 1 mg of simvastatin CRS and dilute to
50.0 ml with acetonitrile R.
gel for chromatography R (5 m),
Mobile phase A. Acetonitrile R,
Mobile phase B. A 0.1 per cent V/V solution of phosphoric acid R,
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
05
60
40
Isocratic
57
60 65
40 35
Linear gradient
713
65 90
35 10
Linear gradient
1315
90
10
Isocratic
1517
90 60
10 40
Linear gradient
1720
60
40
Re-equilibration

Inject 10 l of reference solution (c). The test is not valid unless, in the chromatogram obtained,
the resolution between the peaks corresponding to simvastatin and lovastatin is at least 5.0. When the
chromatograms are recorded in the prescribed conditions the relative retention of simvastatin is about
1.1 (retention time of lovastatin: about 7 min). Inject 10 l of reference solution (a). Adjust the
sensitivity of the system so that the height of the principal peak is at least 50 per cent of the full scale
of the recorder. Inject 10 l of test solution (b).
Calculate the content of C24H36O5 from the peak areas in the chromatograms obtained with test
solution (b) and reference solution (a), and the declared content of C24H36O5 in lovastatin CRS.
STORAGE
Store under nitrogen at a temperature of 2C to 8C.
IMPURITIES
O
R=
H3C
H Me
O H
H
Me
H
Me
HO
O
3C
H Me
H
O H
H
Me
H
A. (1S,7S,8S,8aR)-8-[2-[(2R,4R)-4-hydroxy-6-oxotetrahydro-2H-pyran-2-yl]ethyl]-7-methyl1,2,3,7,8,8a-hexahydronaphthalen-1-yl (2S)-2-methylbutanoate (mevastatin),
24-28
O
COOH
OH
R H
B. (3R,5R)-7-[(1S,2S,6R,8S,8aR)-2,6-dimethyl-8-[[(2S)-2-methylbutanoyl]oxy]1,2,6,7,8,8a-hexahydronaphthalen-1-yl]-3,5-dihydroxyheptanoic acid (hydroxyacid

lovastatin),
O
O
R H
C. (1S,3R,7S,8S,8aR)-3,7-dimethyl-8-[2-[(2R)-6-oxo-3,6-dihydro-2H-pyran-2-yl]ethyl]1,2,3,7,8,8a-hexahydronaphthalen-1-yl (2S)-2-methylbutanoate (dehydrolovastatin),

O
O
O
O
H
R H
O
OH
R H
D. (2R,4R)-2-[2-[(1S,2S,6R,8S,8aR)-2,6-dimethyl-8-[[(2S)-2-methylbutanoyl]oxy]1,2,6,7,8,8a-hexahydronaphthalen-1-yl]ethyl]-6-oxotetrahydro-2H-pyran-4-yl (3R,5R)-7[(1S,2S,6R,8S,8aR)-2,6-dimethyl-8-[[(2S)-2-methylbutanoyl]oxy]-1,2,6,7,8,8ahexahydronaphthalen-1-yl]-3,5-dihydroxyheptanoate (lovastatin dimer).

__________________________________________________________________________________________________________ Ph Eur
24-29
Lymecycline
992-21-2
Definition Lymecycline is a water-soluble combination of tetracycline, lysine and formaldehyde.
The potency is not less than 900 IU per mg, calculated with reference to the anhydrous substance.
Characteristics A yellow powder; very hygroscopic.
Very soluble in water; slightly soluble in ethanol (96%); practically insoluble in acetone, in chloroform
and in ether.
Identification
A. Carry out the method for thin-layer chromatography, Appendix III A, using silica gel H as the coating substance and a mixture of 6 volumes of water, 35 volumes of methanol and 59 volumes of
dichloromethane as the mobile phase. Adjust the pH of a 10% w/v solution of disodium edetate to 8.0
with 10M sodium hydroxide and spray the solution evenly onto the plate (about 10 ml for a plate
100 mm 200 mm). Allow the plate to dry in a horizontal position for at least 1 hour. Before use,
dry the plate in an oven at 110 for 1 hour. Apply separately to the plate 1 l of each of three solutions in methanol containing (1) 0.05% w/v of the substance being examined, (2) 0.05% w/v of
lymecycline BPCRS and (3) 0.05% w/v each of tetracycline hydrochloride EPCRS, chlortetracycline
hydrochloride EPCRS and doxycycline hyclate EPCRS. After removal of the plate, allow it to dry in a
current of air and examine under ultraviolet light (365 nm). The principal spot in the chromatogram
obtained with solution (1) is similar in position, colour and size to that in the chromatogram obtained
with solution (2). The test is not valid unless the chromatogram obtained with solution (3) shows
three clearly separated spots.
B. To 0.5 mg add 2 ml of sulphuric acid. A purplish red colour is produced.
C. Dissolve 50 mg in 5 ml of water, add 50 mg of ninhydrin, boil and add 15 ml of water. A bluish
violet colour is produced.
D. Dissolve 0.2 g in 5 ml of water, add 0.3 ml of orthophosphoric acid and distil. To 1 ml of the
distillate add 10 ml of chromotropicsulphuric acid solution. A violet colour is produced.
Alkalinity pH of a 1% w/v solution, 7.8 to 8.1, Appendix V L.
Light absorption To 10 ml of a 0.01% w/v solution in 0.01M hydrochloric acid add 75 ml of water
and 5 ml of 5M sodium hydroxide, add sufficient water to produce 100 ml and mix immediately. The
absorbance of the resulting solution at the maximum at 380 nm, when measured exactly 6 minutes
after the addition of the sodium hydroxide solution, is 0.27 to 0.31, calculated with reference to the
anhydrous substance, Appendix II B.
Specific optical rotation In a 0.5% w/v solution, 180 to 210, calculated with reference to the
anhydrous substance, Appendix V F.
Light-absorbing impurities The absorbance of a 0.25% w/v solution in 0.01M hydrochloric acid at
430 nm, when measured within 1 hour of preparing the solution, is not more than 0.50, calculated
with reference to the anhydrous substance, Appendix II B.
Free tetracycline To 0.5 g add 50 ml of butyl acetate and allow to stand for 1 hour at 25. Filter and
extract the filtrate with two 25-ml quantities of 0.1M hydrochloric acid. Combine the extracts, add
sufficient 0.1M hydrochloric acid to produce 50 ml and dilute 10 ml of this solution to 100 ml with
0.1M hydrochloric acid. The absorbance of the resulting solution at 355 nm is not more than 0.64,
calculated with reference to the anhydrous substance, Appendix II B.
plate prepared in the following manner. Boil 50 g of keiselguhr G with a mixture of 250 ml of
hydrochloric acid and 250 ml of water for 10 minutes, filter and wash the filter with water until the
washings are alkaline to congo red solution. Dry the residue at 105 and slurry 25 g with a mixture of
2.5 ml of a 20% v/v solution of polyethylene glycol 400 in glycerol and 47.5 ml of 0.1M disodium edetate
previously adjusted to pH 7 with 5M ammonia. After spreading the plate, allow it to dry at room
temperature until the surface acquires a uniform matt appearance (usually after 1 to 2 hours) and
place in a tank the atmosphere of which has been allowed to equilibrate with a saturated solution of
ammonium chloride for at least 24 hours. Allow the plate to remain in the tank for 24 hours and use
immediately after removal. Use as the mobile phase the solution obtained by shaking 5 ml of 0.1M
disodium edetate, previously adjusted to pH 7 with 5M ammonia, with 200 ml of a mixture consisting
of 3 volumes of acetone, 1 volume of chloroform and 1 volume of ethyl acetate, the emulsion being
removed by filtering through absorbent cotton. Apply separately to the plate 1 l of each of the
following solutions. For solution (1) add 5 ml of water and 1 ml of a 4% w/v solution of sodium
metabisulphite to 0.125 g of the substance being examined and allow to stand for 16 hours at room
temperature, without stirring. Add sufficient 0.1M hydrochloric acid to produce 10 ml and filter if
necessary. Solutions (2) to (5) are freshly prepared solutions in methanol containing (2) 0.0050% w/v
of anhydrotetracycline hydrochloride EPCRS, (3) 0.0050% w/v of 4-epianhydrotetracycline hydrochloride
24-30
EPCRS, (4) 0.020% w/v of chlortetracycline hydrochloride EPCRS and (5) 0.050% w/v of 4-epitetracycline hydrochloride EPCRS. After removal of the plate, allow it to dry in air, expose to the vapour of
13.5M ammonia and examine under ultraviolet light (365 nm). Any secondary spot in the chromatogram
obtained with solution (1) is not more intense than the corresponding spot in the chromatograms
obtained with solutions (2) to (5).
Water Not more than 5.0% w/w, Appendix IX C. Use 0.5 g.
Assay Carry out the biological assay of antibiotics, Appendix XIV A. The precision of the assay is such
that the fiducial limits of error are not less than 95% and not more than 105% of the estimated
potency.
Storage Lymecycline should be kept in a well-closed container, protected from light and stored at a
temperature not exceeding 25.
Labelling The label states (1) the number of IU (Units), per mg; (2) the date after which the
material is not intended to be used; (3) the conditions under which it should be stored.
Action and use Antibacterial.
Preparation
Lymecycline Capsules
135 mg of Lymecycline is equivalent to approximately 100 mg of tetracycline.
24-31
Lynestrenol
Me
OH
C
H
H
C20H28O
CH
H
H
284.4
52-76-6
Lynestrenol complies with the requirements of the 3rd edition of the European Pharmacopoeia [0558]. These
Action and use Progestogen.
When lynoestrenol is prescribed or demanded, Lynestrenol shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Lynestrenol contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent
of 19-nor-17-pregn-4-en-20-yn-17-ol, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, practically insoluble in water, soluble in acetone, in
alcohol and in ether.
IDENTIFICATION
obtained with lynestrenol CRS.
C. Examine the chromatograms obtained in the test for related substances in ultraviolet light at
365 nm. The principal spot in the chromatogram obtained with test solution (b) is similar in position,
fluorescence and size to the principal spot in the chromatogram obtained with reference solution (b).
TESTS
Appearance of solution Dissolve 0.2 g in alcohol R and dilute to 10 ml with the same solvent. The
solution is clear (2.2.1) and colourless (Method II, 2.2.2).
Specific optical rotation (2.2.7). Dissolve 0.900 g in alcohol R and dilute to 25.0 ml with the same
solvent. The specific optical rotation is 9.5 to 11, calculated with reference to the dried
substance.
Related substances Examine by thin-layer chromatography (2.2.27), using silica gel G R as the
coating substance.
Test solution (a). Dissolve 0.125 g of the substance to be examined in chloroform R and dilute to 25 ml
Test solution (b). Dilute 5 ml of test solution (a) to 10 ml with chloroform R.
Reference solution (a). Dilute 1 ml of test solution (a) to 100 ml with chloroform R. Dilute 5 ml of the
solution to 10 ml with chloroform R.
Reference solution (b). Dissolve 25 mg of lynestrenol CRS in chloroform R and dilute to 10 ml with the
same solvent.
20 volumes of acetone R and 80 volumes of heptane R. Allow the plate to dry in air, spray with 0.25M
alcoholic sulphuric acid and heat at 105C for 10 min. Examine in ultraviolet light at 365 nm. Any spot
intense than the spot in the chromatogram obtained with reference solution (a) (0.5 per cent).
100C to 105C.
24-32
ASSAY
Dissolve 0.150 g in 40 ml of tetrahydrofuran R and add 5.0 ml of a 100 g/l solution of silver nitrate R.
Titrate with 0.1M sodium hydroxide. Determine the end-point potentiometrically (2.2.20), using a
glass indicator electrode and as comparison electrode a silver-silver chloride double-junction electrode with a saturated solution of potassium nitrate R as junction liquid. Carry out a blank titration.
1 ml of 0.1M sodium hydroxide is equivalent to 28.44 mg of C20H28O.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
24-33
Lysine Hydrochloride
H
H 2N
C6H14N2O2,HCl
NH2
COOH
182.7
,HCl
657-27-2
Lysine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia [0930].
Action and use Amino acid.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Lysine hydrochloride contains not less than 98.5 per cent and not more than the equivalent of
101.0 per cent of (S)-2,6-diaminohexanoic acid hydrochloride, calculated with reference to the dried
substance.
PRODUCTION
When Lysine hydrochloride is produced by a process involving fermentation steps, it complies with
the requirements of the monograph on Products of fermentation (1468).
CHARACTERS
A white, crystalline powder or colourless crystals, freely soluble in water, slightly soluble in alcohol,
practically insoluble in ether.
IDENTIFICATION
First identification: A, B, E.
Second identification: A, C, D, E.
obtained with lysine hydrochloride CRS. Examine the substances prepared as discs. If the spectra
obtained show differences, dissolve the substance to be examined and the reference substance
separately in the minimum volume of water R, evaporate to dryness at 60C, and record new spectra
using the residues.
C. Examine the chromatograms obtained in the test for ninhydrin-positive substances. The principal
spot in the chromatogram obtained with test solution (b) is similar in position, colour and size to the
principal spot in the chromatogram obtained with reference solution (a).
D. To 0.1 ml of solution S (see Tests) add 2 ml of water R and 1 ml of a 50 g/l solution of
phosphomolybdic acid R. A yellowish-white precipitate is formed.
E. To 0.1 ml of solution S add 2 ml of water R. The solution gives reaction (a) of chlorides (2.3.1).
TESTS
Appearance of solution Solution S is clear (2.2.1) and not more intensely coloured than reference
solution B7 or GY7 (Method II, 2.2.2).
Specific optical rotation (2.2.7). Dissolve 2.00 g in hydrochloric acid R1 and dilute to 25.0 ml with
the same acid. The specific optical rotation is +21.0 to +22.5, calculated with reference to the dried
substance.
Ninhydrin-positive substances Examine by thin-layer chromatography (2.2.27), using a TLC silica
gel plate R.
Test solution (a). Dissolve 0.10 g of the substance to be examined in water R and dilute to 10 ml with
the same solvent.
Test solution (b). Dilute 1 ml of test solution (a) to 50 ml with water R.
Reference solution (a). Dissolve 10 mg of lysine hydrochloride CRS in water R and dilute to 50 ml with
the same solvent.
Reference solution (b). Dilute 5 ml of test solution (b) to 20 ml with water R.
Reference solution (c). Dissolve 10 mg of lysine hydrochloride CRS and 10 mg of arginine CRS in water R
24-34
30 volumes of concentrated ammonia R and 70 volumes of 2-propanol R. Dry the plate at 100C to
105C until the ammonia disappears completely. Spray with ninhydrin solution R and heat at 100C
to 105C for 15 min. Any spot in the chromatogram obtained with test solution (a), apart from the
principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (b) (0.5 per cent). The test is not valid unless the chromatogram obtained with reference solution (c) shows two clearly separated spots.
Sulphates (2.4.13). Dilute 5 ml of solution S to 15 ml with distilled water R. The solution complies
with the limit test for sulphates (300 ppm).
Ammonium Prepare a cell consisting of two watch-glasses 60 mm in diameter placed edge to edge.
To the inner wall of the upper watch-glass stick a piece of red litmus paper R 5 mm square and wetted
with a few drops of water R. Finely powder the substance to be examined, place 50 mg in the lower
watch-glass and dissolve in 0.5 ml of water R. To the solution add 0.30 g of heavy magnesium oxide R.
Briefly triturate with a glass rod. Immediately close the cell by putting the two watch-glasses together.
Heat at 40C for 15 min. The litmus paper is not more intensely blue coloured than a standard
prepared at the same time and in the same manner using 0.1 ml of ammonium standard solution
(100 ppm NH4) R, 0.5 ml of water R and 0.30 g of heavy magnesium oxide R (200 ppm).
Iron (2.4.9). In a separating funnel, dissolve 0.33 g in 10 ml of dilute hydrochloric acid R. Shake with
three quantities, each of 10 ml, of methyl isobutyl ketone R1, shaking for 3 min each time. To the
combined organic layers add 10 ml of water R and shake for 3 min. The aqueous layer complies with
the limit test for iron (30 ppm).
100C to 105C.
ASSAY
Dissolve 0.150 g in 5 ml of anhydrous formic acid R. Add 50 ml of anhydrous acetic acid R. Titrate with
0.1M perchloric acid, determining the end-point potentiometrically (2.2.20).
1 ml of 0.1M perchloric acid is equivalent to 18.27 mg of C6H15ClN2O2.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
24-35
Macrogols
revised 1/01
Macrogols comply with the requirements of the 3rd edition of the European Pharmacopoeia [1444]. These
The label states the type of macrogol in terms of a nominal relative molecular mass.
Action and use Pharmaceutical aids.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Macrogols are mixtures of polymers with the general formula H-(OCH2-CH2)n-OH, corresponding
to an average relative molecular mass of the labelled nominal value (n). A suitable stabiliser may be
added.
CHARACTERS
Clear, viscous, colourless or almost colourless, hygroscopic liquids, or white or almost white solids
with a waxy or paraffin-like appearance, miscible with or very soluble in water, in alcohol and in
methylene chloride, practically insoluble in fatty oils and in mineral oils.
IDENTIFICATION
A. It complies with the test for viscosity (see Tests).
B. To 1 g in a test-tube add 0.5 ml of sulphuric acid R, close the test-tube with a stopper fitted with a
bent delivery tube and heat until white fumes are evolved. Collect the fumes via the delivery tube into
1 ml of mercuric chloride solution R. An abundant white, crystalline precipitate is formed.
C. To 0.1 g add 0.1 g of potassium thiocyanate R and 0.1 g of cobalt nitrate R, and mix thoroughly with
a glass rod. Add 5 ml of methylene chloride R and shake. The liquid phase becomes blue.
TESTS
Appearance of solution Dissolve 12.5 g in water R and dilute to 50 ml with the same solvent. The
solution is clear (2.2.1) and not more intensely coloured than reference solution BY6 (2.2.2, Method
II).
Acidity or alkalinity Dissolve 5.0 g in 50 ml of carbon dioxide-free water R and add 0.15 ml of
bromothymol blue solution R1. The solution is yellow or green. Not more than 0.1 ml of 0.1M sodium
hydroxide is required to change the colour of the indicator to blue.
Viscosity (2.2.9). The viscosity is calculated using a density given in Table 1444.-1.
Table 14441
Type of
macrogol
300
400
600
1000
1500
3000
3350
4000
6000
8000
20,000
35,000
Kinematic
viscosity
(mm2sec 1)
Dynamic
viscosity
(mPas)
Density
71 to 94
94 to 116
13.9 to 18.5
20.4 to 27.7
31 to 46
69 to 93
76 to 110
102 to 158
185 to 250
240 to 472
2500 to 3200
10,000 to 13,000
80 to 105
105 to 130
15 to 20
22 to 30
34 to 50
75 to 100
83 to 120
110 to 170
200 to 270
260 to 510
2700 to 3500
11,000 to 14,000
1.120
1.120
1.080
1.080
1.080
1.080
1.080
1.080
1.080
1.080
1.080
1.080
(g/ml)
For macrogols having a relative molecular mass greater than 400, determine the viscosity on a 50 per
cent m/m solution of the substance to be examined.
Freezing point (2.2.18). See Table 1444.-2.
24-36
Table 14442
Type of macrogol
Freezing point (C)
600
15 to 25
35 to 40
42 to 48
50 to 56
53 to 57
53 to 59
55 to 61
55 to 62
not less than 57
not less than 57
1000
1500
3000
3350
4000
6000
8000
20,000
35,000
Hydroxyl value Introduce m g (see Table 1444.-3) into a dry conical flask fitted with a reflux
condenser. Add 25.0 ml of phthalic anhydride solution R, swirl to dissolve and boil under a reflux
condenser on a hot plate for 60 min. Allow to cool. Rinse the condenser first with 25 ml of pyridine R
and then with 25 ml of water R, add 1.5 ml of phenolphthalein solution R and titrate with 1M sodium
hydroxide until a faint pink colour is obtained (n1 ml). Carry out a blank test (n2 ml). Calculate the
hydroxyl value using the expression:
56.1 (n2 n1 )
m
Table 14443
Type of macrogol
300
400
600
1000
1500
3000
3350
4000
6000
8000
20,000
35,000
Hydroxyl value
m (g)
340 to 394
264 to 300
178 to 197
107 to 118
70 to 80
34 to 42
30 to 38
25 to 32
16 to 22
12 to 16
1.5
1.9
3.5
5.0
7.0
12.0
12.0
14.0
18.0
24.0
For macrogols having a relative molecular mass greater than 1000, if the water content is more than
0.5 per cent, dry a sample of suitable mass at 100C to 105C for 2 h and carry out the determination of the hydroxyl value on the dried sample.
Reducing substances Dissolve 1 g in 1 ml of a 10 g/l solution of resorcinol R and warm gently if
necessary. Add 2 ml of hydrochloric acid R. After 5 min the solution is not more intensely coloured
than reference solution R3 (2.2.2, Method I).
Formaldehyde
Test solution. To 1.00 g add 0.25 ml of chromotropic acid, sodium salt solution R, cool in iced water and
add 5.0 ml of sulphuric acid R. Allow to stand for 15 min and complete slowly to 10 ml with water R.
Reference solution. Dilute 0.430 g of formaldehyde solution R to 100 ml with water R. Dilute 1.0 ml of
this solution to 100 ml with water R. In a 10 ml flask, mix 1.00 ml of this solution with 0.25 ml of
chromotropic acid, sodium salt solution R, cool in iced water and add 5.0 ml of sulphuric acid R. Allow to
stand for 15 min and complete slowly to 10 ml with water R.
Blank solution. In a 10 ml flask mix 1.00 ml of water R with 0.25 ml of chromotropic acid, sodium salt
solution R, cool in iced water and add 5.0 ml of sulphuric acid R. Complete slowly to 10 ml with
water R.
Determined at 567 nm against the blank solution, the absorbance of the test solution is not higher
than that of the reference solution (15 ppm).
Ethylene glycol and diethylene glycol Carry out this test if the macrogol has a relative molecular mass
below 1000. Not more than 0.4 per cent calculated as the sum of the contents of ethylene glycol and
diethylene glycol, determined by gas chromatography (2.2.28).
Test solution. Dissolve 5.00 g of the substance to be examined in acetone R and dilute to 100.0 ml with
the same solvent.
24-37
Reference solution. Dissolve 0.10 g of ethylene glycol R and 0.50 g of diethylene glycol R in acetone R and
dilute to 100.0 ml with the same solvent. Dilute 1.0 ml of this solution to 10.0 ml with acetone R.
a glass column 1.8 m long and 2 mm in internal diameter packed with silanised diatomaceous
earth for gas chromatography R, impregnated with 5 per cent m/m of macrogol 20,000 R,
nitrogen for chromatography R as the carrier gas at a flow rate of 30 ml/min,
a flame-ionisation detector.
If necessary, precondition the column by heating at 200C for about 15 h. Maintain the temperature
of the injection port and that of the detector at 250C. Adjust the initial temperature of the column
to obtain a retention time of 14 min to 16 min for diethylene glycol.
Inject 2 l of each solution and raise the temperature of the column by about 30C at a rate of 2C
per minute but without exceeding 170C. Carry out five replicate injections to check the repeatability
of the response.
Measure the areas of the peaks corresponding to ethylene glycol and diethylene glycol in the
chromatograms obtained with the test and reference solutions. Calculate the concentrations of
ethylene glycol and diethylene glycol in the test solution.
Residual ethylene oxide and dioxan (2.4.25). Not more than 1 ppm of ethylene oxide and not
more than 10 ppm of dioxan. When determining the dioxan content, apply a correction factor of 1/5
to the calculation expression.
Heavy metals (2.4.8). Dissolve 2.0 g in water R and dilute to 20 ml with the same solvent. 12 ml of
the solution complies with limit test A for heavy metals (20 ppm). Prepare the standard using lead
standard solution (2 ppm Pb) R.
Water (2.5.12). Determined on 2.00 g by the semi-micro determination of water, the water content
of macrogol with a maximal nominal molecular mass of 1000 is not more than 2.0 per cent, and the
water content of macrogol with a molecular mass greater than 1000 is not more than 1.0 per cent.
LABELLING
The label states:
the name and the concentration of any added stabiliser,
the type of macrogol.
__________________________________________________________________________________________________________ Ph Eur
24-38
Macrogol Cetostearyl Ether

Macrogol Cetostearyl Ether complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Pharmaceutical aid.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Macrogol cetostearyl ether is a mixture of ethers of mixed macrogols with linear fatty alcohols, mainly
cetostearyl alcohol. It may contain some free macrogols and it contains various amounts of free
cetostearyl alcohol. The amount of ethylene oxide reacted with cetostearyl alcohol is from two to
thirty-three units per molecule (nominal value).
CHARACTERS
White or yellowish-white waxy, unctuous mass, pellets, microbeads or flakes.
Macrogol cetostearyl ether with low numbers of ethylene oxide units per molecule is practically
insoluble in water, soluble in alcohol and in methylene chloride.
Macrogol cetostearyl ether with higher numbers of ethylene oxide units per molecule is dispersible
or soluble in water, soluble in alcohol and in methylene chloride.
It solidifies at 32C to 52C.
IDENTIFICATION
A. It complies with the test for hydroxyl value (see Tests).
B. It complies with the test for iodine value (see Tests).
C. It complies with the test for saponification value (see Tests).
D. Examine by thin-layer chromatography (2.2.27), using a suitable silica gel as the coating
substance.
Test solution. Dissolve the amount of substance to be examined given in the following table in a
mixture of 1 volume of water R and 9 volumes of methanol R and dilute to 75 ml with the same
mixture of solvents.
Table 11231
Ethylene oxide units
per molecule
Amount to be
dissolved
26
10 22
25 33
5.0 g
10.0 g
15.0 g
Add 60 ml of hexane R and shake for 3 min. The formation of foam can be reduced by the addition
of some drops of alcohol R. Pass the hexane layer through a filter with anhydrous sodium sulphate R,
wash the filter with three quantities, each of 10 ml, of hexane R and evaporate the combined filtrates
to dryness. Dissolve 0.05 g of the dried hexane extract in 10 ml of methanol R (sometimes the solution is opalescent).
Reference solution. Dissolve 25 mg of stearyl alcohol CRS in methanol R and dilute to 25 ml with the
same solvent.
Apply separately to the plate 20 l of each solution. Develop over a path of 15 cm using ethyl
acetate R. Dry and spray with vanillin-sulphuric acid reagent prepared as follows: dissolve 0.50 g of
vanillin R in 50.0 ml of alcohol R and dilute to 100.0 ml with sulphuric acid R. Allow the plate to dry
in air. Heat the plate at about 130C for 15 min and allow to cool in air. The chromatogram
obtained with the test solution shows several spots; one of these spots corresponds to the principal
spot in the chromatogram obtained with the reference solution.
E. Dissolve or disperse 0.1 g in 5 ml of alcohol R, add 2 ml of water R, 10 ml of dilute hydrochloric
acid R, 10 ml of barium chloride solution R1 and 10 ml of a 100 g/l solution of phosphomolybdic acid R.
A precipitate is formed.
TESTS
Appearance of solution. Dissolve 5.0 g in alcohol R and dilute to 50 ml with the same solvent. The
solution is not more intensely coloured than reference solution BY5 (Method II, 2.2.2).
Alkalinity. Dissolve 2.0 g in a hot mixture of 10 ml of water R and 10 ml of alcohol R. Add 0.1 ml of
bromothymol blue solution R1. Not more than 0.5 ml of 0.1M hydrochloric acid is required to change the
colour of the indicator to yellow.
24-39
Acid value (2.5.1). Not more than 1.0, determined on 5.0 g.
Hydroxyl value (2.5.3, method A). See Table 1123-2.
Table 11232

per molecule
(nominal value)
Hydroxyl value
2
3
5 6
10
12
15
20 - 22
25
30 33
150
135
100
75
67
58
40
36
32
180
155
134
90
77
67
55
46
40
Iodine value (2.5.4) Not more than 2.0.

Saponification value (2.5.6). Not more than 3.0, determined on 10.0 g.
1,4-Dioxan Not more than 10 ppm, determined by the method for residual solvents (2.4.24).
Ethylene oxide and dioxan (2.4.25) Not more than 1 ppm of ethylene oxide and Not more than
100 ppm of dioxan. When determining the dioxan content, apply a correction factor of 1/5 to the
calculation.
Water (2.5.12). Not more than 3.0 per cent, determined on 2.00 g by the semi-micro determination
of water.
Total ash (2.4.16). Not more than 0.2 per cent, determined on 2.0 g.
STORAGE
LABELLING
The label states the amount of ethylene oxide reacted with cetostearyl alcohol (nominal value).
__________________________________________________________________________________________________________ Ph Eur
24-40
Macrogol 6 Glycerol Caprylocaprate

Macrogol 6 Glycerol Caprylocaprate complies with the requirements of the 3rd edition of the European
Pharmacopoeia [1443]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Macrogol 6 glycerol caprylocaprate is a mixture of mainly mono- and diesters of polyoxyethylene
glycerol ethers mainly with caprylic (octanoic) and capric (decanoic) acids. The average content of
the ethylene oxide is 6 units per molecule. Macrogol 6 glycerol caprylocaprate may be obtained by
ethoxylation of glycerol and esterification with distilled coconut or palm kernel fatty acids, or by
ethoxylation of mono- and diglycerides of caprylic and capric acids.
CHARACTERS
A pale yellow liquid, partly soluble in water, freely soluble in castor oil, in glycerol, in isopropanol
and in propylene glycol.
It has a viscosity of about 145 mPas.
IDENTIFICATION
A. Dissolve 1.0 g in 99 g of a mixture of 10 volumes of 2-propanol R and 90 volumes of water R. Heat
the solution obtained to about 40C. A turbidity is produced. Allow to cool until the turbidity
disappears. The cloud point is between 15C and 35C.
B. It complies with the test for saponification value (see Tests).
C. It complies with the test for composition of fatty acids (see Tests).
TESTS
Appearance The substance to be examined is clear (2.2.1) and not more intensely coloured than
reference solution Y2 (Method I, 2.2.2).
Alkalinity Dissolve 2.0 g in a hot mixture of 10 ml of alcohol R and 10 ml of water R. Add 0.1 ml of
Hydroxyl value (2.5.3). 165 to 225 (Method A).
Saponification value (2.5.6). 85 to 105, determined on 2.0 g.
Composition of fatty acids Carry out the test for foreign oils in fatty oils by gas chromatography
(2.4.22, Method A). The fatty acid fraction has the following composition:
caproic acid: not more than 2.0 per cent,
caprylic acid: 50.0 per cent to 80.0 per cent,
capric acid: 20.0 per cent to 50.0 per cent,
lauric acid: not more than 3.0 per cent,
myristic acid: not more than 1.0 per cent.
Residual ethylene oxide and dioxan (2.4.25). Not more than 1 ppm of ethylene oxide and not
more than 10 ppm of dioxan. When determining the dioxan content, apply a correction factor of 1/5
to the calculation.
of water.
__________________________________________________________________________________________________________ Ph Eur
24-41
Macrogol Glycerol Cocoates

Macrogol Glycerol Cocoates comply with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Pharmaceutical aids.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Macrogol glycerol cocoates are mixtures of mono-, di- and triesters of ethoxylated glycerol with fatty
acids of vegetable origin having a composition corresponding to the fatty acid composition of the oil
extracted from the hard, dried fraction of the endosperm of Cocos nucifera L. The average content of
ethylene oxide is either 7 units per molecule or 23 units per molecule (nominal value).
CHARACTERS
A clear, yellowish, oily liquid, soluble in water and in alcohol and practically insoluble in light
petroleum (50C to 70C) for macrogol 7 glycerol cocoate; soluble in water and in alcohol and
practically insoluble in light petroleum (50C to 70C) for macrogol 23 glycerol cocoate. Macrogol 7
glycerol cocoate has a relative density of about 1.05 and macrogol 23 glycerol cocoate has a relative
density of about 1.09.
IDENTIFICATION
A. Dissolve 1.0 g of macrogol 7 glycerol cocoate in 99 g of a mixture of 10 volumes of 2-propanol R
and 90 volumes of water R. Heat the solution to about 65C. A turbidity is produced. Allow to cool
until the turbidity disappears. The cloud point is between 35C and 54C.
Heat a 10 g/l solution of macrogol 23 glycerol cocoate in a 100 g/l solution of sodium chloride R to
about 90C. A turbidity is produced. Allow to cool until the turbidity disappears. The cloud point is
between 65C and 85C.
B. They comply with the test for iodine value (see Tests).
C. They comply with the test for saponification value (see Tests).
TESTS
Alkalinity Dissolve 2.0 g in a hot mixture of 10 ml of water R and 10 ml of alcohol R. Add 0.1 ml of
Hydroxyl value (2.5.3, Method A). See Table 11221 below.
Saponification value (2.5.6). See Table 11221 below.
Table 11221
Number of units
of ethylene oxide
(nominal value)
Hydroxyl value
Saponification value
(determined on 2.0 g)
7
23
170 to 210
80 to 100
85 to 105
40 to 50
Iodine value (2.5.4). Not more than 5.0.

lauric acid: 40.0 per cent to 55.0 per cent,
myristic acid: 14.0 per cent to 23.0 per cent,
palmitic acid: 8.0 per cent to 12.0 per cent,
stearic acid: 1.0 per cent to 5.0 per cent,
oleic acid: 5.0 per cent to 10.0 per cent,
linoleic acid: not more than 3.0 per cent,
Residual ethylene oxide and dioxan (2.4.25). Not more than 1 ppm of residual ethylene oxide
and not more than 10 ppm of residual dioxan. When determining the dioxan content, apply a
correction factor of 1/5 to the calculation expression.
24-42
of water.
LABELLING
The label states the number of ethylene oxide units per molecule (nominal value).
__________________________________________________________________________________________________________ Ph Eur
24-43
Macrogol Lauryl Ether

Macrogol Lauryl Ether complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Macrogol lauryl ether is a mixture of ethers of mixed macrogols with linear fatty alcohols, mainly
lauryl alcohol. It may contain some free macrogols and it contains various amounts of free lauryl
alcohol. The amount of ethylene oxide reacted with lauryl alcohol is from three to twenty-three units
per molecule (nominal value).
CHARACTERS
Macrogol lauryl ether with three to five units of ethylene oxide per molecule is a colourless liquid,
practically insoluble in water, soluble or dispersible in alcohol, practically insoluble in light
petroleum.
Macrogol lauryl ether with nine to twenty-three units of ethylene oxide per molecule is a white,
waxy mass, soluble or dispersible in water, soluble in alcohol, practically insoluble in light petroleum.
IDENTIFICATION
substance.
Test solution. Dissolve 0.20 g of the substance to be examined in methanol R and dilute to 5 ml with
the same solvent.
Reference solution. Dissolve 40 mg of lauryl alcohol CRS in methanol R and dilute to 10 ml with the
same solvent. Dilute 2 ml of this solution to 10 ml with methanol R.
Impregnate the plate by placing it in a chromatography tank containing the necessary quantity of a
30 g/l solution of oxalic acid R in alcohol R. Allow to dry in air and apply separately to the plate 5 l of
each solution. Develop twice over a path of 15 cm, using ethyl acetate R. Dry after each migration and
spray with vanillin-sulphuric acid reagent prepared as follows: dissolve 0.50 g of vanillin R in 50.0 ml
of alcohol R and dilute to 100.0 ml with sulphuric acid R. Allow the plate to dry in air. Heat the plate
at about 130C for 10 min and allow to cool in air. The chromatogram obtained with the test solution shows several spots; one of these spots corresponds to the principal spot in the chromatogram
obtained with the reference solution.
E. Dissolve or disperse 0.1 g in 5 ml of alcohol R, add 10 ml of dilute hydrochloric acid R, 10 ml of
barium chloride solution R1 and 10 ml of a 100 g/l solution of phosphomolybdic acid R. A precipitate is
formed.
TESTS
Hydroxyl value (2.5.3, method A). See Table 1124-1.
24-44
Table 11241
per molecule
(nominal value)
Hydroxyl value
3
4
5
9
10
12
15
20 to 23
165 to 180
145 to 165
130 to 140
90 to 100
85 to 95
73 to 83
64 to 74
40 to 60

1,4-Dioxan. Not more than 10 ppm, determined by the method for residual solvents (2.4.24).
Ethylene oxide. Not more than 1 ppm, determined by head-space gas chromatography (2.2.28).
Test solution. Weigh 1.00 g (mT) of the substance to be examined in a vial of appropriate volume and
add 1 ml of water R. If necessary, heat to 50C for about 10 min.
Reference solution (a). Weigh 1.00 g (mR) of the substance to be examined in a vial of appropriate
volume and add 0.20 g of cooled ethylene oxide solution R and 0.8 ml of water R. If necessary, heat to
50C for about 10 min.
Reference solution (b). To 0.1 g of ethylene oxide solution R in a vial of appropriate volume, add 0.1 ml
of a freshly prepared 10 mg/l solution of acetaldehyde R.
Close the vials with a butyl-rubber membrane stopper, coated with aluminium or
polytetrafluoroethylene and secure with an aluminium crimped cap. Mix to obtain a homogeneous
solution.
The following static head-space injection conditions may be used:
equilibration temperature: at least 70C,
equilibration time: 45 min,
transfer-line temperature: 75C,
carrier gas: helium for chromatography R or nitrogen for chromatography R,
pressurisation time: 30 s,
injection volume: 1 ml.
a glass or quartz capillary column 30 m long and 0.32 mm in internal diameter, the inner surface
of which is coated with a 1.0 m layer of polydimethylsiloxane R,
helium for chromatography R or nitrogen for chromatography R as carrier gas with a linear velocity of
about 20 cm/s and a split ratio of 1:20,
a flame-ionisation detector,
maintaining the temperature of the column at 50C for 5 min, then raising the temperature at a rate
of 30C per minute to 230C and maintaining at 230C for 5 min; maintaining the temperature of
the injection port at 150C and that of the detector at 250C.
Inject 1 ml of the gaseous phase above reference solution (b). Adjust the sensitivity of the system so
that the heights of the two principal peaks in the chromatogram obtained are not less than 15 per
cent of the full scale of the recorder. The test is not valid unless: in the chromatogram obtained with
reference solution (b), the resolution between the peaks corresponding to acetaldehyde and ethylene
oxide is at least 2.0; the peak due to ethylene oxide in the chromatogram obtained with reference
solution (b) has a signal- to-noise ratio of at least 10.
Inject separately suitable volumes (for example 1 ml) of the gaseous phases of the test solution and
reference solution (a). Repeat the procedure twice more. The mean area of the ethylene oxide peak in
the chromatogram obtained with the test solution is not greater than half the mean area of the corresponding peak in the chromatogram obtained with reference solution (a).
The test is not valid unless the relative standard deviation of the area for three injections of reference solution (a) is at most 15 per cent.
Calculate the content of ethylene oxide in parts per million from the expression:
AT
( AR m T ) ( AT m R )
AT = area of the peak corresponding to ethylene oxide in the chromatogram obtained with the
test solution,
AR = area of the peak corresponding to ethylene oxide in the chromatogram obtained with
reference solution (a).
24-45
of water.
STORAGE
LABELLING
The label states the amount of ethylene oxide reacted with lauryl alcohol (nominal value).
__________________________________________________________________________________________________________ Ph Eur
25-1
Macrogol Oleyl Ether

Macrogol Oleyl Ether complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Macrogol oleyl ether is a mixture of ethers of mixed macrogols with linear fatty alcohols, mainly oleyl
alcohol. It may contain some free macrogols and it contains various amounts of free oleyl alcohol.
The amount of ethylene oxide reacted with oleyl alcohol is from two to twenty units per molecule
(nominal value).
CHARACTERS
Macrogol oleyl ether with two to five units of ethylene oxide per molecule is a yellow liquid,
practically insoluble in water, soluble in alcohol, practically insoluble in light petroleum.
Macrogol oleyl ether with ten to twenty units of ethylene oxide per molecule is a yellowish-white
waxy mass, dispersible or soluble in water, soluble in alcohol, practically insoluble in light petroleum.
IDENTIFICATION
substance.
the same solvent.
Reference solution. Dissolve 40 mg of oleyl alcohol CRS in methanol R and dilute to 10 ml with the same
solvent. Dilute 2 ml of this solution to 10 ml with methanol R.
Impregnate the plate by placing it in a chromatography tank containing the necessary quantity of a
30 g/l solution of oxalic acid R in alcohol R. Allow to dry in air and apply separately to the plate 5 l of
each solution. Develop twice over a path of 15 cm using ethyl acetate R. Dry after each migration and
spray with a vanillin-sulphuric acid reagent prepared as follows: dissolve 0.50 g of vanillin R in
50.0 ml of alcohol R and dilute to 100.0 ml with sulphuric acid R. Allow the plate to dry in air. Heat
the plate at about 130C for 10 min and allow to cool in air. The chromatogram obtained with the
test solution shows several spots; one of these spots corresponds to the principal spot in the chromatogram obtained with the reference solution.
TESTS
Hydroxyl value (2.5.3, Method A). See Table 11251.
Iodine value (2.5.4). See Table 11251.
Table 11251
per molecule
(nominal value)
Hydroxyl value
Iodine value
2
5
10
20
158 to 178
110 to 125
75 to 95
40 to 65
48 to 74 *
48 to 56
24 to 38
14 to 24
*This broad range is needed since two different grades of oleyl

alcohol may be used for the synthesis.
25-2
1,4-Dioxan Not more than 10 ppm, determined by the method for residual solvents (2.4.24).
Ethylene oxide. Not more than 1 ppm, determined by head-space gas chromatography (2.2.28).
Test solution. Weigh 1.00 g (mT) of the substance to be examined in a vial of appropriate volume and
add 1 ml of water R. If necessary, heat to 50C for about 10 min.
Reference solution (a). Weigh 1.00 g (mR) of the substance to be examined in a vial of appropriate
volume and add 0.20 g of cooled ethylene oxide solution R and 0.8 ml of water R. If necessary, heat to
50C for about 10 min.
Reference solution (b). To 0.1 g of ethylene oxide solution R in a vial of appropriate volume add 0.1 ml of
a freshly prepared 10 mg/l solution of acetaldehyde R.
Close the vials with a butyl-rubber membrane stopper, coated with aluminium or
polytetrafluoroethylene and secure with an aluminium crimped cap. Mix to obtain a homogeneous
solution.
The following static head-space injection conditions may be used:
equilibration temperature: at least 70C,
equilibration time: 45 min,
transfer-line temperature: 75C,
carrier gas: helium for chromatography R or nitrogen for chromatography R,
pressurisation time: 30 s,
injection volume: 1 ml.
a glass or quartz capillary column 30 m long and 0.32 mm in internal diameter, the inner surface
of which is coated with a 1.0 m layer of polydimethylsiloxane R,
helium for chromatography R or nitrogen for chromatography R as the carrier gas with a linear
velocity of about 20 cm/s and a split ratio of 1:20,
of 30C per minute to 230C and maintaining at 230C for 5 min; maintaining the temperature of
the injection port at 150C and that of the detector at 250C.
Inject 1 ml of the gaseous phase above reference solution (b). Adjust the sensitivity of the system so
that the heights of the two principal peaks in the chromatogram obtained are not less than 15 per
cent of the full scale of the recorder. The test is not valid unless: in the chromatogram obtained with
reference solution (b), the resolution between the peaks corresponding to acetaldehyde and ethylene
oxide is at least 2.0; the peak due to ethylene oxide in the chromatogram obtained with reference
solution (b) has a signal- to-noise ratio of at least 10.
Inject separately suitable volumes (for example, 1 ml) of the gaseous phases above the test solution
and reference solution (a). Repeat the procedure twice more. The mean area of the ethylene oxide
peak in the chromatogram obtained with the test solution is not greater than half the mean area of the
corresponding peak in the chromatogram obtained with reference solution (a).
The test is not valid unless the relative standard deviation of the area for three injections of reference solution (a) is at most 15 per cent.
Calculate the content of ethylene oxide in parts per million from the expression:
AT
( AR m T ) ( AT m R )
AT = area of the peak corresponding to ethylene oxide in the chromatogram obtained with the
test solution,
AR = area of the peak corresponding to ethylene oxide in the chromatogram obtained with
of water.
STORAGE
LABELLING
The label states the amount of ethylene oxide reacted with oleyl alcohol (nominal value).
__________________________________________________________________________________________________________ Ph Eur
25-3
Macrogol Stearate
Macrogol Stearate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1234].
Ph Eur ___________________________________________________________________________________________________________
25-4
DEFINITION
Macrogol stearate is a mixture of the monoesters and diesters of mainly stearic acid and macrogols.
It may be obtained by ethoxylation of stearic acid or by esterification of macrogols with stearic
acid. It may contain free macrogols. The average polymer length is equivalent to 6 to 100 ethylene
oxide units per molecule (nominal value).
PRODUCTION
Where applicable, it complies with the monograph on Products with risk of transmitting agents of animal
spongiform encephalopathies (1483).
CHARACTERS
White or slightly yellowish waxy mass, soluble in alcohol and in 2-propanol. Macrogol stearate
corresponding to a product with 6 to 9 units of ethylene oxide per molecule is practically insoluble
but freely dispersible in water and miscible with fatty oil and with waxes. Macrogol stearate corresponding to a product with 20 to 100 units of ethylene oxide per molecule is soluble in water and
practically insoluble in fatty oils and in waxes.
IDENTIFICATION
A. It complies with the test for saponification value (see Tests).
B. Examine by thin-layer chromatography (2.2.27), using a suitable octadecylsilyl silica gel for high
performance chromatography as the coating substance.
Test solution. To 1 g add 100 ml of a 100 g/l solution of potassium hydroxide R and boil under a reflux
condenser for 30 min. Acidify the warm solution with 15 ml of hydrochloric acid R, dissolve in the
mixture 10 g of sodium chloride R and allow to cool. Shake the mixture with 50 ml of ether R and allow
to stand until a separation of the layers is visible. Transfer 20 ml of the upper, cloudy layer to a
suitable centrifuge tube and centrifuge for 10 min. Transfer 10 ml of the clear ether solution to a
suitable tube and evaporate to dryness on a water-bath. Dissolve the residue in 2.0 ml of ether R.
Reference solution. Dissolve 30 mg of palmitic acid R and 30 mg of stearic acid R in 2.0 ml of ether R.
10 volumes of methylene chloride R, 40 volumes of glacial acetic acid R and 50 volumes of acetone R.
Dry the plate in a current of cold air, spray with a 200 g/l solution of phosphomolybdic acid R in
alcohol R and heat at 120C for about 10 min. The chromatogram obtained with the reference
solution shows two principal, clear spots on a greenish-blue ground, corresponding, in order of
increasing Rf value, to stearic and palmitic acid. A crescent-shaped blue spot may be visible at the top
of the spot due to stearic acid. The chromatogram obtained with the test solution shows spots which
are similar in position and colour to the spots in the chromatogram obtained with the reference
solution. Disregard any other spots.
TESTS
Acidity or alkalinity Dissolve 1.0 g in 10.0 ml of a 90 per cent V/V solution of ethanol R. To 2 ml
of the solution add 0.05 ml of methyl red solution R. The solution is orange. To a further 2 ml of the
solution add 0.05 ml of bromothymol blue solution R1. The solution is not blue.
Melting point (2.2.15). Ensure that the column of powdered substance is at least 10 cm from the
end of the capillary tube. See Table 1234.-1.
Hydroxyl value (2.5.3 Method A). See Table 1234.-1.
Saponification value (2.5.6). See Table 1234.-1.
Table 12341
Ethylene oxide
units per
molecule
(nominal value)
6
89
20
40 50
100
Melting point
26C to 35C
33C to 40C
38C to 52C
48C to 55C
Hydroxyl
value
Saponification
value
90 to 110
80 to 105
50 to 62
23 to 40
15 to 30
85 to 105
88 to 100
46 to 56
20 to 35
5 to 20
Reducing substances Dissolve or disperse 2.0 g in water R and dilute to 20 ml with the same
solvent. Mix 1.0 ml of the solution with 9 ml of 0.1M sodium hydroxide and 0.5 ml of triphenyltetrazolium chloride solution R. Heat in a water-bath at 70C. After 5 min, the solution is not more
25-5
intensely coloured than a mixture of 0.15 ml of yellow primary solution, 0.9 ml of red primary
solution and 8.95 ml of a 10 g/l solution of hydrochloric acid R (2.2.2, Method II).
correction factor of 1/5 in the calculation.
Heavy metals (2.4.8). 2.0 g complies with the limit test C for heavy metals (10 ppm). Prepare the
of water. Use as the solvent a mixture of equal volumes of methylene chloride R and anhydrous
methanol R.
STORAGE
LABELLING
The label states the number of ethylene oxide units per molecule (nominal value).
__________________________________________________________________________________________________________ Ph Eur
Macrogol Stearyl Ether

Macrogol Stearyl Ether complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Macrogol stearyl ether is a mixture of ethers obtained by ethoxylation of stearyl alcohol. It may
contain some free macrogols and various amounts of free stearyl alcohol. The amount of ethylene
oxide reacted with stearyl alcohol is two to twenty units per molecule (nominal value).
PRODUCTION
25-6
CHARACTERS
A white to yellowish-white, waxy, unctuous mass, pellets, microbeads or flakes.
Macrogol stearyl ether with two units of ethylene oxide per molecule is practically insoluble in water,
soluble in alcohol with heating and in methylene chloride. Macrogol stearyl ether with ten units of
ethylene oxide per molecule is soluble in water and in alcohol. Macrogol stearyl ether with twenty
units of ethylene oxide per molecule is soluble in water, in alcohol and in methylene chloride.
After melting, it solidifies at about 45C.
IDENTIFICATION
D. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel plate R.
Test solution. Dissolve 10.0 g in a mixture of 1 volume of water R and 9 volumes of methanol R and
dilute to 75 ml with the same mixture of solvents. Add 60 ml of heptane R and shake for 3 min. The
formation of foam can be reduced by the addition of a few drops of alcohol R. Filter the upper layer
through anhydrous sodium sulphate R, wash the filter with three quantities, each of 10 ml, of heptane R
and evaporate the combined filtrates to dryness. Dissolve 50 mg of the residue in 10 ml of methanol R
(the solution may be opalescent).
Reference solution. Dissolve 25 mg of stearyl alcohol CRS in methanol R and dilute to 25 ml with the
same solvent.
Apply to the plate 20 l of each solution. Develop over a path of 15 cm using ethyl acetate R. Dry and
spray with vanillin-sulphuric acid reagent prepared as follows: dissolve 0.5 g of vanillin R in 50 ml of
alcohol R and dilute to 100 ml with sulphuric acid R. Allow the plate to dry in air. Heat the plate at
about 130C for 15 min and allow to cool in air. The chromatogram obtained in the test solution
shows several spots; one of these spots corresponds to the principal spot in the chromatogram
TESTS
solution is not more intensely coloured than reference solution BY5 (2.2.2, Method II).
Alkalinity Dissolve 2.0 g in a hot mixture of 10 ml of alcohol R and 10 ml of water R. Add 0.1 ml of
Hydroxyl value (2.5.3, Method A). See Table 1340.-1.
Table 13401
Ethylene oxide
units per
molecule
(nominal value)
Hydroxyl value
2
10
20
150 to 180
75 to 90
40 to 60

correction factor of 1/5 to the calculation.
of water.
STORAGE
25-7
LABELLING
The label states the number of ethylene oxide units reacted with stearyl alcohol (nominal value).
__________________________________________________________________________________________________________ Ph Eur
Magaldrate
1/01
Al5Mg10(OH)31(SO4)2,xH2O
1097 (anhydrous)
74978-16-8
Magaldrate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1539]. These
Action and use Antacid.
Preparation
Magaldrate Oral Suspension
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Magaldrate is composed of aluminium and magnesium hydroxides and sulphates. Its composition
corresponds approximately to the formula Al5Mg10(OH)31(SO4)2,xH2O. It contains not less than
90.0 per cent and not more than the equivalent of 105.0 per cent of Al5Mg10(OH)31(SO4)2, calculated with reference to the dried substance.
25-8
CHARACTERS
A white or almost white, crystalline powder, practically insoluble in water and in alcohol, soluble in
dilute mineral acids.
IDENTIFICATION
A. Dissolve 0.6 g in 20 ml of 3M hydrochloric acid, add about 30 ml of water R and heat to boiling.
Adjust to pH 6.2 with dilute ammonia R1, continue boiling for a further 2 min, filter and retain the
precipitate and the filtrate. To 2 ml of the filtrate add 2 ml of ammonium chloride solution R and
neutralise with a solution prepared by dissolving 2 g of ammonium carbonate R and 2 ml of dilute
ammonia R1 in 20 ml of water R; no precipitate is produced. Add disodium hydrogen phosphate solution R; a white, crystalline precipitate is produced which does not dissolve in dilute ammonia R1.
B. The precipitate retained in identification test A gives the reaction of aluminium (2.3.1).
C. The filtrate retained in identification test A gives reaction (a) of sulphates (2.3.1).
TESTS
Soluble chlorides Disperse 1 g in 50 ml of water R, boil for 5 min, cool, dilute again to 50.0 ml, mix
and filter. To 25.0 ml of the filtrate add 0.2 ml of potassium chromate solution R and titrate with 0.1M
silver nitrate until a persistent violet-red colour is obtained. Not more than 5.0 ml of 0.1M silver nitrate
is required (3.5 per cent).
Soluble sulphates To 2.5 ml of the filtrate obtained in the test for soluble chlorides, add 30 ml of
water R, neutralise to blue litmus paper R with hydrochloric acid R, add 3 ml of 1M hydrochloric acid,
3 ml of a 120 g/l solution of barium chloride R and dilute to 50 ml with water R. Mix and allow to
stand for 10 min. Any opalescence in the test solution is not more intense than that of a standard
prepared at the same time in the same manner using 1 ml of 0.01M sulphuric acid instead of 2.5 ml of
filtrate (1.9 per cent).
Sulphates 16.0 per cent to 21.0 per cent, calculated with reference to the dried substance. Dissolve
0.875 g in a mixture of 5 ml of glacial acetic acid R and 10 ml of water R and dilute to 25 ml with
water R. Prepare a chromatographic column of 1 cm in internal diameter containing 15 ml of cation
exchange resin R (150 m to 300 m), previously washed with 30 ml of water R. Transfer 5 ml of the
solution to be examined to the column and elute with 15 ml of water R. To the eluate add 5 ml of a
536 g/l solution of magnesium acetate R, 32 ml of methanol R and 0.2 ml of alizarin S solution R. Add
from a burette about 5.0 ml of 0.05M barium chloride, add a further 0.2 ml of alizarin S solution R and
slowly complete the titration until the yellow colour disappears and a violet-red tinge is visible.
1 ml of 0.05M barium chloride is equivalent to 4.803 mg of SO4.
Aluminium hydroxide 32.1 per cent to 45.9 per cent, calculated with reference to the dried
substance. Dissolve 0.800 g in 10 ml of dilute hydrochloric acid R1, heating on a water-bath. Cool and
dilute to 50.0 ml with water R. To 10.0 ml of the solution, add dilute ammonia R1 until a precipitate
begins to appear. Add the smallest quantity of dilute hydrochloric acid R needed to dissolve the
precipitate and dilute to 20 ml with water R. Carry out the complexometric titration of aluminium
(2.5.11).
1 ml of 0.1M sodium edetate is equivalent to 7.80 mg of Al(OH)3.
Magnesium hydroxide 49.2 per cent to 66.6 per cent, calculated with reference to the dried
substance. Dissolve 0.100 g in 2 ml of dilute hydrochloric acid R and carry out the complexometric
titration of magnesium (2.5.11).
1 ml of 0.1M sodium edetate is equivalent to 5.832 mg of Mg(OH)2.
Sodium Not more than 0.10 per cent of Na, determined by atomic absorption spectrometry (Method
I, 2.2.23).
Test solution. Weigh 2.00 g of the substance to be examined into a 100 ml volumetric flask, place in
an ice-bath, add 5 ml of nitric acid R and swirl to mix. Allow to warm to room temperature and dilute
to 100 ml with water R. Filter, if necessary, to obtain a clear solution. Dilute 10.0 ml of the filtrate to
100.0 ml with water R.
Reference solutions. Prepare the reference solutions using sodium standard solution (200 ppm Na) R,
diluted as necessary with dilute nitric acid R.
Measure the absorbance at 589 nm using a sodium hollow-cathode lamp as a source of radiation
and an air-acetylene flame.
Heavy metals (2.4.8). Dissolve 2.0 g in 30 ml of hydrochloric acid R1 and shake with 50 ml of methyl
isobutyl ketone R for 2 min. Allow to stand, separate the aqueous layer and evaporate to dryness.
Dissolve the residue in 30 ml of water R.12 ml of the solution complies with limit test A for heavy
metals (30 ppm). Prepare the standard using lead standard solution (2 ppm Pb) R.
Loss on drying (2.2.32).10.0 per cent to 20.0 per cent, determined on 1.000 g by drying in an oven
at 200C for 4 h.
ASSAY
25-9
To 1.500 g add 50.0 ml of 1M hydrochloric acid. Titrate the excess hydrochloric acid with 1M sodium
hydroxide to pH 3.0, determining the end-point potentiometrically (2.2.3). Carry out a blank titration.
1 ml of 1M hydrochloric acid is equivalent to 35.40 mg of Al5Mg10(OH)31(SO4)2.
__________________________________________________________________________________________________________ Ph Eur
Magnesium Acetate
C4H6MgO4,4H2O
214.5
16674-78-5
Definition Magnesium Acetate contains not less than 98.0% and not more than 100.5% of
C4H6MgO4,4H2O.
Characteristics Colourless crystals or a white, crystalline powder; odourless or almost odourless.
Freely soluble in water and in ethanol (96%).
Identification Yields the reactions characteristic of magnesium salts and of acetates, Appendix VI.
Aluminium Dissolve 4.0 g in 100 ml of water and add 10 ml of acetate buffer pH 6.0. The resulting
solution complies with the limit test for aluminium, Appendix VII (1 ppm).
Calcium 1.0 g dissolved in sufficient water to produce 15 ml complies with the limit test for calcium,
Appendix VII (100 ppm).
Heavy metals Dissolve 1.0 g in 20 ml of water. 12 ml of the resulting solution complies with limit
test A for heavy metals, Appendix VII. Use lead standard solution (2 ppm Pb) to prepare the standard
(40 ppm).
25-10
Potassium Not more than 0.1% of K when determined by Method II for atomic emission spectrophotometry, Appendix II D, measuring at 766.7 nm. Prepare a 0.5% w/v solution and use potassium
standard solution (600 ppm K), suitably diluted with water, to prepare the standard solutions.
Sodium Not more than 0.5% of Na when determined by Method II for atomic emission spectrophotometry, Appendix II D, measuring at 589.0 nm. Prepare a 1.0% w/v solution and use sodium
solution (200 ppm Na), suitably diluted with water, to prepare the standard solutions.
Chloride Dissolve 1.0 g in 100 ml of water. 15 ml of the resulting solution complies with the limit test
for chlorides, Appendix VII (330 ppm).
Nitrate Dissolve 1.0 g in 10 ml of water, add 5 mg of sodium chloride, 0.05 ml of indigo carmine
solution and, with stirring, 10 ml of nitrogen-free sulphuric acid. A blue colour is produced which
persists for at least 10 minutes.
Sulphate 0.25 g complies with the limit test for sulphates, Appendix VII (600 ppm).
Readily oxidisable substances Dissolve 2.0 g in 100 ml of boiling water, add 6 ml of 5M sulphuric
acid and 0.3 ml of 0.02M potassium permanganate, mix and boil gently for 5 minutes. The pink colour
is not completely discharged.
Assay Carry out the complexometric titration of magnesium, Appendix VIII D, using 0.5 g. Each ml of
0.1M disodium edetate VS is equivalent to 21.45 mg of C4H6MgO4, 4H2O.
Storage Magnesium Acetate should be kept in a well-closed container.
Action and use Used in dialysis solutions.
Magnesium Aspartate
1/01
H
g2+
NH2
HOOC
COO
C8H12MgN2O8,2H2O
324.5
72231-13-1
Magnesium Aspartate complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Magnesium Aspartate Dihydrate [1445]. These requirements are reproduced after the heading Definition
below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
25-11
Magnesium aspartate dihydrate contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent of magnesium di[(S)-2-aminohydrogenobutane-1,4-dioate], calculated with
reference to the anhydrous substance.
PRODUCTION
When magnesium aspartate dihydrate is produced by a process involving fermentation steps, it
complies with the requirements of the monograph on Products of fermentation (1468).
CHARACTERS
A white, crystalline powder or colourless crystals, freely soluble in water.
IDENTIFICATION
B. Examine the chromatograms obtained in the test for ninhydrin-positive substances. The principal
C. Ignite about 15 mg until a white residue is obtained. Dissolve the residue in 1 ml of dilute hydrochloric acid R, neutralise to red litmus paper R by the addition of dilute sodium hydroxide solution R and
filter if necessary. The solution gives the reaction of magnesium (2.3.1).
TESTS
pH (2.2.3). The pH of solution S is 6.0 to 8.0.
Specific optical rotation (2.2.7). Dissolve 0.50 g in 5M hydrochloric acid and dilute to 25.0 ml with
the same acid. The specific optical rotation is +20.5 to +23.0, calculated with reference to the
gel plate R.
the same solvent.
Reference solution (a). Dissolve 10 mg of magnesium aspartate dihydrate CRS in water R and dilute to
Reference solution (c). Dissolve 10 mg of magnesium aspartate dihydrate CRS and 10 mg of glutamic acid
CRS in 2 ml of water R and dilute to 25 ml with the same solvent.
Apply to the plate 5 l of each solution. Allow the plate to dry in air. Develop over a path of 15 cm
using a mixture of 20 volumes of glacial acetic acid R, 20 volumes of water R and 60 volumes of
butanol R. Allow the plate to dry in air and spray with ninhydrin solution R. Heat at 100C to 105C
for 15 min. Any spot in the chromatogram obtained with test solution (a), apart from the principal
spot, is not more intense than the spot in the chromatogram obtained with reference solution (b)
(0.5 per cent). The test is not valid unless the chromatogram obtained with reference solution (c)
shows two clearly separated principal spots.
with the limit test for sulphates (500 ppm). Carry out the evaluation of the test after 30 min.
Ammonium (2.4.1). 50 mg complies with limit test B for ammonium (200 ppm). Prepare the
standard using 0.1 ml of ammonium standard solution (100 ppm NH4) R.
Heavy metals (2.4.8). Dissolve 2.0 g with gentle heating in 20 ml of water R. 12 ml of the solution
complies with limit test A for heavy metals (10 ppm). Prepare the standard using lead standard
solution (1 ppm Pb) R.
Water (2.5.12). 10.0 per cent to 14.0 per cent, determined on 0.100 g by the semi-micro determination of water. Dissolve the substance in 10 ml of formamide R1 at 50C protected from moisture, add
10 ml of anhydrous methanol R and allow to cool. Carry out a blank determination.
25-12
ASSAY
Dissolve 0.260 g in 10 ml of water R and carry out the complexometric titration of magnesium
(2.5.11).
1 ml of 0.1M sodium edetate is equivalent to 28.85 mg of C8H12MgN2O8.
STORAGE
Store in a well-closed container.
IMPURITIES
A. (S)-2-aminobutane-1,4-dioic acid (aspartic acid).
__________________________________________________________________________________________________________ Ph Eur
Heavy Magnesium Carbonate

546-93-0
Heavy Magnesium Carbonate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0043]. These requirements are reproduced after the heading Definition below.
Action and use Antacid; osmotic laxative.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Heavy magnesium carbonate is a hydrated basic magnesium carbonate. It contains the equivalent of
not less than 40.0 per cent and not more than 45.0 per cent, calculated as MgO (Mr 40.30).
CHARACTERS
A white powder, practically insoluble in water. It dissolves in dilute acids with strong effervescence.
Apparent volume: 15 g occupies a volume of about 30 ml.
25-13
IDENTIFICATION
A. It gives the reaction of carbonates (2.3.1).
B. Dissolve about 15 mg in 2 ml of dilute nitric acid R and neutralise with dilute sodium hydroxide
solution R. The solution gives the reaction of magnesium (2.3.1).
TESTS
Solution S Dissolve 5.0 g in 100 ml of dilute acetic acid R. When the effervescence has ceased, boil
for 2 min, cool and dilute to 100 ml with the same acid. Filter, if necessary, through a previously
ignited and tared porcelain or silica filter crucible of suitable porosity to give a clear filtrate.
Appearance of solution Solution S is not more intensely coloured than reference solution B4
(Method II, 2.2.2).
Soluble substances Mix 2.00 g with 100 ml of water R and boil for 5 min. Filter whilst hot through
a sintered-glass filter (40), allow to cool and dilute to 100 ml with water R. Evaporate 50 ml of the
filtrate to dryness and dry at 100C to 105C. The residue weighs not more than 10 mg (1.0 per
cent).
Substances insoluble in acetic acid Any residue obtained during the preparation of solution S,
washed, dried, and ignited at 600C, weighs not more than 2.5 mg (0.05 per cent).
chlorides (0.07 per cent).
Sulphates (2.4.13). 0.5 ml of solution S diluted to 15 ml with distilled water R complies with the
limit test for sulphates (0.6 per cent).
Arsenic (2.4.2). 10 ml of solution S complies with limit test A for arsenic (2 ppm).
Calcium (2.4.3). Dilute 2.6 ml of solution S to 150 ml with distilled water R. 15 ml of the solution
complies with the limit test for calcium (0.75 per cent).
Heavy metals (2.4.8). To 20 ml of solution S add 15 ml of hydrochloric acid R1 and shake with
25 ml of methyl isobutyl ketone R for 2 min. Allow to stand, separate the aqueous layer and evaporate
to dryness. Dissolve the residue in 1 ml of acetic acid R and dilute to 20 ml with water R. 12 ml of the
solution complies with limit test A for heavy metals (20 ppm). Prepare the standard using lead
Iron (2.4.9). Dissolve 0.1 g in 3 ml of dilute hydrochloric acid R and dilute to 10 ml with water R.
2.5 ml of the solution diluted to 10 ml with water R complies with the limit test for iron (400 ppm).
ASSAY
Dissolve 0.150 g in a mixture of 20 ml of water R and 2 ml of dilute hydrochloric acid R. Carry out the
complexometric titration of magnesium (2.5.11).
1 ml of 0.1M sodium edetate is equivalent to 4.030 mg of MgO.
__________________________________________________________________________________________________________ Ph Eur
Light Magnesium Carbonate

546-93-0
Light Magnesium Carbonate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Antacid; Osmotic laxative.
Preparation
Aromatic Magnesium Carbonate Mixture
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Light magnesium carbonate is a hydrated basic magnesium carbonate. It contains the equivalent of
not less than 40.0 per cent and not more than 45.0 per cent, calculated as MgO (Mr 40.30).
CHARACTERS
A white powder, practically insoluble in water. It dissolves in dilute acids with strong effervescence.
25-14
IDENTIFICATION
A. It gives the reaction of carbonates (2.3.1).
B. Dissolve about 15 mg in 2 ml of dilute nitric acid R and neutralise with dilute sodium hydroxide
TESTS
Solution S Dissolve 5.0 g in 100 ml of dilute acetic acid R. When the effervescence has ceased, boil
for 2 min, cool and dilute to 100 ml with the same acid. Filter, if necessary, through a previously
ignited and tared porcelain or silica filter crucible of suitable porosity to give a clear filtrate.
(Method II, 2.2.2).
cent).
washed, dried and ignited at 600C, weighs not more than 2.5 mg (0.05 per cent).
Sulphates (2.4.13). 1 ml of solution S diluted to 15 ml with distilled water R complies with the limit
test for sulphates (0.3 per cent).
to dryness. Dissolve the residue in 1 ml of acetic acid R and dilute to 20 ml with water R. 12 ml of the
Iron (2.4.9) Dissolve 0.1 g in 3 ml of dilute hydrochloric acid R and dilute to 10 m with water R.
2.5 ml of the solution diluted to 10 ml with water R complies with the limit test for iron (400 ppm).
ASSAY
Dissolve 0.150 g in a mixture of 20 ml of water R and 2 ml of dilute hydrochloric acid R. Carry out the
__________________________________________________________________________________________________________ Ph Eur
Partially Hydrated Magnesium Chloride

MgCl2,~4.5H2O
95.21 (anhydrous)
Partially Hydrated Magnesium Chloride comlies with the requirements of the 3rd edition of the European
Pharmacopoeia for Magnesium Chloride 4.5 - Hydrate [1341]. These requirements are reproduced after the
heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Magnesium chloride 4.5-hydrate contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of MgCl2, calculated with reference to the anhydrous substance.
CHARACTERS
A white or almost white granular powder, hygroscopic, very soluble in water, freely soluble in alcohol.
IDENTIFICATION
A. It complies with the test for water (see Tests).
B. It gives reaction (a) of chlorides (2.3.1).
25-15
C. It gives the reaction of magnesium (2.3.1).
TESTS
Acidity or alkalinity To 5 ml of solution S add 0.05 ml of phenol red solution R. Not more than
indicator.
Bromides Dilute 2.0 ml of solution S to 10.0 ml with water R. To 1.0 ml of the solution add 4.0 ml
of water R, 2.0 ml of phenol red solution R3 and 1.0 ml of chloramine solution R2 and mix immediately.
After exactly 2 min add 0.30 ml of 0.1M sodium thiosulphate, mix and dilute to 10.0 ml with water R.
The absorbance (2.2.25) of the solution measured at 590 nm, using water R as the compensation
liquid, is not greater than that of a standard prepared at the same time and in the same manner using
5.0 ml of a 3 mg/l solution of potassium bromide R (500 ppm).
Sulphates (2.4.13). 15 ml of solution S complies with the limit test for sulphates (100 ppm).
Aluminium (2.4.17). If intended for use in the manufacture of peritoneal dialysis solutions,
haemodialysis solutions, or haemofiltration solutions it complies with the test for aluminium.
Dissolve 4 g in 100 ml of water R and add 10 ml of acetate buffer solution pH 6.0 R. The solution
complies with the limit test for aluminium (1 ppm). Use as the reference solution a mixture of 2 ml of
aluminium standard solution (2 ppm Al) R, 10 ml of acetate buffer solution pH 6.0 R and 98 ml of
water R. To prepare the blank use a mixture of 10 ml of acetate buffer solution pH 6. 0 R and 100 ml of
water R.
Calcium (2.4.3). Dilute 1 ml of solution S to 15 ml with distilled water R. The solution complies with
the limit test for calcium (0.1 per cent).
Iron (2.4.9). 10 ml of solution S complies with the limit test for iron (10 ppm).
Potassium If intended for use in the manufacture of parenteral dosage forms, not more than
500 ppm of K, determined by atomic emission spectrometry (Method I, 2.2.22).
Test solution. Dissolve 1.00 g of the substance to be examined in water R and dilute to 100.0 ml with
the same solvent.
Reference solution. Dissolve 1.144 g of potassium chloride R in water R, previously dried at 100C to
105C for 3 h, and dilute to 1000.0 ml with the same solvent (600 g of K per millilitre). Dilute as
required.
Water (2.5.12). 44.0 per cent to 48.0 per cent, determined on 50.0 mg by the semi-micro determination of water.
ASSAY
Dissolve 0.250 g in 50 ml of water R. Carry out the complexometric titration of magnesium (2.5.11).
1 ml of 0.1M sodium edetate is equivalent to 9.521 mg of MgCl2.
STORAGE
LABELLING
The label states:
where applicable, that the substance is suitable for use in the manufacture of peritoneal dialysis
solutions, haemodialysis solutions or haemofiltration solutions,
where applicable, that the substance is suitable for use in the manufacture of parenteral dosage
forms.
__________________________________________________________________________________________________________ Ph Eur
Magnesium Chloride Hexahydrate

MCl2,6H2O
203.3
7791-18-6
Magnesium Chloride Hexahydrate complies with the requirements of the 3rd edition of the European Pharma-
25-16
copoeia [0044]. These requirements are reproduced after the heading Definition below.
Action and use Used in the treatment of electrolyte deficiencies and in dialysis solutions.
Preparation
Magnesium Chloride Injection
When magnesium chloride is prescribed or demanded, Magnesium Chloride Hexahydrate shall be
dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Magnesium chloride hexahydrate contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent of MgCl2,6H2O.
CHARACTERS
25-17
Colourless crystals, hygroscopic, very soluble in water, freely soluble in alcohol.
IDENTIFICATION
A. It complies with the test for water (see Tests).
TESTS
indicator.
Bromides Dilute 2.0 ml of solutin S to 10.0 ml with water R. To 1.0 ml of the solution add 4.0 ml
of water R, 2.0 ml of phenol red solution R3 and 1.0 ml of chloramine solution R2 and mix immediately.
After exactly 2 min, add 0.30 ml of 0.1M sodium thiosulphate, mix and dilute to 10.0 ml with water R.
The absorbance (2.2.25) of the solution measured at 590 nm, using water R as the compensation
liquid, is not greater than that of a standard prepared at the same time and in the same manner using
5.0 ml of a 3 mg/l solution of potassium bromide R (500 ppm).
haemodialysis solutions, or haemofiltration solutions, it complies with the test for aluminium.
Dissolve 4 g in 100 ml of water R and add 10 ml of acetate buffer solution pH 6.0 R. The solution
complies with the limit test for aluminium (1 ppm). Use as the reference solution a mixture of 2 ml of
aluminium standard solution (2 ppm Al) R, 10 ml of acetate buffer solution pH 6.0 R and 98 ml of
water R. To prepare the blank use a mixture of 10 ml of acetate buffer solution pH 6.0 R and 100 ml of
water R.
Calcium (2.4.3). Dilute 1 ml of solution S to 15 ml with distilled water R. The solution complies with
the limit test for calcium (0.1 per cent).
Potassium If intended for use in the manufacture of parenteral dosage forms, not more than
500 ppm of K, determined by atomic emission spectrometry (Method I, 2.2.22).
the same solvent.
Reference solution. Dissolve 1.144 g of potassium chloride R, previously dried at 100C to 105C for 3 h
in water R and dilute to 1000.0 ml with the same solvent (600 g of K per millilitre). Dilute as
required.
ASSAY
Dissolve 0.300 g in 50 ml of water R. Carry out the complexometric titration of magnesium
(2.5.11).
1 ml of 0.1M sodium edetate is equivalent to 20.33 mg of MgCl2,6H2O.
STORAGE
LABELLING
The label states:
solutions, haemodialysis solutions or haemofiltration solutions,
forms.
__________________________________________________________________________________________________________ Ph Eur
25-18
Magnesium Glycerophosphate
O
g2+
O
HO
O
O
_
_
H OH
2+
and Mg
HO
OH
and enantiomer
C3H7MgO6P
194.4
927-20-8
Magnesium Glycerophosphate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1446]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Magnesium glycerophosphate is a mixture, in variable proportions, of magnesium (RS)-2,3dihydroxypropyl phosphate, and of magnesium 2-hydroxy-1-(hydroxymethyl)ethyl phosphate, which
25-19
may be hydrated. Magnesium glycerophosphate contains not less than 11.0 per cent and not more
than 12.5 per cent of Mg, calculated with reference to the dried substance.
CHARACTERS
A white powder, hygroscopic, practically insoluble in alcohol. It dissolves in dilute solutions of acids.
IDENTIFICATION
A. Mix 1 g with 1 g of potassium hydrogen sulphate R in a test tube fitted with a glass tube. Heat
strongly and direct the white vapour towards a piece of filter paper impregnated with a freshly
prepared 10 g/l solution of sodium nitroprusside R. The filter paper develops a blue colour in contact
with piperidine R.
B. Ignite 0.1 g in a crucible. Take up the residue with 5 ml of nitric acid R and heat on a water-bath
for 1 min. Filter. The filtrate gives reaction (b) of phosphates (2.3.1).
TESTS
Solution S Dissolve 2.5 g in carbon dioxide-free water R prepared from distilled water R, and dilute to
Appearance of solution Solution S is not more opalescent than reference suspension III (2.2.1).
Acidity Dissolve 1.0 g in 100 ml of carbon dioxide-free water R. Add 0.1 ml of phenolphthalein solution R. Not more than 1.5 ml of 0.1M sodium hydroxide is required to change the colour of the
indicator.
Glycerol and alcohol-soluble substances Shake 1.0 g with 25 ml of alcohol R for 2 min. Filter and
wash the residue with 5 ml of alcohol R. Combine the filtrate and the washings, evaporate to dryness
on a water-bath and dry the residue at 70C for 1 h. The residue weighs not more than 15 mg
(1.5 per cent).
Chlorides (2.4.4). Dissolve 1.0 g in water R and dilute to 100 ml with the same solvent. Dilute
3.5 ml of the solution to 15 ml with water R. The solution complies with the limit test for chlorides
(0.15 per cent).
Phosphates (2.4.11). Dilute 4 ml of solution S to 100 ml with water R. Dilute 1 ml of the solution to
100 ml with water R. The solution complies with the limit test for phosphates (0.5 per cent).
with the limit test for sulphates (0.1 per cent).
Iron (2.4.9). Dissolve 67 mg in water R and dilute to 10 ml with the same solvent. The solution
complies with the limit test for iron (150 ppm).
Heavy metals (2.4.8). To 20 ml of solution S add 15 ml of hydrochloric acid R and shake with 25 ml
of methyl isobutyl ketone R for 2 min. Allow to stand, separate the aqueous layer and evaporate to
dryness. Dissolve the residue in 2.5 ml of acetic acid R and dilute to 20 ml with water R. 12 ml of the
Loss on drying (2.2.32). Not more than 12.0 per cent, determined on 1.000 g by drying in an oven
at 150C for 4 h.
ASSAY
Dissolve 0.200 g in 40 ml of water R. Carry out the complexometric titration of magnesium
(2.5.11).
1 ml of 0.1M sodium edetate is equivalent to 2.431 mg of Mg.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
Magnesium Hydroxide
Mg(OH)2
58.32
1309-42-8
Magnesium Hydroxide complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparations
25-20
Co-magaldrox Oral Suspension
Co-magaldrox Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Magnesium hydroxide contains not less than 95.0 per cent and not more than the equivalent of
100.5 per cent of Mg(OH)2.
CHARACTERS
A white, fine, amorphous powder, practically insoluble in water, to which it gives a reaction alkaline
to phenolphthalein. It dissolves in dilute acids.
25-21
IDENTIFICATION
Dissolve about 15 mg in 2 ml of dilute nitric acid R and neutralise with dilute sodium hydroxide solution R. The solution gives the reaction of magnesium (2.3.1).
TESTS
Solution S Dissolve 5.0 g in a mixture of 50 ml of acetic acid R and 50 ml of distilled water R. Not
more than slight effervescence is produced. Boil for 2 min, cool and dilute to 100 ml with dilute acetic
acid R. Filter, if necessary, through a previously ignited and tared porcelain or silica filter crucible of
suitable porosity to give a clear filtrate.
(Method II, 2.2.2).
cent).
washed, dried, and ignited at 600C, weighs not more than 5 mg (0.1 per cent).
Chlorides (2.4.4). 1 ml of solution S diluted to 15 ml with water R complies with the limit test for
Arsenic (2.4.2). 5 ml of solution S complies with limit test A for arsenic (4 ppm)
Heavy metals (2.4.8). Dissolve 1.3 g in 15 ml of hydrochloric acid R1 and shake with 25 ml of methyl
isobutyl ketone R for 2 min. Allow to stand, separate the aqueous layer and evaporate to dryness.
Dissolve the residue in 20 ml of water R. 12 ml of the solution complies with limit test A for heavy
1 ml of this solution diluted to 10 ml with water R complies with the limit test for iron (0.07 per
cent).
Loss on ignition 30.0 per cent to 32.5 per cent. Heat 0.5 g gradually to 900C and ignite to
constant mass.
ASSAY
Dissolve 0.100 g in 2 ml of dilute hydrochloric acid R and carry out the complexometric titration of
magnesium (2.5.11).
1 ml of 0.1M sodium edetate is equivalent to 5.832 mg of Mg(OH)2.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
Heavy Magnesium Oxide

MgO
40.30
1309-48-4
Heavy Magnesium Oxide complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Heavy magnesium oxide contains not less than 98.0 per cent and not more than the equivalent of
100.5 per cent of MgO, calculated with reference to the ignited substance.
CHARACTERS
A fine, white powder, practically insoluble in water to which it gives a reaction alkaline to phenolphthalein. It dissolves in dilute acids with at most slight effervescence.
25-22
IDENTIFICATION
TESTS
Solution S Dissolve 5.0 g in a mixture of 70 ml of acetic acid R and 30 ml of distilled water R, boil for
2 min, cool and dilute to 100 ml with dilute acetic acid R. Filter, if necessary, through a previously
ignited and tared porcelain or silica filter crucible of suitable porosity to give a clear solution.
(Method II, 2.2.2).
cent).
25 ml of methyl isobutyl ketone R for 2 min. Separate the layers, evaporate the aqueous layer to
dryness, dissolve the residue in 1 ml of acetic acid R and dilute to 30 ml with water R. 12 ml of the
1 ml of the solution diluted to 10 ml with water R complies with the limit test for iron (0.07 per cent).
Loss on ignition Not more than 8.0 per cent, determined on 1.00 g at 900C.
ASSAY
Dissolve 0.700 g in 20 ml of dilute hydrochloric acid R and dilute to 100.0 ml with water R. Using
10.0 ml of the solution, carry out the complexometric titration of magnesium (2.5.11).
STORAGE
__________________________________________________________________________________________________________ Ph Eur
Light Magnesium Oxide

Light Magnesia
MgO
40.30
1309-48-4
Light Magnesium Oxide complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Magnesium Hydroxide Mixture
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Light magnesium oxide contains not less than 98.0 per cent and not more than the equivalent of
100.5 per cent of MgO, calculated with reference to the ignited substance.
CHARACTERS
25-23
A fine, white, amorphous powder, practically insoluble in water to which it gives a reaction alkaline
to phenolphthalein. It dissolves in dilute acids with at most slight effervescence.
IDENTIFICATION
TESTS
Solution S Dissolve 5.0 g in a mixture of 70 ml of acetic acid R and 30 ml of distilled water R, boil for
2 min, cool and dilute to 100 ml with dilute acetic acid R. Filter if necessary through a previously
ignited and tared porcelain or silica filter crucible of a suitable porosity to give a clear filtrate.
(Method II, 2.2.2).
Soluble substances To 2.00 g add 100 ml of water R and heat to boiling for 5 min. Filter whilst hot
through a sintered-glass filter (40), allow to cool and dilute to 100 ml with water R. Evaporate 50 ml
of the filtrate to dryness and dry at 100C to 105C. The residue weighs not more than 20 mg
(2.0 per cent).
to dryness. Dissolve the residue in 1.5 ml of acetic acid R and dilute to 30 ml with water R. 12 ml of
Iron (2.4.9). Dissolve 50 mg in 5 ml of dilute hydrochloric acid R and dilute to 10 ml with water R.
2 ml of this solution diluted to 10 ml with water R complies with the limit test for iron (0.1 per cent).
ASSAY
Dissolve 0.700 g in 20 ml of dilute hydrochloric acid R and dilute to 100.0 ml with water R. Using
10.0 ml of the solution, carry out the complexometric titration of magnesium (2.5.11).
STORAGE
__________________________________________________________________________________________________________ Ph Eur
Magnesium Peroxide
1/01
Magnesium Peroxide complies with the requirements of the 3rd edition of the European Pharmacopoeia [1540].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Magnesium peroxide is a mixture of magnesium peroxide and magnesium oxide. It contains not less
than 22.0 per cent and not more than 28.0 per cent of MgO2 (56.30).
CHARACTERS
A white or slightly yellow, amorphous, light powder, practically insoluble in water and in alcohol. It
dissolves in dilute mineral acids.
25-24
IDENTIFICATION
A. Dissolve about 15 mg in 2 ml of dilute nitric acid R and neutralise with dilute sodium hydroxide
B. Dissolve 0.1 g in 2 ml of dilute sulphuric acid R and dilute to 10 ml with water R. Shake 1 ml of the
solution with 5 ml of ether R and 0.5 ml of potassium dichromate solution R1. The ether layer is blue.
TESTS
Solution S1 Dissolve cautiously 5.0 g in 40 ml of hydrochloric acid R1. Cautiously evaporate the
solution to a volume of 10 ml and dilute to 100 ml with a mixture of equal volumes of acetic acid R
and distilled water R. Filter, if necessary, through a previously ignited and tared porcelain or silica
filter crucible of suitable porosity to give a clear filtrate. Keep the residue for the test for acid
insoluble substances.
Solution S2 Dilute 5 ml of solution S1 to 25 ml with distilled water R.
Appearance of solution Solution S1 is not more intensely coloured than reference solution B4
(Method II, 2.2.2).
Acid insoluble substances Any residue obtained during the preparation of solution S1, washed,
dried and ignited at 600C, weighs not more than 5 mg (0.1 per cent).
Acidity or alkalinity To 2.0 g add 100 ml of carbon dioxide-free water R and heat to boiling for
5 min. Filter whilst hot through a sintered-glass filter (40), allow to cool and dilute to 100 ml with
carbon dioxide-free water R. To 15 ml of the filtrate, add 0.1 ml of phenolphthalein solution R. The
solution is red. Not more than 0.2 ml of 0.1M hydrochloric acid is necessary to change the colour of the
indicator. Keep the filtrate for the test for soluble substances.
Soluble substances Take 50 ml of the filtrate obtained in the test for acidity or alkalinity, evaporate
to dryness and dry at 100C to 105C. The residue weighs not more than 15 mg (1.5 per cent).
Chlorides (2.4.4). Dissolve 50 mg in 5 ml of dilute nitric acid R and dilute to 15 ml with water R.
The solution complies with the limit test for chlorides (0.1 per cent).
Sulphates (2.4.13). 3 ml of solution S2 diluted to 15 ml with distilled water R complies with the limit
test for sulphates (0.5 per cent).
Arsenic (2.4.2). 5 ml of solution S1 complies with limit test A for arsenic (4 ppm).
Calcium (2.4.3). 1 ml of solution S2 diluted to 15 ml with distilled water R complies with the limit
test for calcium (1.0 per cent).
Iron (2.4.9).2 ml of solution S2 diluted to 10 ml with water R complies with the limit test for iron
(500 ppm).
Heavy metals (2.4.8). To 20 ml of solution S1 add 15 ml of hydrochloric acid R1 and shake with
to dryness. Dissolve the residue in 1.5 ml of acetic acid R and dilute to 30 ml with water R. 12 ml of
ASSAY
Dissolve 80.0 mg, shaking cautiously, in a mixture, previously cooled to 20C, of 10 ml of sulphuric
acid R and 90 ml of water R. Titrate with 0.02M potassium permanganate until a pink colour is
obtained.
1 ml of 0.02M potassium permanganate is equivalent to 2.815 mg of MgO2.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
Magnesium Stearate
Magnesium Stearate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0229].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Magnesium stearate is a mixture of magnesium salts of different fatty acids consisting mainly of
stearic acid [(C17H35COO)2Mg; 591.3] and palmitic acid [(C15H31COO)2Mg; 535.1] and in minor
proportions other fatty acids. It contains not less than 4.0 per cent and not more than 5.0 per cent
of Mg (Ar 24.30), calculated with reference to the dried substance. The fatty acid fraction contains
25-25
not less than 40.0 per cent of stearic acid and the sum of stearic acid and palmitic acid is not less
than 90.0 per cent.
PRODUCTION
CHARACTERS
A white, very fine, light powder, greasy to the touch, practically insoluble in water and in ethanol.
IDENTIFICATION
First identification: C, D.
Second identification: A, B, D.
A. The residue obtained in the preparation of solution S (see Tests) has a freezing point (2.2.18) not
lower than 53C.
B. The acid value of the fatty acids (2.5.1) is 195 to 210, determined on 0.200 g of the residue
obtained in the preparation of solution S dissolved in 25 ml of the prescribed mixture of solvents.
C. Examine the chromatograms obtained in the test for fatty acid composition. The retention times
of the principal peaks in the chromatogram obtained with the test solution are approximately the
same as those of the principal peaks in the chromatogram obtained with the reference solution.
D. 1 ml of solution S (see Tests) gives the reaction of magnesium (2.3.1).
TESTS
Solution S To 5.0 g add 50 ml of peroxide-free ether R, 20 ml of dilute nitric acid R and 20 ml of
distilled water R and heat under a reflux condenser until dissolution is complete. Allow to cool. In a
separating funnel, separate the aqueous layer and shake the ether layer with two quantities, each of
4 ml, of distilled water R. Combine the aqueous layers, wash with 15 ml of peroxide-free ether R and
dilute to 50 ml with distilled water R (solution S). Evaporate the organic layer to dryness and dry the
residue at 100C to 105C. Keep the residue for identification A and B.
Acidity or alkalinity To 1.0 g add 20 ml of carbon dioxide-free water R and boil for 1 min with
continuous shaking. Cool and filter. To 10 ml of the filtrate add 0.05 ml of bromothymol blue solution R1. Not more than 0.5 ml of 0.01M hydrochloric acid or 0.01M sodium hydroxide is required to
change the colour of the indicator.
Cadmium Not more than 3 ppm of Cd, determined by atomic absorption spectrometry (Method II,
2.2.23).
Test solution. Place 50.0 mg of the substance to be examined in a polytetrafluoroethylene digestion
bomb and add 0.5 ml of a mixture of 1 volume of hydrochloric acid R and 5 volumes of cadmium- and
lead-free nitric acid R. Allow to digest at 170C for 5 h. Allow to cool. Dissolve the residue in water R
Reference solutions. Prepare the reference solutions using cadmium standard solution (10 ppm Cd) R,
diluted if necessary with a 1 per cent V/V solution of hydrochloric acid R.
Measure the absorbance at 228.8 nm, using a cadmium hollow-cathode lamp as a source of
radiation and an air-acetylene flame.
Lead Not more than 10 ppm of Pb, determined by atomic absorption spectrometry (Method II,
2.2.23).
Test solution. Use the solution described in the test for cadmium.
Reference solutions. Prepare the reference solutions using lead standard solution (10 ppm Pb) R, diluted
if necessary with water R.
Measure the absorbance at 283.3 nm, using a lead hollow-cathode lamp as a source of radiation
and an air-acetylene flame, depending on the apparatus the line at 217.0 nm may be used.
Nickel Not more than 5 ppm of Ni, determined by atomic absorption spectrometry (Method II,
2.2.23).
Test solution. Use the solution described in the test for cadmium.
Reference solutions. Prepare the reference solutions using nickel standard solution (10 ppm Ni) R, diluted
if necessary with water R.
Measure the absorbance at 232.0 nm, using a nickel hollow-cathode lamp as a source of radiation
25-26
at 100C to 105C.
Microbial contamination Total viable aerobic count (2.6.12) not more than 103 micro-organisms
per gram, determined by plate count. It complies with the test for Escherichia coli (2.6.13).
ASSAY
Magnesium To 0.500 g in a 250 ml conical flask add 50 ml of a mixture of equal volumes of
butanol R and ethanol R, 5 ml of concentrated ammonia R, 3 ml of ammonium chloride buffer solution pH
10.0 R, 30.0 ml of 0.1M sodium edetate and 15 mg of mordant black 11 triturate R. Heat to 45C to
50C until the solution is clear and titrate with 0.1M zinc sulphate until the colour changes from blue
to violet. Carry out a blank titration.
1 ml of 0.1M sodium edetate is equivalent to 2.431 mg of Mg.
Fatty acid composition Examine by gas chromatography (2.2.28).
Test solution. In a conical flask fitted with a reflux condenser, dissolve 0.10 g of the substance to be
examined in 5 ml of boron trifluoride-methanol solution R. Boil under a reflux condenser for 10 min.
Add 4 ml of heptane R through the condenser and boil again under a reflux condenser for 10 min.
Allow to cool. Add 20 ml of a saturated solution of sodium chloride R. Shake and allow the layers to
separate. Remove about 2 ml of the organic layer and dry over 0.2 g of anhydrous sodium sulphate R.
Dilute 1.0 ml of the solution to 100.0 ml with heptane R.
Reference solution. Prepare the reference solution in the same manner as the test solution using
50.0 mg of palmitic acid CRS and 50.0 mg of stearic acid CRS instead of magnesium stearate.
a fused-silica column 30 m long and 0.32 mm in internal diameter coated with macrogol
20,000 R (film thickness 0.5 m),
helium for chromatography R as the carrier gas at a flow rate of 2.4 ml/min,
with the following temperature programme:
Column
Time (min) Temperature

(oC)
Rate
Comment
(oC/min)
02
2 36
70 240
36 41
240
70
Injection port
220
Detector
260
isothermal
linear
gradient
isothermal
Inject 1 l of the reference solution. When the chromatograms are recorded in the prescribed
conditions, the retention time of methyl palmitate relative to that of methyl stearate is about 0.88.
The test is not valid unless, in the chromatogram obtained with the reference solution, the resolution
between the peaks corresponding to methyl stearate and methyl palmitate is at least 5.0.
Inject 1 l of the test solution. Calculate the percentage content of stearic acid and palmitic acid
from the areas of the peaks in the chromatogram obtained with the test solution by the normalisation
procedure, disregarding the peak due to the solvent.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
Magnesium Sulphate Heptahydrate

Epsom Salts
MgSO4,7H2O
246.5
10034-99-8
Magnesium Sulphate Heptahydrate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0044]. These requirements are reproduced after the heading Definition below.
Action and use Osmotic laxative; used in treatment of electrolyte deficiency.
Preparations
25-27
Magnesium Sulphate Injection
Magnesium Sulphate Mixture
When magnesium sulphate is prescribed or demanded, Magnesium Sulphate Heptahydrate shall be
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Magnesium sulphate heptahydrate contains not less than 99.0 per cent and not more than the
equivalent of 100.5 per cent of MgSO4, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder or brilliant, colourless crystals, freely soluble in water, very soluble in
boiling water, practically insoluble in alcohol.
IDENTIFICATION
A. It gives the reactions of sulphates (2.3.1).
B. It gives the reaction of magnesium (2.3.1).
TESTS
Solution S Dissolve 5.0 g in water R and dilute to 50 ml with the same solvent.
indicator.
(20 ppm).
Loss on drying (2.2.32). 48.0 per cent to 52.0 per cent, determined on 0.500 g by drying in an oven
at 110C to 120C for 1 h and then at 400C to constant mass.
ASSAY
Dissolve 0.450 g in 100 ml of water R and carry out the complexometric titration of magnesium
(2.5.11).
1 ml of 0.1M sodium edetate is equivalent to 12.04 mg of MgSO4.
__________________________________________________________________________________________________________ Ph Eur
Dried Magnesium Sulphate

Dried Epsom Salts
Definition Dried Magnesium Sulphate contains not less than 62.0% and not more than 70.0% of
MgSO4. It may be prepared by drying magnesium sulphate at 100 until it has lost approximately
25% of its weight.
Characteristics A white powder; odourless or almost odourless.
Freely soluble in water; dissolves more rapidly in hot water.
Identification Yields the reactions characteristic of magnesium salts and of sulphates, Appendix VI.
Acidity or alkalinity To 10 ml of a 7.5% w/v solution in carbon dioxide-free water add 0.05 ml of
phenol red solution. Not more than 0.2 ml of either 0.01M hydrochloric acid VS or 0.01M sodium
25-28
hydroxide VS is required to change the colour of the solution.
Arsenic 0.33 g dissolved in 25 ml of water complies with the limit test for arsenic, Appendix VII
(3 ppm).
Heavy metals Dissolve 1.3 g in 20 ml of water and add 1 g of ammonium chloride. 12 ml of the
resulting solution complies with limit test A for heavy metals, Appendix VII. Use lead standard solution
(1 ppm Pb) to prepare the standard (15 ppm).
Iron 0.33 g dissolved in 10 ml of water complies with the limit test for iron, Appendix VII (30 ppm).
Chloride 0.13 g dissolved in 15 ml of water complies with the limit test for chlorides, Appendix VII
(400 ppm).
Insoluble matter 7.5 g dissolves in 20 ml of water, producing a solution which may be slightly
turbid at first but which becomes clear in a few minutes.
Assay Dissolve 0.3 g in 50 ml of water and carry out the complexometric titration of magnesium,
Appendix VIII D. Each ml of 0.1M disodium edetate VS is equivalent to 12.04 mg of MgSO4.
Storage Dried Magnesium Sulphate should be kept in a well-closed container.
Preparation
Magnesium Sulphate Paste
Magnesium Trisilicate
Magnesium Trisilicate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Antacid.
Preparations
Magnesium Trisilicate Mixture
Compound Magnesium Trisilicate Oral Powder
Compound Magnesium Trisilicate Tablets
Ph Eur ___________________________________________________________________________________________________________
25-29
DEFINITION
Magnesium trisilicate has a variable composition corresponding approximately to Mg2Si3O8,xH2O
and contains not less than the equivalent of 29.0 per cent of magnesium oxide (MgO; Mr 40.30) and
not less than the equivalent of 65.0 per cent of silicon dioxide (SiO2; Mr 60.1), both calculated with
reference to the ignited substance.
CHARACTERS
A white powder, practically insoluble in water and in alcohol.
IDENTIFICATION
A. 0.25 g gives the reaction of silicates (2.3.1).
B. 1 ml of solution S (see Tests) neutralised with dilute sodium hydroxide solution R gives the reaction
of magnesium (2.3.1).
TESTS
Solution S To 2.0 g add a mixture of 4 ml of nitric acid R and 4 ml of distilled water R. Heat to
boiling with frequent shaking. Add 12 ml of distilled water R and allow to cool. Filter or centrifuge to
obtain a clear solution and dilute to 20 ml with distilled water R.
Alkalinity To 10.0 g in a 200 ml conical flask, add 100.0 g of water R and heat on a water-bath for
30 min. Allow to cool and make up to the initial mass with water R. Allow to stand and filter or
centrifuge until a clear liquid is obtained. To 10 ml of the liquid add 0.1 ml of phenolphthalein solution R. Not more than 1.0 ml of 0.1M hydrochloric acid is required to change the colour of the
indicator.
Water-soluble salts In a platinum dish, evaporate to dryness on a water-bath 20.0 ml of the liquid
obtained in the test for alkalinity. The residue, ignited to constant mass at 900C, weighs not more
than 30 mg (1.5 per cent).
chlorides (500 ppm). Prepare the standard using a mixture of 5 ml of chloride standard solution (5 ppm
Cl) R and 10 ml of water R.
Arsenic (2.4.2). 2.5 ml of solution S complies with limit test A for arsenic (4 ppm).
Heavy metals (2.4.8). Neutralise 10 ml of solution S with dilute ammonia R1, using metanil yellow
solution R as an external indicator. Dilute to 20 ml with water R and filter if necessary. 12 ml of the
Loss on ignition 17 per cent to 34 per cent, determined on 0.5 g ignited to constant mass at 900C
in a platinum crucible.
Acid-absorbing capacity Suspend 0.25 g in 0.1M hydrochloric acid, dilute to 100.0 ml with the
same acid and allow to stand for 2 h in a water-bath at 37 0.5C, with frequent shaking. Allow to
cool. To 20.0 ml of the supernatant solution add 0.1 ml of bromophenol blue solution R and titrate with
0.1M sodium hydroxide until a blue colour is obtained. The acid-absorbing capacity is not less than
100.0 ml of 0.1M hydrochloric acid per gram.
ASSAY
Magnesium oxide To 1.000 g in a 200 ml conical flask, add 35 ml of hydrochloric acid R and 60 ml
of water R and heat in a water-bath for 15 min. Allow to cool, filter, wash the conical flask and the
residue with water R and dilute the combined filtrate and washings to 250.0 ml with water R.
Neutralise 50.0 ml of the solution with strong sodium hydroxide solution R (about 8 ml). Carry out the
Silicon dioxide To 0.700 g add 10 ml of dilute sulphuric acid R and 10 ml of water R. Heat for 1.5 h
on a water-bath with frequent shaking, replacing the evaporated water. Allow to cool and decant onto
an ashless filter paper (diameter 7 cm). Wash the precipitate by decantation with three quantities,
each of 5 ml, of hot water R, transfer it to the filter and wash it with hot water R until 1 ml of the
filtrate remains clear after the addition of 0.05 ml of dilute hydrochloric acid R and 2 ml of barium
chloride solution R1. Ignite the filter and its contents (SiO2) in a platinum crucible at 900C to
constant mass.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
25-30
Refined Maize Oil

Refined Maize Oil complies with the requiements of the 3rd edition of the European Pharmacopoeia [1342].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Maize oil is the fatty oil obtained from the seeds of Zea mays L. by expression or by extraction. It is
then refined.
CHARACTERS
A clear, light yellow or yellow oil, practically insoluble in water and in alcohol, miscible with light
25-31
petroleum (bp: 40C to 60C) and with methylene chloride.
It has a relative density of about 0.920 and a refractive index of about 1.474.
IDENTIFICATION
A. Carry out the identification of fatty oils by thin-layer chromatography (2.3.2). The chromatogram obtained with the test solution is similar to that obtained with the reference solution.
B. It complies with the test for composition of fatty acids (see Tests).
TESTS
Acid value (2.5.1). Not more than 0.5, determined on 10.0 g. If intended for use in the
manufacture of parenteral dosage forms, not more than 0.3.
Peroxide value (2.5.5). Not more than 10.0. If intended for use in the manufacture of parenteral
dosage forms, not more than 5.0.
Unsaponifiable matter (2.5.7). Not more than 2.8 per cent, determined on 5.0 g.
Alkaline impurities (2.4.19). It complies with the test for alkaline impurities in fatty oils.
(2.4.22). The fatty-acid fraction of the oil has the following composition:
fatty acids of chain length less than C16: not more than 0.6 per cent,
palmitic acid: 8.6 per cent to 16.5 per cent
stearic acid: not more than 3.3 per cent,
oleic acid: 20.0 per cent to 42.2 per cent (equivalent chain length on polyethylene glycol adipate
18.3),
linoleic acid: 39.4 per cent to 65.6 per cent (equivalent chain length on polyethylene glycol
adipate 18.9),
linolenic acid: 0.5 per cent to 1.5 per cent (equivalent chain length on polyethylene glycol
adipate 19.7),
arachidic acid: not more than 0.8 per cent,
eicosenoic acid: not more than 0.5 per cent (equivalent chain length on polyethyleneglycol
adipate 20.3),
behenic acid: not more than 0.5 per cent,
other fatty acids: not more than 0.5 per cent.
Sterols Determined by gas chromatography (2.4.23), the sterol fraction of the oil contains not more
than 0.3 per cent of brassicasterol.
Water (2.5.32). If intended for use in the manufacture of parenteral dosage forms, not more than
0.1 per cent, determined on 5.00 g by the micro-determination of water. Use a mixture of equal
volumes of decanol R and anhydrous methanol R as the solvent.
STORAGE
Store protected from light, at a temperature not exceeding 25C.
LABELLING
The label states:
forms,
whether the oil is obtained by mechanical expression or by extraction.
__________________________________________________________________________________________________________ Ph Eur
Maize Starch
Maize Starch complies with the requirements of the 3rd edition of the European Pharmacopoeia [0344]. These
When Starch is specified and the type is not indicated, Maize Starch, Potato Starch, Rice Starch,
Wheat Starch or, in tropical countries where these are not available, Tapioca Starch may be supplied
or used.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Maize starch is obtained from the caryopsis of Zea mays L.
25-32
CHARACTERS
A matt, white to slightly yellowish, very fine powder which creaks when pressed between the fingers, tasteless, practically insoluble in cold water and in alcohol. The presence of granules with
cracks or irregularities on the edge is exceptional.
DESCRIPTION
Examined under a microscope it presents granules either angular polyhedral 2 m to 23 m in size,
or rounded 25 m to 32 m in size. The central hilum consists of a distinct cavity or two- to fiverayed cleft and there are no concentric striations. Between crossed nicol prisms, the starch
granules show a distinct black cross intersecting at the hilum.
IDENTIFICATION
A. Suspend 1 g in 50 ml of water R, boil for 1 min and cool. A thin, cloudy mucilage is formed.
B. To 1 ml of the mucilage obtained in identification test A add 0.05 ml of iodine solution R1. A darkblue colour is produced which disappears on heating and reappears on cooling.
TESTS
Acidity Add 10 g to 100 ml of alcohol (70 per cent V/V) R previously neutralised to 0.5 ml of phenolphthalein solution R and shake for 1 h. Filter and take 50 ml of the filtrate. Not more than 2.0 ml of
0.1M sodium hydroxide is required to change the colour of the indicator.
Foreign matter Not more than traces of cell membranes and protoplasm are present.
100C to 105C.
Microbial contamination. Total viable aerobic count (2.6.12) not more than 103 bacteria and 102
fungi per gram, determined by plate-count. It complies with the test for Escherichia coli (2.6.13).
STORAGE
__________________________________________________________________________________________________________ Ph Eur
Malathion
OMe
P
OMe
S
H
EtOOC
S
COOEt
and enantiomer
C10H19O6PS2
330.4
121-75-5
Malathion complies with the requirements of the 3rd edition of the European Pharmacopoeia [1343]. These
Action and use Insecticide.
25-33
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Malathion contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent
of diethyl (2RS)-2-(dimethoxyphosphinodithioyl)butanedioate, calculated with reference to the
CHARACTERS
A clear, colourless or slightly yellowish liquid, slightly soluble in water, miscible with alcohol and
with acetone, with cyclohexane and with vegetable oils.
It freezes at about 3C.
IDENTIFICATION
Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum
obtained with malathion CRS.
TESTS
Relative density (2.2.5): 1.220 to 1.240.
Optical rotation (2.2.7). Dissolve 2.50 g in alcohol R and dilute to 25.0 ml with the same solvent.
The angle of optical rotation is 0.1 to +0.1.
Related substances Examine by liquid chromatography (2.2.29) as prescribed under Assay.
Inject 20 l of test solution (a) and 20 l of reference solution (d). In the chromatogram obtained
with test solution (a): the area of any peak due to impurity A is not greater than three times the area
of the corresponding peak in the chromatogram obtained with reference solution (d) (0.3 per cent);
the area of any peak due to impurity B is not greater than the area of the corresponding peak in the
chromatogram obtained with reference solution (d) (0.1 per cent); the sum of the areas of all the
peaks apart from the principal peak and any peak due to impurity A or impurity B is not greater than
twice the area of the principal peak in the chromatogram obtained with reference solution (b) (1 per
cent). Disregard any peak with an area less than 0.1 times that of the area of the principal peak in the
chromatogram obtained with reference solution (b).
Water (2.5.12). Not more than 0.1 per cent, determined on 2.000 g by the semi-micro determination of water.
ASSAY
Test solution (a). Dissolve 0.10 g of the substance to be examined in a mixture of 1 volume of water R
and 3 volumes of acetonitrile R and dilute to 5.0 ml with the same mixture of solvents.
Test solution (b). Take 1.0 ml of test solution (a) and dilute to 10.0 ml with a mixture of 1 volume of
water R and 3 volumes of acetonitrile R.
Reference solution (a). Dissolve 0.100 g of malathion CRS in a mixture of 1 volume of water R and 3
volumes of acetonitrile R and dilute to 50.0 ml with the same mixture of solvents.
Reference solution (b). Take 0.5 ml of test solution (a) and dilute to 100.0 ml with a mixture of 1
volume of water R and 3 volumes of acetonitrile .
Reference solution (c). Dissolve 5.0 mg of malathion impurity A CRS and 5.0 mg of malathion impurity
B CRS in a mixture of 1 volume of water R and 3 volumes of acetonitrile R and dilute to 50.0 ml with
the same mixture of solvents.
Reference solution (d). Take 2.0 ml of reference solution (c) and dilute to 10.0 ml with a mixture of 1
volume of water R and 3 volumes of acetonitrile R.
as mobile phase at a flow rate of 1 ml/min a mixture of 45 volumes of acetonitrile R and 55
volumes of water R,
Inject 20 l of reference solution (b) and 20 l of reference solution (c).
When the chromatograms are recorded in the prescribed conditions, the retention times are: impurity
B about 3.5 min; impurity A about 5 min and malathion about 16 min. Adjust the sensitivity of the
system so that the height of the principal peak in the chromatogram obtained with reference solution
(b) is at least 50 per cent of the full scale of the recorder. The test is not valid unless in the chromatogram obtained with reference solution (c) the resolution between the peaks corresponding to impurity
A and impurity B is at least 2.0. Inject reference solution (a) six times. The assay is not valid unless
the relative standard deviation of the peak area for malathion is at most 1.0 per cent.
Inject alternately test solution (b) and reference solution (a). Calculate the percentage content of
malathion.
25-34
STORAGE
IMPURITIES
XMe
EtOOC
P OMe
O
and enantiomer
COOEt
A. X = S: diethyl (2RS)-2-[(methoxy)(methylsulfanyl)-S-phosphinothioyl]butanedioate
(isomalathion),
B. X = O: diethyl (2RS)-2-(dimethoxy-S-phosphinothioyl)butanedioate (maloxon),
OMe
EtOOC
P OMe
S
and enantiomer
COOMe
C. ethyl and methyl (2RS)-2-(dimethoxyphosphinothioyl)butanedioate (methyl analogue).

__________________________________________________________________________________________________________ Ph Eur
Maleic Acid
H
COOH
COOH
C4H4O4
116.1
110-16-7
Maleic Acid complies with the requirements of the 3rd edition of the European Pharmacopoeia [0365]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Maleic acid contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of (Z)-butenedioic acid, calculated with reference to the anhydrous substance.
25-35
CHARACTERS
A white, crystalline powder, freely soluble in water and in alcohol, sparingly soluble in ether.
IDENTIFICATION
A. Dilute 5 ml of solution S (see Tests) to 10 ml with water R. The pH of the dilution is less than 2.
B. Examine the chromatograms obtained in the test for fumaric acid. The principal spot in the
chromatogram obtained with test solution (b) is similar in position and size to the principal spot in
the chromatogram obtained with reference solution (a).
C. Dissolve 0.1 g in 10 ml of water R (solution a). To 0.3 ml of solution (a) add a solution of 10 mg
of resorcinol R in 3 ml of sulphuric acid R. Heat on a water-bath for 15 min; no colour develops. To
3 ml of solution (a) add 1 ml of bromine water R. Heat on a water-bath to remove the bromine
(15 min), heat to boiling and cool. To 0.2 ml of this solution add a solution of 10 mg of resorcinol R
in 3 ml of sulphuric acid R. Heat on a water-bath for 15 min. A violet-pink colour develops.
TESTS
solution Y7 (Method II, 2.2.2).
Fumaric acid Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating
substance.
Test solution (a). Dissolve 0.5 g of the substance to be examined in acetone R and dilute to 5 ml with
the same solvent.
Reference solution (a). Dissolve 20 mg of maleic acid CRS in acetone R and dilute to 10 ml with the
same solvent.
Reference solution (b). Dissolve 15 mg of fumaric acid CRS in acetone R and dilute to 10 ml with the
same solvent.
Reference solution (c). Mix 5 ml of reference solution (a) and 5 ml of reference solution (b).
Apply separately to the plate 5 l of test solutions (a) and (b), 5 l of reference solutions (a) and (b)
and 10 l of reference solution (c). Develop in a non-saturated tank over a path of 10 cm using a
mixture of 12 volumes of anhydrous formic acid R, 16 volumes of chloroform R, 32 volumes of
butanol R and 44 volumes of heptane R. Dry the plate at 100C for 15 min and examine in ultraviolet
light at 254 nm. Any spot corresponding to fumaric acid in the chromatogram obtained with test
solution (a) is not more intense than the spot in the chromatogram obtained with reference solution
(b) (1.5 per cent). The test is not valid unless the chromatogram obtained with reference solution (c)
Heavy metals (2.4.8). 1.0 g complies with limit test D for heavy metals (10 ppm). Prepare the
Iron To 10 ml of solution S add 2 ml of dilute hydrochloric acid R and 0.05 ml of bromine water R.
After 5 min, remove the excess of bromine by passing a current of air and add 3 ml of potassium
thiocyanate solution R. Shake. Prepare a standard at the same time and in the same manner, using a
mixture of 5 ml of iron standard solution (1 ppm Fe) R, 1 ml of dilute hydrochloric acid R, 6 ml of
water R and 0.05 ml of bromine water R. Allow both solutions to stand for 5 min. Any red colour in
the test solution is not more intense than that in the standard (5 ppm).
of water.
ASSAY
Dissolve 0.500 g in 50 ml of water R. Titrate with 1M sodium hydroxide using 0.5 ml of phenolphthalein
solution R as indicator.
1 ml of 1M sodium hydroxide is equivalent to 58.04 mg of C4H4O4.
STORAGE
Store in a well-closed glass container, protected from light.
__________________________________________________________________________________________________________ Ph Eur
Mallow Flower
25-36
1/01
Mallow Flower complies with the requirements of the 3rd edition of the European Pharmacopoeia [1541].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Mallow flower consists of the whole or fragmented dried flower of Malva sylvestris L. or its cultivated
varieties.
CHARACTERS
IDENTIFICATION
25-37
A. The flower consists of an epicalyx with three oblong or elliptical-lanceolate parts that are shorter
than those of the calyx and situated immediately below it; a calyx with five pubescent triangular
lobes, gamosepalous at the base; a corolla three to four times longer than the calyx with five wedgeshaped, notched petals fused to the staminal tube at their base; numerous stamens, the filaments of
which fuse into a staminal tube covered by small star-shaped trichomes and occasional simple
trichomes visible using a lens; numerous wrinkled carpels, glabrous or sometimes pubescent,
enclosed in the staminal tube and arranged into a circle around a central style ending with numerous
filiform stigmas. In cultivated varieties, the epicalyx is 3 to 7 partite, the calyx 5 to 8 partite and the
corolla 5 to 10 partite.
B. Reduce to a powder (355). The powder is bluish-grey. Examine under a microscope using chloral
hydrate solution R. The powder shows unicellular, thick-walled, stiff trichomes, up to 2 mm in length;
small unicellular covering trichomes, somewhat curved, either isolated or in small star-shaped groups
of 2 to 6; capitate glandular trichomes with multicellular heads; mesophyll fragments with vessels
accompanied by cluster crystals of calcium oxalate; spherical pollen grains, about 150 m in
diameter, with a roughly spiny exine.
When mounted with alcohol R, numerous elongated cells containing mucilage are seen in the petal
fragments.
C. Examine by thin-layer chromatography (2.2.27) using a TLC silica gel plate R.
Test solution. To 1 g of the powdered drug (355) add 10 ml of alcohol (60 per cent V/V) R. Stir for
15 min and filter.
Reference solution. A 0.5 g/l solution of quinaldine red R in alcohol R.
Apply to the plate as bands 10 l of the test solution and 5 l of the reference solution.
Develop over a path of 10 cm using a mixture of 15 volumes of acetic acid R, 30 volumes of water R
and 60 volumes of butanol R. Allow the plate to dry in air. Examine the chromatograms in daylight.
The chromatogram obtained with the reference solution shows an orange-red zone in the upper part
of the middle third. The chromatogram obtained with the test solution shows, below the zone in the
chromatogram obtained with the reference solution, two violet zones in the middle third; the principal zone (6-malonyl malvin) is situated just below the other violet zone (malvin).
TESTS
Swelling index (2.8.4). Not less than 15, determined on 0.2 g of the powdered drug (710)
humidified with 0.5 ml of ethanol R.
by drying in an oven at 100C to 105C.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
Maltitol
1/01
CH2OH
O
CH2OH
H
OH
OH
HO
HO
OH
O
H
H
OH
CH2OH
C12H24O11
344.3
585-88-6
25-38
Maltitol complies with the requirements of the 3rd edition of the European Pharmacopoeia [1235]. These
Action and use Sweetening agent.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Maltitol contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent of 4O--D-glucopyranosyl-D-glucitol (D-maltitol), calculated with reference to the anhydrous substance.
CHARACTERS
A white, crystalline powder, very soluble in water, practically insoluble in ethanol.
IDENTIFICATION
First identification: A.
Second identification: B, C, D.
obtained with maltitol CRS. Examine the substances prepared as discs.
B. Melting point (2.2.14): 148C to 151C.
C. Dissolve 5.00 g in water R and dilute to 100.0 ml with the same solvent. The specific optical
rotation (2.2.7) is +105.5 to +108.5, calculated with reference to the anhydrous substance.
D. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel G plate R.
Test solution. Dissolve 25 mg of the substance to be examined in water R and dilute to 10 ml with the
same solvent.
Reference solution (a). Dissolve 25 mg of maltitol CRS in water R and dilute to 10 ml with the same
solvent.
Reference solution (b). Dissolve 25 mg of maltitol CRS and 25 mg of sorbitol CRS in water R and dilute
to 10 ml with the same solvent.
Apply to the plate 2 l of each solution. Develop over a path of 17 cm using a mixture of 10 volumes
of water R, 20 volumes of ethyl acetate R and 70 volumes of propanol R. Allow the plate to dry in air
and spray with 4-aminobenzoic acid solution R. Dry the plate in a current of cold air until the acetone is
removed. Heat the plate at 100C to 105C for 15 min. Allow to cool and spray with a 2 g/l solution
of sodium periodate R. Dry the plate in a current of cold air. Heat the plate at 100C for 15 min. The
principal spot in the chromatogram obtained with the test solution is similar in position, colour and
size to the principal spot in the chromatogram obtained with the reference solution (a). The test is
not valid unless the chromatogram obtained with reference solution (b) shows two clearly separated
spots.
TESTS
Conductivity (2.2.38). Not more than 20 Scm-1.
Dissolve 20.0 g in carbon dioxide-free water R prepared from distilled water R and dilute to 100.0 ml
with the same solvent. Measure the conductivity of the solution while gently stirring with a magnetic
stirrer at a temperature of 20C.
Reducing sugars Dissolve 5.0 g in 6 ml of water R with the aid of gentle heat. Cool and add 20 ml
of cupri-citric solution R and a few glass beads. Heat so that boiling begins after 4 min and maintain
boiling for 3 min. Cool rapidly and add 100 ml of a 2.4 per cent V/V solution of glacial acetic acid R
and 20.0 ml of 0.025M iodine. With continuous shaking, add 25 ml of a mixture of 6 volumes of
hydrochloric acid R and 94 volumes of water R and, when the precipitate has dissolved, titrate the
excess of iodine with 0.05M sodium thiosulphate using 1 ml of starch solution R, added towards the end
of the titration, as indicator. Not less than 12.8 ml of 0.05M sodium thiosulphate is required (0.2 per
cent, calculated as glucose equivalent).
Related substances Examine by liquid chromatography (2.2.29) as described under Assay. Inject
20 l of reference solution (b). Adjust the sensitivity of the system so that the height of the peak due
to maltitol is at least 50 per cent of the full scale of the recorder. Inject 20 l of the test solution and
continue the chromatogram for three times the retention time of maltitol. In the chromatogram
obtained with the test solution: the area of any peak, apart from the principal peak, is not greater than
the area of the principal peak in the chromatogram obtained with reference solution (b) (1 per cent);
the sum of the areas of all the peaks, apart from the principal peak, is not greater than twice the area
of the principal peak in the chromatogram obtained with reference solution (b) (2 per cent).
Disregard any peak with an area less than the area of the principal peak in the chromatogram
obtained with reference solution (c) (0.1 per cent).
25-39
Lead (2.4.10). It complies with the limit test for lead in sugars (0.5 ppm).
Nickel (2.4.15). It complies with the limit test for nickel in polyols (1 ppm).
Microbial contamination If intended for use in the manufacture of parenteral dosage forms the
total viable aerobic count (2.6.12) is not more than 102 bacteria and 102 fungi per gram, determined
by plate count; it complies with the tests for Escherichia coli and Salmonella (2.6.13).
Bacterial endotoxins (2.6.14). If intended for use in the manufacture of parenteral dosage forms
without a further appropriate procedure for the removal of bacterial endotoxins, not more than 4 I.U.
of endotoxin per gram for parenteral dosage forms having a concentration of less than 100 g/l of
maltitol, and not more than 2.5 I.U. of endotoxin per gram for parenteral dosage forms having a
concentration of 100 g/l or more of maltitol.
ASSAY
Test solution. Dissolve 5.0 g of the substance to be examined in 20 ml of water R and dilute to
Reference solution (a). Dissolve 0.5 g of maltitol CRS in 2.0 ml of water R and dilute to 10.0 ml with
the same solvent.
Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with water R.
Reference solution (c). Dilute 10.0 ml of reference solution (b) to 100.0 ml with water R.
Reference solution (d). Dissolve 0.5 g of maltitol CRS and 0.5 g of sorbitol CRS in 5 ml of water R and
dilute to 10.0 ml with the same solvent.
The chromatography may be carried out using:
a stainless steel column 0.3 m long and 7.8 mm in internal diameter packed with strong cation
exchange resin (calcium form) R (9 m) and maintained at 85C 1C,
as mobile phase at a flow rate of 0.5 ml/min, degassed water R,
as detector a refractometer maintained at a constant temperature.
Inject 20 l of reference solution (d). Continue the chromatography for three times the retention
time of maltitol.
When the chromatograms are recorded in the prescribed conditions, the retention time of maltitol is
about 16 min and the relative retentions with reference to maltitol are: sorbitol about 1.8 and
maltotriitol about 0.8. The test is not valid unless the resolution between the peaks due to maltitol
and to sorbitol is at least 2 in the chromatogram obtained with reference solution (d).
Inject 20 l of the test solution and 20 l of reference solution (a). Continue the chromatography
for three times the retention time of maltitol.
Calculate the percentage content of D-maltitol from the areas of the peaks and the declared content
of maltitol CRS.
LABELLING
The label states:
where applicable, the maximum concentration of bacterial endotoxins,
forms.
IMPURITIES
A. sorbitol,
OH
OH
OH
H
OH
H
H
OH
OH
HO
OH
OH
O
OH
OH
B. maltotriitol.
__________________________________________________________________________________________________________ Ph Eur
25-40
Liquid Maltitol
1/01
Liquid Maltitol complies with the requirements of the 3rd edition of the European Pharmacopoeia [1236].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Liquid maltitol is an aqueous solution of a hydrogenated, partly hydrolysed starch. It contains not
less than 68.0 per cent m/m and not more than 85.0 per cent m/m of anhydrous substance composed
of a mixture of mainly 4-O--D-glucopyranosyl-D-glucitol (D-maltitol) with D-glucitol (D-sorbitol) and
hydrogenated oligo- and polysaccharides. It contains not less than 50.0 per cent m/m of D-maltitol
(C12H24O11) and not more than 8.0 per cent m/m of D-sorbitol (C6H14O6), both calculated with
25-41
reference to the anhydrous substance. It contains not less than 95.0 per cent and not more than
105.0 per cent of the content of D-maltitol stated on the label.
CHARACTERS
A clear, colourless, syrupy liquid, miscible with water and with glycerol.
IDENTIFICATION
A. Examine the chromatograms obtained in the assay. The principal peak in the chromatogram
obtained with the test solution is similar in retention time to the principal peak in the chromatogram
obtained with reference solution (a).
B. To 3 ml of a freshly prepared 100 g/l solution of pyrocatechol R, add 6 ml of sulphuric acid R while
cooling in iced water. To 3 ml of the cooled mixture, add 0.3 ml of solution S (see Tests). Heat
gently over a naked-flame for about 30 s. A pink colour develops.
C. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel G plate R.
Test solution. Dilute 0.35 g of the substance to be examined to 100 ml with water R.
Reference solution (a). Dissolve 20 mg of maltitol CRS in water R and dilute to 10 ml with the same
solvent.
Reference solution (b). Dissolve 20 mg of maltitol CRS and 20 mg of sorbitol CRS in water R and dilute
removed. Heat the plate at 100C to 105C for 15 min. Allow to cool and spray with a 2 g/l solution
of sodium periodate R. Dry the plate in a current of cold air. Heat the plate at 100C for 15 min. The
principal spot in the chromatogram obtained with the test solution is similar in position and colour to
the principal spot in the chromatogram obtained with the reference solution (a). The test is not valid
unless the chromatogram obtained with reference solution (b) shows two clearly separated spots.
TESTS
Solution S Dilute 7.0 g to 50 ml with water R.
Conductivity (2.2.38). Not more than 10 S.cm-1 measured at a temperature of 20C on the
undiluted liquid maltitol while gently stirring with a magnetic stirrer.
Reducing sugars To 5.0 g add 6 ml of water R, 20 ml of cupri-citric solution R and a few glass beads.
Heat so that boiling begins after 4 min and maintain boiling for 3 min. Cool rapidly and add 100 ml
of a 2.4 per cent V/V solution of glacial acetic acid R and 20.0 ml of 0.025M iodine. With continuous
shaking, add 25 ml of a mixture of 6 volumes of hydrochloric acid R and 94 volumes of water R and,
when the precipitate has dissolved, titrate the excess of iodine with 0.05M sodium thiosulphate using
1 ml of starch solution R, added towards the end of the titration, as indicator. Not less than 12.8 ml of
0.05M sodium thiosulphate is required (0.2 per cent calculated as glucose equivalent).
Water (2.5.12). Not less than 15.0 per cent and not more than 32.0 per cent m/m determined on
0.100 g by the semi-micro determination of water.
ASSAY
Test solution. Mix 1.00 g of the solution to be examined with 20 ml of water R and dilute to 50.0 ml
Reference solution (a). Dissolve 50.0 mg of maltitol CRS in 2 ml of water R and dilute to 5.0 ml with
the same solvent.
Reference solution (b). Dissolve 8.0 mg of sorbitol CRS in 2 ml of water R and dilute to 5.0 ml with the
same solvent.
Reference solution (c). Dissolve 50 mg of maltitol CRS and 50 mg of sorbitol CRS in 2 ml of water R and
a stainless steel column 0.3 m long and 7.8 mm in internal diameter packed with a strong cation
as mobile phase at a flow rate of 0.5 ml/min degassed water R,
25-42
Inject 20 l of reference solution (b). Continue the chromatography for three times the retention
time of maltitol.
When the chromatogram is recorded in the prescribed conditions, the retention time of maltitol is
about 16 min and the relative retention of sorbitol is about 1.8. The test is not valid unless the
resolution between the peaks due to sorbitol and to maltitol is at least 2 in the chromatogram
obtained with reference solution (c).
Inject 20 l of the test solution, 20 l of reference solution (a) and 20 l of reference solution (b).
Continue the chromatography for three times the retention time of maltitol.
Calculate the percentage contents of D-maltitol and D-sorbitol from the areas of the peaks and the
declared contents of maltitol CRS and sorbitol CRS.
LABELLING
The label states the content of D-maltitol.
__________________________________________________________________________________________________________ Ph Eur
Maltodextrin
1/01
Maltodextrin complies with the requirements of the 3rd edition of the European Pharmacopoeia [1542]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Maltodextrin is a mixture of glucose, disaccharides and polysaccharides, obtained by the partial
hydrolysis of starch. The degree of hydrolysis, expressed as dextrose equivalent (DE), is not more
than 20 and is within 2 DE units of the value stated on the label.
25-43
CHARACTERS
A white or almost white, slightly hygroscopic powder or granules, freely soluble in water.
IDENTIFICATION
A. Dissolve 0.1 g in 2.5 ml of water R and heat with 2.5 ml of cupri-tartaric solution R. A red precipitate is formed.
B. Dip, for 1 s, a suitable stick with a reactive pad containing glucose-oxidase, peroxidase and a
hydrogen-donating substance, such as tetramethylbenzidine, in a 5 g/l solution of the substance to be
examined. Observe the colour of the reactive pad; within 60 s the colour changes from yellow to
green or blue.
C. It is a powder or granules.
D. It complies with the test for dextrose equivalent (see Tests).
TESTS
Solution S Dissolve 12.5 g in carbon dioxide-free water R and dilute to 50.0 ml with the same solvent.
pH (2.2.3). The pH of a mixture of 30 ml of solution S and 1 ml of a 223.6 g/l solution of potassium
chloride R is 4.0 to 7.0.
Sulphur dioxide (2.5.29). Not more than 20 ppm.
Heavy metals (2.4.8). Dilute 4 ml of solution S to 30 ml with water R. The solution complies with
limit test E for heavy metals (10 ppm). Prepare the standard using 10 ml of lead standard solution
(1 ppm) R.
100C to 105C.
Dextrose equivalent Accurately weigh an amount of the sample to be examined equivalent to
2.85 g to 3.15 g of reducing carbohydrates, calculated as dextrose equivalent, into a 500 ml
volumetric flask. Dissolve in water R and dilute to 500.0 ml with the same solvent. Transfer the
solution to a 50 ml burette.
Pipette 25.0 ml of cupri-tartaric solution R into a 250 ml flask and add 18.5 ml of the sample solution
from the burette, mix and add boiling chips. Place the flask on a hot plate, previously adjusted so that
the solution begins to boil within 2 min 15 s. Allow to boil for exactly 120 s, add 1 ml of a 1 g/l
solution of methylene blue R and titrate with the sample solution (V1) until the blue colour disappears.
Maintain the solution at boiling throughout the titration.
Standardise the cupri-tartaric solution using a 6.00 g/l solution of glucose R (V0).
Calculate the dextrose equivalent (DE) from the equation:
DE =
300 V0 100
V1 M D
V0 = total volume of glucose standard solution, in millilitres,

V1 = total volume of sample solution, in millilitres,
M = sample weight, in grams,
D = percentage content of dry matter in the substance.
Microbial contamination Total viable aerobic count (2.6.12) not more than 103 bacteria and 102
fungi per gram, determined by plate count. It complies with the test for Escherichia coli and Salmonella
(2.6.13).
LABELLING
The label states the dextrose equivalent (DE).
__________________________________________________________________________________________________________ Ph Eur
Manganese Sulphate
MnSO4,4H2O
223.1
7785-87-7
Definition Manganese Sulphate is manganese(II) sulphate tetrahydrate. It contains not less than
98.0% and not more than 100.5% of MnSO4, calculated with reference to the substance ignited at
450 to 500.
Characteristics Pale pink crystals or crystalline powder; odourless or almost odourless.
Freely soluble in water; practically insoluble in ethanol (96%).
Identification
A. Dissolve 0.5 g in 10 ml of water and add 1 ml of sodium sulphide solution. A pink precipitate is
produced which is soluble in 6M acetic acid.
25-44
B. To 0.1 g add 2 g of lead(IV) oxide and 5 ml of nitric acid, boil gently for a few minutes, add 100 ml
of water and filter. A purple solution is produced.
C. Yields the reactions characteristic of sulphates, Appendix VI.
Arsenic 0.25 g complies with the limit test for arsenic, Appendix VII (4 ppm).
Heavy metals Dissolve 0.50 g in 50 ml of water, add 1 ml of 1M acetic acid and pass hydrogen
sulphide through the solution for 20 seconds. The colour produced within 2 minutes is not more
intense than that obtained by treating 10 ml of lead standard solution (2 ppm Pb) diluted to 50 ml in
the same manner (40 ppm).
Iron Dissolve 1.0 g in 10 ml of water and add 2 ml of 1M hydrochloric acid and, dropwise, 0.05M
potassium permanganate until a permanent pink colour is produced. Add 5 ml of a 10% w/v solution
of ammonium thiocyanate and 20 ml of a mixture of equal volumes of isoamyl alcohol and amyl acetate,
shake well and allow to separate. Any colour in the upper layer is not more intense than that obtained
by treating 2 ml of iron standard solution (20 ppm Fe) diluted to 10 ml in the same manner (40 ppm).
Zinc Dissolve 2.0 g in 10 ml of water, add 3 ml of 1M hydrochloric acid and 0.3 ml of a freshly
prepared 3% w/v solution of potassium hexacyanoferrate(II), mix and allow to stand for 15 minutes.
Any turbidity produced is not more intense than that obtained by treating 10 ml of zinc standard
solution (100 ppm Zn) in the same manner (500 ppm).
Chloride 15 ml of a 1.0% w/v solution complies with the limit test for chlorides, Appendix VII
(330 ppm).
Loss on ignition When ignited to constant weight at 450 to 500, loses 31.0 to 34.0% of its weight.
Use 1 g.
Assay Dissolve 0.15 g in 40 ml of water, add 8 ml of freshly boiled and cooled nitric acid, cool, add
1.5 g of sodium bismuthate and shake for 2 minutes. Add 25 ml of a mixture of 3 volumes of nitric acid
and 97 volumes of water, filter, wash the residue with 40 ml of the mixture, collecting the filtrate and
washings in 50 ml of 0.1M ammonium iron(II) sulphate VS, and titrate immediately with 0.02M
potassium permanganate VS. Repeat the operation without the substance being examined. The difference between the titrations represents the amount of ammonium iron(II) sulphate required. Each ml
of 0.1M ammonium iron(II) sulphate VS is equivalent to 3.020 mg of MnSO4.
Manganese Sulphate Monohydrate

1/01
MnSO4,H2O
169.0
10034-96-5
Manganese Sulphate Monohydrate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1543]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Manganese sulphate monohydrate contains not less than 99.0 per cent and not more than the
25-45
equivalent of 101.0 per cent of MnSO4, calculated with reference to the ignited substance.
CHARACTERS
A pale pink crystalline powder, slightly hygroscopic, freely soluble in water, practically insoluble in
alcohol.
IDENTIFICATION
A. Solution S (see Tests) gives reaction (a) of sulphates (2.3.1).
B. Dissolve 50 mg in 5 ml of water R. Add 0.5 ml of ammonium sulphide solution R. A pale pink
precipitate is formed which dissolves on the addition of 1 ml of anhydrous acetic acid R.
C. It complies with the test for loss on ignition (see Tests).
TESTS
Solution S Dissolve 10.0 g in distilled water R and dilute to 100 ml with the same solvent.
Appearance of solution Solution S is not more opalescent than reference suspension II (2.2.1).
Iron (2.4.9).10 ml of solution S complies with the limit test for iron (10 ppm).
Zinc To 10 ml of solution S add 1 ml of sulphuric acid R and 0.1 ml of potassium ferrocyanide solution R. After 30 s, any opalescence in the solution is not more intense than that in a mixture of 5 ml
of zinc standard solution (10 ppm Zn) R, 5 ml of water R, 1 ml of sulphuric acid R and 0.1 ml of
potassium ferrocyanide solution R (50 ppm).
Heavy metals (2.4.8).12 ml of solution S complies with limit test A for heavy metals (20 ppm).
Loss on ignition 10.0 per cent to 12.0 per cent, determined on 1.00 g at 500C.
ASSAY
Dissolve 0.150 g in 50 ml of water R. Add 10 mg of ascorbic acid R, 20 ml of ammonium chloride buffer
solution pH 10.0 R and 0.2 ml of a 2 g/l solution of mordant black 11 R in triethanolamine R. Titrate
with 0.1M sodium edetate until the colour changes from violet to pure blue.
1 ml of 0.1M sodium edetate is equivalent to 15.10 mg of MnSO4.
__________________________________________________________________________________________________________ Ph Eur
Mannitol
1/01
25-46
CH2OH
HO
HO
OH
OH
CH2OH
C6H14O6
182.2
69-65-8
Mannitol complies with the requirements of the 3rd edition of the European Pharmacopoeia [0559]. These
Action and use Diuretic.
Preparation
Mannitol Intravenous Infusion
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Mannitol contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent of
D-mannitol, calculated with reference to the anhydrous substance.
CHARACTERS
White or almost white, crystalline powder or free-flowing granules, freely soluble in water, very
slightly soluble in alcohol.
IDENTIFICATION
obtained with mannitol CRS. Examine the substances prepared as discs. If the spectra obtained show
differences, dissolve the substance to be examined and the reference substance separately in water R,
evaporate to dryness and record new spectra using the residues.
same solvent.
Reference solution (a). Dissolve 25 mg of mannitol CRS in water R and dilute to 10 ml with the same
solvent.
Reference solution (b). Dissolve 25 mg of mannitol CRS and 25 mg of sorbitol CRS in water R and dilute
removed. Heat the plate at 100C for 15 min. Allow to cool and spray with a 2 g/l solution of sodium
periodate R. Dry the plate in a current of cold air. Heat the plate at 100C for 15 min. The principal
spot in the chromatogram obtained with the test solution is similar in position, colour and size to the
principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless
the chromatogram obtained with reference solution (b) shows two clearly separated spots.
D. Dissolve 2.00 g of the substance to be examined and 2.6 g of disodium tetraborate R in about 20 ml
of water R at a temperature of 30C; shake continuously for 15 min to 30 min without further
heating. Dilute the resulting clear solution to 25.0 ml with water R. The specific optical rotation
(2.2.7) is +23 to +25, calculated with reference to the anhydrous substance.
TESTS
Conductivity (2.2.38). Not more than 20 Scm1.
25-47
cent calculated as glucose equivalent).
to mannitol is at least 50 per cent of the full scale of the recorder. Inject 20 l of the test solution and
of reference solution (c) and continue the chromatography for twice the retention time of mannitol.
In the chromatogram obtained with the test solution: the area of any peak, apart from the principal
peak, is not greater than the area of the principal peak in the chromatogram obtained with reference
solution (b) (2 per cent); the sum of the areas of all the peaks, apart from the principal peak, is not
greater than the area of the principal peak in the chromatogram obtained with reference solution (b)
(2 per cent). Disregard any peak with an area less than the area of the principal peak in the chromatogram obtained with reference solution (c) (0.1 per cent).
Lead (2.4.10). It complies with the limit test for lead in sugars (0.5 ppm). Dissolve the substance to
be examined in 150.0 ml of the prescribed mixture of solvents.
Nickel (2.4.15). It complies with the limit test for nickel in polyols (1 ppm). Dissolve the substance
to be examined in 150.0 ml of the prescribed mixture of solvents.
of water.
Microbial contamination If intended for use in the manufacture of parenteral dosage forms, the
by plate count. It complies with the tests for Escherichia coli and Salmonella (2.6.13).
mannitol, and not more than 2.5 I.U. of endotoxin per gram for parenteral dosage forms having a
concentration of 100 g/l or more of mannitol.
ASSAY
Reference solution (a). Dissolve 0.5 g of mannitol CRS in 2.5 ml of water R and dilute to 10.0 ml with
the same solvent.
Reference solution (d). Dissolve 0.5 g of mannitol CRS and 0.5 g of sorbitol CRS in 5 ml of water R and
times of mannitol.
When the chromatograms are recorded in the prescribed conditions, the retention time of mannitol
is about 22 min and the relative retention of sorbitol with reference to mannitol is about 1.25. The
test is not valid unless the resolution between the peaks due to mannitol and to sorbitol is at least 2 in
the chromatogram obtained with reference solution (d).
for twice the retention time of mannitol.
Calculate the percentage content of D-mannitol from the areas of the peaks and the declared
content of mannitol CRS.
LABELLING
The label states:
25-48
forms.
IMPURITIES
A. sorbitol.
__________________________________________________________________________________________________________ Ph Eur
Maprotiline Hydrochloride
25-49
NHMe
,HCl
C20H23N,HCl
313.9
10347-81-6
Maprotiline Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Maprotiline hydrochloride contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of 3-(9,10-dihydro-9,10-ethanoanthracen-9-yl)-N-methylpropan-1-amine hydrochloride, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, slightly soluble in water, freely soluble in methanol,
soluble in alcohol, sparingly soluble in methylene chloride, very slightly soluble in acetone.
IDENTIFICATION
First identification: B, D.
Second identification: A, C, D.
A. Dissolve 10 mg in 1M hydrochloric acid and dilute to 100 ml with the same acid. Examined
between 250 nm and 300 nm (2.2.25), the solution shows two absorption maxima, at 265 nm and
272 nm, and an absorption minimum at 268 nm. The ratio of the absorbance measured at the
maximum at 272 nm to that measured at the maximum at 265 nm is 1.1 to 1.3.
obtained with maprotiline hydrochloride CRS. Examine the substances prepared as discs. If the spectra
obtained show differences, dissolve the substance to be examined and the reference substance
separately in methanol R, evaporate to dryness and record new spectra using the residues.
C. Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable silica gel
Test solution. Dissolve 25 mg of the substance to be examined in methanol R and dilute to 5 ml with
the same solvent.
Reference solution (a). Dissolve 25 mg of maprotiline hydrochloride CRS in methanol R and dilute to
Reference solution (b). Dissolve 10 mg of maprotiline impurity D CRS in reference solution (a) and
dilute to 2 ml with the same solution.
of ethyl acetate R, 5 volumes of dilute ammonia R1 and 14 volumes of 2-butanol R. Dry the plate in a
current of warm air and examine at 254 nm. The principal spot in the chromatogram obtained with
the test solution is similar in position and size to the principal spot in the chromatogram obtained
with reference solution (a). The test is not valid unless the chromatogram obtained with reference
solution (b) shows two clearly separated principal spots.
D. Dilute 0.5 ml of solution S (see Tests) to 2 ml with methanol R. The solution gives reaction (a) of
chlorides (2.3.1).
TESTS
Solution S Dissolve 1.0 g in methanol R and dilute to 20 ml with the same solvent.
solution BY6 (Method II, 2.2.2).
Test solution. Dissolve 0.10 g of the substance to be examined in the mobile phase and dilute to
100.0 ml with the mobile phase.
Reference solution (a). Dilute 1.0 ml of the test solution to 10.0 ml with the mobile phase. Dilute
2.0 ml of the solution to 100.0 ml with the mobile phase.
Reference solution (b). Dissolve 1.0 mg of maprotiline impurity D CRS in the test solution and dilute to
25-50
10.0 ml with the same solution.
a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with silica gel for
chromatography R (5 m),
as mobile phase a flow rate of 1 ml/min a mixture as prepared as follows: dissolve about 0.580 g
of ammonium acetate R in 200 ml of water R and add 2 ml of a 70 g/l solution of concentrated
ammonia R; add 150 ml of 2-propanol R and 650 ml of methanol R; the resulting apparent pH
value is between 8.2 and 8.4,
When the chromatograms are recorded in the prescribed conditions, the retention time of
maprotiline is about 10.3 min and retention times relative to maprotiline are: impurity A about 0.3,
impurity B about 0.47, impurity C about 0.74, impurity D about 0.81, impurity E about 1.26.
principal peak (maprotiline) in the chromatogram is at least 50 per cent of the full scale of the
recorder. The test is not valid unless, in the chromatogram obtained, the resolution between the
peaks corresponding to impurity D and maprotiline is between 1.8 and 3.2. If necessary adjust the
mobile phase by adding a 50 per cent V/V solution of acetic acid R if the resolution is less than 1.8, or
by adding a 70 g/l solution of concentrated ammonia R if the resolution is greater than 3.2, to adjust
the pH in steps of 0.1 pH units. Inject 20 l of the test solution and 20 l of reference solution (a).
Continue the chromatography of the test solution for 1.5 times the retention time of the principal
peak. In the chromatogram obtained with the test solution: the area of any peak, apart from the
principal peak, is not greater than the area of the principal peak in the chromatogram obtained with
reference solution (a) (0.2 per cent); the sum of the areas of all the peaks, apart from the principal
peak, is not greater than five times the area of the principal peak in the chromatogram obtained with
reference solution (a) (1 per cent). Disregard any peak with an area less than 0.1 times that of the
principal peak in the chromatogram obtained with reference solution (a).
80C at a pressure not exceeding 2.5 kPa for 6 h.
ASSAY
Dissolve 0.250 g in a mixture of 5 ml of 0.1M hydrochloric acid and 50 ml of alcohol R. Carry out a
potentiometric titration (2.2.20), using 0.1M sodium hydroxide. Read the volume added between the
two points of inflexion.
1 ml of 0.1M sodium hydroxide is equivalent to 31.39 mg of C20H24ClN.
IMPURITIES
CHO
A. 3-(9,10-dihydro-9,10-ethanoanthracen-9-yl)prop-2-enal,
N
Me
B. methylbis[3-(9,10-dihydro-9,10-ethanoanthracen-9-yl)propyl]amine,
C. R = CH2CH2CH2NH2: 3-(9,10-dihydro-9,10-ethanoanthracen-9-yl)propan-1-amine,
D. R = CH=CHCH2NHCH3: 3-(9,10-dihydro-9,10-ethanoanthracen-9-yl)-N-methylprop-2-en-1amine (dehydromaprotiline),
E. R = CH2CH2CH2N(CH3) 2: 3-(9,10-dihydro-9,10-ethanoanthracen-9-yl)-N,N-dimethylpropan-
25-51
1-amine.
__________________________________________________________________________________________________________ Ph Eur
Marshmallow Root
25-52
Marshmallow Root complies with the requirements of the 3rd edition of the European Pharmacopoeia [1126].
Action and use Demulcent.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Marshmallow root consists of the peeled or unpeeled, whole or cut, dried root of Althaea officinalis L.
CHARACTERS
IDENTIFICATION
A. The unpeeled, non-fragmented drug consists of cylindrical, slightly twisted roots, up to 2 cm
thick, with deep longitudinal furrows. The outer surface is greyish-brown and bears numerous rootlet
scars. The fracture is fibrous externally, rugged and granular internally. The section shows a more or
less thick, whitish bark with brownish periderm, separated by the well-marked, brownish cambium
from a white xylem. The stratified structure of the bark and the radiate structure of xylem become
more distinct when moistened.
The peeled drug has a greyish-white finely fibrous outer surface. Cork and external cortical parenchyma are absent.
B. Reduce to a powder (355). The powder is brownish-grey (unpeeled root) or whitish (peeled root).
Examine under a microscope using chloral hydrate solution R. The powder shows the following
diagnostic characters: fragments of colourless, mainly unlignified, thick-walled fibres with pointed or
split ends; fragments of bordered, pitted or scalariformly thickened vessels; cluster crystals of calcium
oxalate about 20 m to 35 m, mostly 25 m to 30 m in size; parenchymatous cells containing
mucilage; fragments of cork with thin-walled, tabular cells in the unpeeled root. Examine under a
microscope using water R. The powder shows numerous starch granules about 3 m to 25 m in size
occasionally with a longitudinal hilum. The starch grains are mostly simple, a few being two to four
compound.
TESTS
Foreign matter (2.8.2). Not more than 2 per cent of brown deteriorated drug. Not more than 2 per
cent of cork in the peeled root.
Swelling index (2.8.4). Not less than 10, determined on the powdered drug (710).
Total ash (2.4.16). Not more than 6.0 per cent for peeled root and not more than 8.0 per cent for
the unpeeled root.
STORAGE
Store in well-closed container, protected from light.
__________________________________________________________________________________________________________ Ph Eur
Matricaria Flowers
25-53
Matricaria Flowers comply with the requirements of the 3rd edition of the European Pharmacopoeia for
Matricaria Flower [0404]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Matricaria flower consists of the dried-flower heads of Matricaria recutita L. (Chamomilla recutita
(L.) Rauschert). It contains not less than 4 ml/kg of blue essential oil.
CHARACTERS
Matricaria flower has a characteristic, pleasant and aromatic odour.
The capitula, when spread out, consist of an involucre made up of many bracts arranged in one to
three rows; an elongated-conical receptacle, occasionally hemispherical (young capitula); twelve to
twenty marginal ligulate florets with a white ligule; several dozen yellow central tubular florets.
IDENTIFICATION
A. The bracts of the involucre are obovate to lanceolate, with a brownish-grey scarious margin. The
receptacle is essentially conical and hollow, without paleae. The base of the corolla of ligulate florets
consists of a light yellow to brownish-yellow tube extending to an elongated-oval, white ligule. The
corolla of tubular florets is yellow and broadens at the apex, where it splits into five teeth; its base is
yellowish-brown to brown.
B. Separate the capitulum into its different parts. Examine under a microscope using chloral hydrate
solution R. The outer epidermis (abaxial) of the involucral bracts shows a scarious margin with a
single layer of radially elongated cells and a central part made up of chlorophyll tissue covered with
elongated epidermal cells with sinuous lateral walls, stomata and secretory trichomes. Surrounding
the vascular bundles are numerous elongated, pitted sclereids with a fairly large lumen. In surface
view, the corolla of ligulate florets and tubular florets show isodiametric or elongated cells with a
more or less wavy wall and a few glandular trichomes. The outer part of the epidermis of the ligulate
florets consists of papillary cells with cuticular striations radiating from their tips. In the mesophyll,
very small clusters of calcium oxalate are sometimes seen. Four main veins run lengthwise through
the entire mesophyll, sometimes accompanied by one or two other veins, which are shorter and run
parallel to the main veins. The two main median veins both split into two near the tip and, with the
lateral veins, anastomose two by two, forming three arcs at the three terminal teeth of the ligule. The
ovaries, oval to spherical, of both kinds of florets, have at their base a sclerous ring consisting of a
single row of cells. The epidermis of the ovary is made up of elongated cells with sinuous walls
between which are inserted secretory trichomes. The ovaries contain numerous very small clusters of
calcium oxalate. In the tubular florets, the lower part of each stamen filament is surrounded by thickwalled cells. The epidermal cells of the ends of the two stigmata are very papillose. The pollen grains
have a diameter of about 30 m and are rounded and triangular, with three germinal pores and a
spiny exine.
C. Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating substance.
Test solution. In a porcelain mortar, coarsely pound 1.0 g of the drug, transfer to a chromatography
column 150 mm long and 15 mm in internal diameter and tap lightly with a glass rod. Rinse the
mortar and the pestle with two quantities, each of 10 ml, of methylene chloride R and pour the rinsings
into the column. Collect the percolate in a flask with a long, narrow neck and remove the solvent by
evaporation on a water-bath. Dissolve the residue in 0.5 ml of toluene R.
Reference solution. Dissolve 10 mg of borneol R, 20 mg of bornyl acetate R and 4 mg of guaiazulene R in
toluene R and dilute to 10 ml with the same solvent.
Apply separately to the plate as bands 20 mm by 3 mm 10 l of each solution. Develop over a path of
10 cm using chloroform R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm.
The chromatogram obtained with the test solution shows a number of quenching zones. The largest
zone (en-yne-dicycloether) is situated at the same level as the zone due to bornyl acetate in the
chromatogram obtained with the reference solution; a further zone is seen near the starting-point
(matricin). Spray the plate with anisaldehyde solution R, using 10 ml for a plate 200 mm square, and
examine in daylight while heating at 100C to 105C for 5 min to 10 min. The chromatogram
obtained with the reference solution shows: in the lower third, a brownish-yellow zone (borneol)
which becomes violet-grey after a few hours; in the middle a yellowish-brown to grey zone (bornyl
acetate); and in the upper third a deep red zone with a blue edge (guaiazulene). The chromatogram
obtained with the test solution shows: a blue zone (matricin) near the starting-point; several violetred zones (one of which is due to bisabolol) with Rf values between those of borneol and bornyl
acetate; a brownish zone (en-yne-dicycloether) with an Rf value similar to that of bornyl acetate; red
zones (terpenes) with Rf values similar to that of guaiazulene; other zones appear in the middle and
lower parts of the chromatogram.
D. Place 0.1 ml of the test solution used in identification test C in a test-tube, add 2.5 ml of a solu-
25-54
tion prepared by dissolving 0.25 g of dimethylaminobenzaldehyde R in a mixture of 5 ml of phosphoric
acid R, 45 ml of acetic acid R and 45 ml of water R. Heat on a water-bath for 2 min and allow to cool.
Add 5 ml of light petroleum R and shake. The aqueous layer has a distinct greenish-blue or blue
colour.
TESTS
Broken drug Not more than 25 per cent passes through a no. 710 sieve.
ASSAY
Carry out the determination of essential oils in vegetable drugs (2.8.12). Use 30 g of whole drug, a
1000 ml flask, 300 ml of water R as distillation liquid and 0.50 ml of xylene R in the graduated tube.
Distil at a rate of 3 ml to 4 ml per minute for 4 h.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
Mebendazole
25-55
NH
N
O
N
H
OMe
O
C16H13N3O3
295.3
31431-39-7
Mebendazole complies with the requirements of the 3rd edition of the European Pharmacopoeia [0845]. These
Action and use Anthelmintic.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Mebendazole contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent
of methyl N-(5-benzoyl-1H-benzimidazol-2-yl)carbamate, calculated with reference to the dried
substance.
CHARACTERS
A white or faintly yellowish powder, practically insoluble in water, in alcohol, in ether and in
methylene chloride.
IDENTIFICATION
A. Dissolve 30.0 mg in 2 ml of anhydrous formic acid R and dilute to 100.0 ml with 2-propanol R.
Dilute 2.5 ml of this solution to 100.0 ml with 2-propanol R. Examined between 230 nm and 320 nm
(2.2.25), the solution shows two absorption maxima, at 247 nm and 312 nm. The specific absorbances at these maxima are 940 to 1040 and 485 to 535, respectively. Use a 0.05 per cent V/V solution of anhydrous formic acid R in 2-propanol R as the compensation liquid.
obtained with mebendazole CRS. Examine the substances prepared as discs, without recrystallisation.
C. To about 10 mg add 5 ml of alcohol R, 1 ml of dinitrobenzene solution R and 1 ml of dilute sodium
hydroxide solution R. An intense yellow colour is produced.
TESTS
Appearance of solution Dissolve 0.10 g in a mixture of 1 volume of anhydrous formic acid R and 9
volumes of methylene chloride R and dilute to 10 ml with the same mixture of solvents. The solution is
clear (2.2.1) and not more intensely coloured than reference solution BY4 (Method II, 2.2.2).
Related substances Examine by thin-layer chromatography (2.2.27), using silica gel HF254 R as the
coating substance.
Test solution. Dissolve 50 mg of the substance to be examined in a mixture of 1 volume of anhydrous
formic acid R and 9 volumes of methylene chloride R and dilute to 5 ml with the same mixture of
solvents.
Reference solution (a). Dilute 0.5 ml of the test solution to 100 ml with a mixture of 1 volume of
anhydrous formic acid R and 9 volumes of methylene chloride R.
Reference solution (b). Dilute 10 ml of reference solution (a) to 20 ml with a mixture of 1 volume of
anhydrous formic acid R and 9 volumes of methylene chloride R.
Apply separately to the plate 10 l of each solution. Develop over a path of 15 cm with a mixture of 5
volumes of methanol R, 5 volumes of anhydrous formic acid R and 90 volumes of methylene chloride R.
Allow the plate to dry in a current of warm air. Examine in ultraviolet light at 254 nm. Any spot in
the chromatogram obtained with the test solution, apart from the principal spot, is not more intense
than the spot in the chromatogram obtained with reference solution (a) (0.5 per cent) and at most
one such spot is more intense than the spot in the chromatogram obtained with reference solution (b)
(0.25 per cent).
100C to 105C for 4 h.
ASSAY
Dissolve 0.250 g in 3 ml of anhydrous formic acid R and add 40 ml of anhydrous acetic acid R. Titrate
with 0.1M perchloric acid, determining the end-point potentiometrically (2.2.20).
1 ml of 0.1M perchloric acid is equivalent to 29.53 mg of C16H13N3O3.
25-56
STORAGE
__________________________________________________________________________________________________________ Ph Eur
Mebeverine Hydrochloride
25-57
O
O
Et
OMe
N
,HCl
H
Me
MeO
MeO
and enantiomer
C25H35NO5,HCl
466.0
2753-45-9
Definition Mebeverine Hydrochloride is (RS)-4-[ethyl(4-methoxy-methylphenethyl)amino]butyl veratrate hydrochloride. It contains not less than 99.0% and not
more than 101.0% of C25H35NO5,HCl, calculated with reference to the dried substance.
Characteristics A white or almost white, crystalline powder.
Very soluble in water; freely soluble in ethanol (96%); practically insoluble in ether.
Identification
mebeverine hydrochloride (RS 209).
B. Dissolve 25 mg in 2 ml of water, acidify with 2M nitric acid and centrifuge. The supernatant liquid
yields reaction A characteristic of chlorides, Appendix VI, beginning at the words add 0.4 ml of ...'.
Acidity pH of a 2% w/v solution, 4.5 to 6.5, Appendix V L.
Ether-soluble extractive Dissolve 40 mg in 25 ml of 2M hydrochloric acid and shake with 50 ml of
ether for 1 minute. Wash the ether layer with three 25-ml quantities of water, evaporate the ether to
dryness using a rotary evaporator and dissolve the residue in sufficient methanol to produce 20 ml.
The absorbance of the resulting solution at 260 nm is not more than 0.23, Appendix II B.
Non-tertiary amine Dissolve 0.5 g in 5 ml of pyridine, add 5 ml of copper chloridepyridine reagent
and heat at 50 for 30 minutes. Cool, add sufficient acetone to produce 50 ml and measure the absorbance of the resulting solution at 405 nm, Appendix II B, using in the reference cell a solution
obtained by treating 5 ml of pyridine in the same manner. The absorbance is not more than that
obtained by repeating the test using 5 ml of a 0.0060% w/v solution of di-n-butylamine in pyridine and
beginning at the words add 5 ml of copper chloridepyridine reagent ....
silica gel F254 precoated plate (Merck silica gel 60 F254 plates are suitable) and a mixture of 50
volumes of absolute ethanol, 50 volumes of chloroform and 1 volume of 18M ammonia as the mobile
phase. Apply separately to the plate 10 l of each of three solutions in acetone containing (1) 2.0%
w/v of the substance being examined, (2) 0.010% w/v of the substance being examined and (3)
0.0020% w/v of veratric acid. After removal of the plate, allow it to dry in air and examine under
ultraviolet light (254 nm). Expose the plate to iodine vapour for 1 hour. When viewed under ultraviolet light any spot corresponding to veratric acid in the chromatogram obtained with solution (1) is
not more intense than the spot in the chromatogram obtained with solution (3). By each method of
visualisation any other secondary spot in the chromatogram obtained with solution (1) is not more
intense than the spot in the chromatogram obtained with solution (2).
Loss on drying When dried at 105 for 1 hour, loses not more than 0.5% of its weight. Use 1 g.
Assay Carry out Method I for non-aqueous titration, Appendix VIII A, using 0.4 g, adding 7 ml of
mercury(II) acetate solution and determining the end point potentiometrically. Each ml of 0.1M
perchloric acid VS is equivalent to 46.60 mg of C25H35NO5,HCl.
Storage Mebeverine Hydrochloride should be kept in an airtight container, protected from light and
stored at a temperature not exceeding 30.
Action and use Antispasmodic.
Preparation
Mebeverine Tablets
Meclozine Hydrochloride
25-58
N
N
,2HCl
H
Me
Cl
and enantiomer
C25H27ClN2,2HCl
463.9
1104-22-9
Meclozine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Anti-emetic.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Meclozine hydrochloride contains not less than 98.0 per cent and not more than the equivalent of
102.0 per cent of (RS)-1-[(4-chlorophenyl)phenylmethyl]-4-[(3-methylphenyl)methyl]piperazine
dihydrochloride, calculated with reference to the anhydrous substance.
CHARACTERS
A yellow or yellowish-white, crystalline powder, slightly soluble in water, soluble in alcohol and in
methylene chloride, practically insoluble in ether.
IDENTIFICATION
A. Dissolve 15.0 mg in 0.1M hydrochloric acid and dilute to 100.0 ml with the same acid. Dilute
10.0 ml of this solution to 100.0 ml with 0.1M hydrochloric acid. Examined between 220 nm and
350 nm (2.2.25), the solution shows an absorption maximum at 232 nm and weak absorbance
without a defined maximum between 260 nm and 300 nm. The specific absorbance at the maximum
is 345 to 380, calculated with reference to the anhydrous substance.
obtained with meclozine hydrochloride CRS. Examine the substances prepared as discs using potassium
chloride R.
D. Dissolve about 15 mg in 2 ml of alcohol R. The solution gives reaction (a) of chlorides (2.3.1).
TESTS
solution is clear (2.2.1) and not more intensely coloured than reference solution Y6 (Method II,
2.2.2).
Acidity or alkalinity Calculate the acidity or alkalinity from the titration volumes noted in the Assay
using the equation:
A = V2 2V1
V1 = volume of 0.1M sodium hydroxide added at the first point of inflexion,
V2 = volume of 0.1M sodium hydroxide added at the second point of inflexion.
A is 0.3 ml to 0.3 ml for 0.3500 g of the substance to be examined.
coating substance.
Test solution (a). Dissolve 0.50 g of the substance to be examined in a mixture of equal volumes of
methanol R and methylene chloride R and dilute to 10 ml with the same mixture of solvents.
Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with a mixture of equal volumes of
methanol R and methylene chloride R.
Reference solution (a). Dissolve 50 mg of meclozine hydrochloride CRS in a mixture of equal volumes of
25-59
Reference solution (b). Dilute 0.5 ml of test solution (b) to 10 ml with a mixture of equal volumes of
0.5 volumes of concentrated ammonia R, 5 volumes of methanol R, 30 volumes of toluene R and 60
volumes of methylene chloride R. Allow the plate to dry in air and spray with dilute potassium
iodobismuthate solution R. Any spot in the chromatogram obtained with test solution (a), apart from
the principal spot, is not more intense than the spot in the chromatogram obtained with reference
solution (b) (0.5 per cent). Disregard any yellowish-white spot at the starting point.
ASSAY
Dissolve 0.3500 g in 50 ml of alcohol R. Carry out a potentiometric titration (2.2.20), using 0.1M
sodium hydroxide. Read the volume added between the two points of inflexion.
1 ml of 0.1M sodium hydroxide is equivalent to 46.39 mg of C25H29Cl3N2.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
25-60
Medroxyprogesterone Acetate
O
Me
Me
OAc
Me
O
H Me
C24H34O4
386.5
71-58-9
Medroxyprogesterone Acetate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Medroxyprogesterone acetate contains not less than 97.0 per cent and not more than the equivalent of 103.0 per cent of 6-methyl-3,20-dioxopregn-4-en-17-yl acetate, calculated with reference
to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, practically insoluble in water, freely soluble in
methylene chloride, soluble in acetone and in dioxan, sparingly soluble in alcohol.
IDENTIFICATION
First identification: A, B.
obtained with medroxyprogesterone acetate CRS.
C. Examine the chromatogram obtained in the test for impurity F after spraying at 365 nm. The
principal spot in the chromatogram obtained with test solution (b) is similar in position, fluorescence
in ultraviolet light at 365 nm and size to the principal spot in the chromatogram obtained with
TESTS
Specific optical rotation (2.2.7). Dissolve 0.250 g in dioxan R and dilute to 25.0 ml with the same
solvent. The specific optical rotation is +45 to +51, calculated with reference to the dried
substance.
Impurity F (6
-methyl-3,20-dioxo-5
-pregnan-17-yl acetate) Examine by thin-layer chromatography (2.2.27), using a TLC silica gel plate R.
Test solution (a). Dissolve 0.200 g of the substance to be examined in methylene chloride R and dilute
to 10.0 ml with the same solvent.
Test solution (b). Dilute 1 ml of test solution (a) to 20 ml with methylene chloride R.
Reference solution (a). Add to 20.0 mg of medroxyprogesterone acetate for performance test CRS (containing 0.5 per cent of impurity F) 1.0 ml of methylene chloride R and dissolve.
Reference solution (b). Dissolve 10 mg of medroxyprogesterone acetate CRS in methylene chloride R and
dilute to 10 ml with the same solvent.
10 volumes of tetrahydrofuran R, 45 volumes of 1,1-dimethylethyl methyl ether R and 45 volumes of
hexane R. Allow the plate to dry in air and carry out a second development in the same direction over
a path of 10 cm with the same mixture. Allow the plate to dry at 120C for 10 min. Spray with a
200 g/l solution of toluenesulphonic acid R in alcohol R. Heat at 120C for 10 min. Allow to cool.
Examine the chromatograms in ultraviolet light at 365 nm. In the chromatogram obtained with test
solution (a) any blue fluorescent spot with a Rf value higher than the principal spot due to
medroxyprogesterone acetate is not more intense than the corresponding blue fluorescent spot of
impurity F in the chromatogram obtained with reference solution (a) (0.5 per cent). The test is not
valid unless the chromatogram obtained with reference solution (a) shows two clearly separated
spots.
25-61
Test solution. Dissolve 25.0 mg of the substance to be examined in 1 ml of tetrahydrofuran R and
dilute to 10.0 ml with the mobile phase.
Reference solution (a). Dissolve 2 mg of medroxyprogesterone acetate CRS and 5 mg of megestrol acetate
CRS in the mobile phase and dilute to 100.0 ml with the mobile phase.
Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase.
a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with basedeactivated end-capped octadecylsilyl silica gel for chromatography R (5 m),
as mobile phase at a flow rate of 2 ml/min a mixture prepared as follows: in a 1000 ml
volumetric flask mix 100 ml of tetrahydrofuran R with 350 ml of acetonitrile R and 500 ml of
water R; allow to equilibrate and adjust the volume to 1000 ml with water R and mix again,
Equilibrate the column with the mobile phase at a flow rate of 2 ml/min for about 30 min.
Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram
obtained with 20 l of reference solution (b) is at least 50 per cent of the full scale of the recorder.
Inject 20 l of reference solution (a). When the chromatograms are recorded in the prescribed
conditions, the retention times are: megestrol acetate about 14.5 min and medroxyprogesterone
acetate about 16.5 min. The test is not valid unless the resolution between the peaks corresponding
to megestrol acetate and medroxyprogesterone acetate is at least 3.3. If necessary, adjust the concentration of acetonitrile and/or tetrahydrofuran in the mobile phase.
Inject 20 l of the solvent mixture of the test solution as a blank, 20 l of the test solution and 20 l
of reference solution (b). Continue the chromatogram of the test solution for 1.5 times the retention
time of the principal peak. In the chromatogram obtained with the test solution: the area of any peak,
apart from the principal peak, is not greater than the area of the principal peak in the chromatogram
obtained with reference solution (b) (1 per cent); the sum of the areas of all the peaks, apart from the
principal peak, is not greater than 1.5 times of the area of the principal peak in the chromatogram
obtained with reference solution (b) (1.5 per cent). Disregard any peak due to the blank and any
peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained
with reference solution (b).
100C to 105C for 3 h.
ASSAY
Dissolve 50.0 mg in alcohol R and dilute to 50.0 ml with the same solvent. Dilute 2.0 ml of the
solution to 100.0 ml with alcohol R. Measure the absorbance (2.2.25) at the maximum at 241 nm.
Calculate the content of C24H34O4 taking the specific absorbance to be 420.
STORAGE
IMPURITIES
O
Me
CH3
OAc
Me
H
H
O
HO Me
A. 6-hydroxy-6-methyl-3,20-dioxopregn-4-en-17-yl acetate (6-hydroxymedroxyprogesterone

acetate),
O
Me
CH3
OH
Me
H
H
O
H Me
B. 6-methyl-17-hydroxypregn-4-en-3,20-dione (medroxyprogesterone),
25-62
Me
Me
Me
OAc
O
H
H
O
H Me
C. 6,17-dimethyl-3,17-dioxo-D-homopregn-4-en-17-yl acetate,
O
Me
CH3
OAc
Me
H
H
O
H Me
D. 6-methyl-3,20-dioxopregn-4-en-17-yl acetate (6-epimedroxyprogesterone acetate),

O
Me
CH3
OAc
Me
H
H
O
CH2
E. 6-methylene-3,20-dioxopregn-4-en-17-yl acetate (6-methylenehydroxyprogesterone acetate),

O
Me
CH3
OAc
Me
H
O
H
H Me
F. 6-methyl-3,20-dioxo-5-pregnan-17-yl acetate (4,5-dihydromedroxyprogesterone acetate),

O
Me
CH3
OAc
Me
H
H
O
Me
G. 6-methyl-3,20-dioxopregna-4,6-dien-17-yl acetate.
__________________________________________________________________________________________________________ Ph Eur
25-63
Mefenamic Acid
COOH
C15H15NO2
H
N
Me
Me
241.3
61-68-7
Mefenamic Acid complies with the requirements of the 3rd edition of the European Pharmacopoeia [1240].
Action and use Analgesic; anti-inflammatory.
Preparation
Mefenamic Acid Capsules
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Mefenamic acid contains not less than 99.0 per cent and not more than the equivalent of 100.5 per
cent of 2-[(2,3-dimethylphenyl)aminobenzoic acid, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, microcrystalline powder, practically insoluble in water, slightly soluble in
alcohol and in methylene chloride. It dissolves in dilute solutions of alkali hydroxides.
IDENTIFICATION
A. Dissolve 20 mg in a mixture of 1 volume of 1M hydrochloric acid and 99 volumes of methanol R and
dilute to 100 ml with the same mixture of solvents. Dilute 5 ml of the solution to 50 ml with a
mixture of 1 volume of 1M hydrochloric acid and 99 volumes of methanol R. Examined between
250 nm and 380 nm (2.2.25), the solution shows two absorption maxima, at 279 nm and 350 nm.
The ratio of the absorbance measured at the maximum at 279 nm to that measured at 350 nm is 1.1
to 1.3.
obtained with mefenamic acid CRS. Examine the substances prepared as discs. If the spectra obtained
show differences, dissolve the substance to be examined and the reference substance separately in
alcohol R, evaporate to dryness and record new spectra using the residues.
C. Dissolve about 25 mg in 15 ml of methylene chloride R and examine in ultraviolet light at 365 nm;
the solution exhibits a strong greenish-yellow fluorescence. Carefully add 0.5 ml of a saturated
solution of trichloroacetic acid R dropwise and examine in ultraviolet light at 365 nm. The solution
does not exhibit fluorescence.
D. Dissolve about 5 mg in 2 ml of sulphuric acid R and add 0.05 ml of potassium dichromate solution R1. An intense blue colour is produced, turning rapidly to brownish-green.
TESTS
Related substances Examine by thin-layer chromatography (2.2.27), using a TLC silica gel GF254
plate R.
Test solution. Dissolve 0.125 g of the substance to be examined in a mixture of 1 volume of
methanol R and 3 volumes of methylene chloride R and dilute to 5 ml with the same mixture of
solvents.
Reference solution (a). Dilute 1 ml of the test solution to 50 ml with a mixture of 1 volume of
methanol R and 3 volumes of methylene chloride R. Dilute 1 ml of the solution to 10 ml with a mixture
of 1 volume of methanol R and 3 volumes of methylene chloride R.
Reference solution (b). Dissolve 5 mg of flufenamic acid R and 5 mg of mefenamic acid CRS in a mixture
of 1 volume of methanol R and 3 volumes of methylene chloride R and dilute to 10 ml with the same
Apply to the plate 20 l of each solution. Develop over a path of 15 cm using a mixture of 1 volume
of glacial acetic acid R, 25 volumes of dioxan R and 90 volumes of toluene R. Dry the plate in a current
of warm air. Expose the plate to iodine vapour for 5 min and examine in ultraviolet light at 254 nm.
25-64
Any spot in the chromatogram obtained with the test solution, apart from the principal spot, is not
more intense than the spot in the chromatogram obtained with reference solution (a) (0.2 per cent).
The test is not valid unless the chromatogram obtained with reference solution (b) shows two clearly
separated spots.
2,3-Dimethylaniline
Solution (a). Dissolve 0.250 g of the substance to be examined in a mixture of 1 volume of
solvents. This solution is used to prepare the test solution.
Solution (b). Dissolve 50 mg of 2,3-dimethylaniline R in a mixture of 1 volume of methanol R and 3
volumes of methylene chloride R and dilute to 100 ml with the same mixture of solvents. Dilute 1 ml of
the solution to 100 ml with a mixture of 1 volume of methanol R and 3 volumes of methylene
chloride R. This solution is used to prepare the standard.
Using three flat-bottomed tubes, place in the first 2 ml of solution (a), in the second 1 ml of solution
(b) and 1 ml of a mixture of 1 volume of methanol R and 3 volumes of methylene chloride R and in the
third 2 ml of a mixture of 1 volume of methanol R and 3 volumes of methylene chloride R (used to
prepare a blank). To each tube add 1 ml of a freshly prepared 10 g/l solution of dimethylaminobenzaldehyde R in methanol R and 2 ml of glacial acetic acid R and allow to stand at room temperature for
10 min. The intensity of the yellow colour of the test solution is between that of the blank and that of
the standard (100 ppm).
Copper Not more than 10 ppm of Cu, determined by atomic absorption spectrometry (Method I,
2.2.23).
Test solution. Place 1.00 g of the substance to be examined in a silica crucible, moisten with sulphuric
acid R, heat cautiously on a flame for 30 min and then progressively to 650C. Continue ignition
until all black particles have disappeared. Allow to cool, dissolve the residue in 0.1M hydrochloric acid
and dilute to 25.0 ml with the same acid.
Reference solutions. Prepare the reference solutions using copper standard solution (0.1 per cent Cu) R,
diluted as necessary with 0.1M nitric acid.
Measure the absorbance at 324.8 nm using a copper hollow-cathode lamp as a source of radiation
Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying at 100C to
105C.
ASSAY
Dissolve with the aid of ultrasound 0.200 g in 100 ml of warm ethanol R, previously neutralised to
phenol red solution R. Add 0.1 ml of phenol red solution R and titrate with 0.1M sodium hydroxide.
1 ml of 0.1M sodium hydroxide is equivalent to 24.13 mg of C15H15NO2.
IMPURITIES
Me
H2N
Me
A. 2,3-dimethylaniline,
Me
Me
O
NH
H
N
Me
Me
B. N-(2,3-dimethyphenyl)-2-[(2,3-dimethylphenyl)amino]benzamide.
__________________________________________________________________________________________________________ Ph Eur
25-65
Mefloquine Hydrochloride
1/01
H
OH
H
N
3C
, HCl
N
CF3
and enantiomer
C17H16F6N2O,HCl
414.8
51773-92-3
Mefloquine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Antimalarial.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Mefloquine hydrochloride contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of (RS)-[2,8-bis(trifluoromethyl)quinolin-4-yl][(2SR)-piperidin-2-yl]methanol
hydrochloride, calculated with reference to the anhydrous substance.
CHARACTERS
A white or slightly yellow, crystalline powder, very slightly soluble in water, freely soluble in
methanol, soluble in alcohol.
IDENTIFICATION
First identification: A, E.
Second identification: B, C, D, E.
obtained with mefloquine hydrochloride CRS. If the spectra obtained show differences, dissolve the
substance to be examined and the reference substance separately in methanol R, evaporate to dryness
and record new spectra using the residues.
B. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel F254 plate R. Predevelop the
plate before use with a mixture of 20 volumes of methanol R and 80 volumes of methylene chloride R,
and dry at 100C to 105C for 15 min before use.
Test solution. Dissolve 8 mg of the substance to be examined in methanol R and dilute to 5 ml with the
same solvent.
Reference solution (a). Dissolve 8 mg of mefloquine hydrochloride CRS in methanol R and dilute to 5 ml
Reference solution (b). Dilute 2.5 ml of the test solution to 100 ml with methanol R.
Reference solution (c). To 1 ml of reference solution (b) add 1 ml of a 0.016 g/l solution of quinidine
sulphate R in methanol R.
Apply to the plate 20 l of each solution. Develop over a path of 10 cm using a mixture of 10
volumes of anhydrous acetic acid R, 10 volumes of methanol R and 80 volumes of methylene chloride R.
Dry the plate in a current of warm air for 15 min and examine in ultraviolet light at 254 nm. Lightly
spray with a mixture prepared immediately before use of 1 volume of sulphuric acid R and 40 volumes
of iodoplatinate reagent R. Spray with strong hydrogen peroxide solution R. The principal spot in the
chromatogram obtained with the test solution is similar in position, colour and size to the principal
spot in the chromatogram obtained with reference solution (a). The test is not valid unless the
chromatogram obtained with reference solution (c) shows two clearly separated spots.
C. Mix about 10 mg with 45 mg of heavy magnesium oxide R and ignite in a crucible until a practically
white residue is obtained. Allow to cool, add 2 ml of water R, 0.05 ml of phenolphthalein solution R1
and about 1 ml of dilute hydrochloric acid R to make the solution colourless. Filter. To the filtrate add
a freshly prepared mixture of 0.1 ml of alizarin S solution R and 0.1 ml of zirconyl nitrate solution R.
Mix, allow to stand for 5 min and compare the colour of the solution with a blank prepared in the
same manner. The colour of the test solution is yellow and that of the blank is red.
25-66
D. To about 20 mg add 0.2 ml of sulphuric acid R. Blue fluorescence appears in ultraviolet light at
365 nm.
E. It gives reaction (b) of chlorides (2.3.1).
TESTS
Solution S Dissolve 2.50 g in methanol R and dilute to 50.0 ml with the same solvent.
solution BY7 (Method I, 2.2.2).
Optical rotation (2.2.7). 0.2 to +0.2, determined on solution S.
1.0 ml of the solution to 20.0 ml the mobile phase.
Reference solution (b). Dissolve 8 mg of mefloquine hydrochloride CRS and 8 mg of quinidine sulphate R
in the mobile phase and dilute to 50.0 ml with the mobile phase. Dilute 5.0 ml of the solution to
a stainless steel precolumn 0.025 m long and 4 mm in internal diameter and a stainless steel
column 0.25 m long and 4 mm in internal diameter, both packed with end-capped octadecylsilyl
as mobile phase at a flow rate of 0.8 ml/min a mixture prepared as follows: dissolve 1 g of
tetraheptylammonium bromide R in a mixture of 200 volumes of methanol R, 400 volumes of a
1.5 g/l solution of sodium hydrogen sulphate R and 400 volumes of acetonitrile R,
Inject 20 l of reference solution (a). Adjust the sensitivity of the system so that the height of the
principal peak is at least 50 per cent of the full scale of the recorder.
Inject 20 l of reference solution (b).When the chromatograms are recorded in the prescribed
conditions, the retention times are: quinidine about 2 min, mefloquine about 4 min, impurity B
about 15 min, impurity A about 36 min.
The test is not valid unless, in the chromatogram obtained with reference solution (b), the resolution between quinidine and mefloquine is at least 8.5.
for 10 times the retention time of the principal peak. In the chromatogram obtained with the test
solution: the area of any peak with a relative retention with reference to mefloquine of about 0.7 is
not greater than twice the area of the principal peak in the chromatogram obtained with reference
solution (a) (0.2 per cent); the area of any peaks apart from the principal peak is not greater than the
area of the principal peak in the chromatogram obtained with reference solution (a) (0.1 per cent)
and the sum of the areas of any such peaks is not greater than five times the area of the principal peak
in the chromatogram obtained with reference solution (a) (0.5 per cent). Disregard any peak with an
area less than 0.2 times that of the principal peak in the chromatogram obtained with reference
solution (a) (0.02 per cent).
Heavy metals (2.4.8). 1.0 g complies with limit test C for heavy metals (20 ppm). Prepare the
ASSAY
Dissolve 0.350 g in 15 ml of anhydrous formic acid R and add 40 ml of acetic anhydride R. Titrate with
1 ml of 0.1M perchloric acid is equivalent to 41.48 mg of C17H17ClF6N2O.
STORAGE
25-67
IMPURITIES
CF3
N
CF3
O
N
A. [2,8-bis(trifluoromethyl)quinolin-4-yl](pyridin-2-yl)methanone,
CF3
N
CF3
and enantiomer
HO
H
B. (RS)-[2,8-bis(trifluoromethyl)quinolin-4-yl](pyridin-2-yl)methanol,
CF3
CF3
N
H
HO
H
and enantiomer
HN
C. (RS)-[2,8-bis(trifluoromethyl)quinolin-4-yl][(2RS)-piperidin-2-yl]methanol.
__________________________________________________________________________________________________________ Ph Eur
25-68
Megestrol Acetate
O
Me
Me
OAc
Me
H
O
Me
C24H32O4
384.5
595-33-5
Definition Megestrol Acetate is 6-methyl-3,20-dioxopregna-4,6-dien-17-yl acetate. It contains

not less than 97.0% and not more than 103.0% of C24H32O4, calculated with reference to the dried
substance.
Characteristics A white to creamy white, crystalline powder; odourless or almost odourless.
Practically insoluble in water; very soluble in chloroform; sparingly soluble in ethanol (96%); slightly
soluble in ether and fixed oils.
Identification
megestrol acetate (RS 211).
B. Complies with the test for identification of steroids, Appendix III A, using impregnating solvent II and
mobile phase D.
C. Yields the reaction characteristic of acetyl groups, Appendix VI.
D. Melting point, about 217, Appendix V A.
Light absorption The light absorption, Appendix II B, in the range 230 to 350 nm of the solution
obtained in the Assay exhibits a maximum only at 287 nm. The ratio of the absorbance at 240 nm to
that at the maximum at 287 nm is not more than 0.17.
Specific optical rotation In a 5% w/v solution in chloroform, +9 to +12, calculated with reference
to the dried substance, Appendix V F.
Related foreign steroids Carry out the method for thin-layer chromatography, Appendix III A, using
silica gel G as the coating substance and a mixture of 92 volumes of 1,2-dichloroethane, 8 volumes of
methanol and 0.5 volume of water as the mobile phase. Apply separately to the plate 1 l of each of
two solutions in a mixture of 9 volumes of chloroform and 1 volume of methanol containing (1) 5.0%
w/v of the substance being examined and (2) 0.025% w/v of megestrol BPCRS. After removal of the
plate, allow it to dry in air until the solvent has evaporated, spray with ethanolic sulphuric acid (20%),
heat at 110 for 10 minutes and examine under ultraviolet light (365 nm). Any secondary spot in the
chromatogram obtained with solution (1) is not more intense than the spot in the chromatogram
obtained with solution (2).
Loss on drying When dried to constant weight at 105, loses not more than 0.5% of its weight. Use
1 g.
Assay Dissolve 15 mg in sufficient absolute ethanol to produce 100 ml, dilute 5 ml to 50 ml with
absolute ethanol and measure the absorbance of the resulting solution at the maximum at 287 nm,
Appendix II B. Calculate the content of C24H32O4 taking 630 as the value of A(1%, 1 cm) at the
maximum at 287 nm.
Storage Megestrol Acetate should be protected from light.
25-69
Meglumine
CH2NHMe
H
OH
HO
OH
OH
CH2OH
C7H17NO5
195.2
6284-40-8
Definition Meglumine is 1-deoxy-1-methylamino-D-glucitol. It contains not less than 99.0% and

not more than 100.5% of C7H17NO5, calculated with reference to the dried substance.
Characteristics A white, microcrystalline powder; odour, slight.
Freely soluble in water; slightly soluble in ethanol (96%); practically insoluble in chloroform and in
ether.
Identification
A. Dissolve 0.2 g in 2 ml of water and neutralise with 0.5M sulphuric acid using 0.05 ml of methyl red
solution as indicator. To 1 ml of the solution add 2 ml of a freshly prepared mixture of 1 ml of
acetaldehyde and 10 ml of a 1% w/v solution of sodium nitroprusside and then 2 ml of dilute sodium
carbonate solution. A blue colour develops.
B. To the remainder of the solution used in test A add 0.5 ml of 0.1M sodium hydroxide and 0.5 g of
boric acid. The solution becomes distinctly acidic.
Melting point 128 to 131, Appendix V A.
Specific optical rotation In a 10% w/v solution, 16.0 to 17.0, calculated with the reference to
the dried substance, Appendix V F.
Reducing sugars Dissolve 0.25 g in 5 ml of water, add 5 ml of cupri-tartaric solution, boil for 1
minute and filter. No red colour is visible on the filter.
1 g.
Assay Dissolve 0.8 g in 20 ml of water and titrate with 0.05M sulphuric acid VS using methyl red mixed
solution as indicator. Each ml of 0.05M sulphuric acid VS is equivalent to 19.52 mg of C7H17NO5.
Meglumine intended for use in the manufacture of a parenteral dosage form complies with the following
additional requirement.
Pyrogens Complies with the test for pyrogens, Appendix XIV D. Use per kg of the rabbits weight
0.6 g dissolved in not more than 5 ml of water for injections.
25-70
Meloxicam
O
O
S
NMe
H
N
OH
N
S
Me
C14H13N3O4S2
351.4
71125-38-7
Definition Meloxicam is 4-hydroxy-2-methyl-N-(5-methyl-1,3-thiazol-2-yl)-2H-1,2-benzothiazine3-carboxamide 1,1-dioxide. It contains not less than 99.0% and not more than 100.5% of
C14H13N3O4S2, calculated with reference to the dried substance.
Characteristics A pale yellow powder.
Practically insoluble in water; slightly soluble in acetone; soluble in dimethylformamide; very slightly
soluble in ethanol (96%) and in methanol.
Identification
meloxicam (RS 374).
B. The light absorption, Appendix II B, in the range 240 nm to 450 nm of a 0.0015% w/v solution in
methanol exhibits a maximum at 354 nm. The absorbance at 354 nm is about 0.8.
Clarity of solution A 5.0% w/v solution in dimethylformamide is clear, Appendix IV A.
following solutions. For solution (1) prepare a 0.4% w/v of the substance being examined in a
mixture of 50 volumes of methanol (40%) and 3 volumes of 0.4M sodium hydroxide and dilute a portion of this solution with an equal volume of methanol (40%). For solution (2) dilute 1 volume of
solution (1) to 100 volumes with methanol (40%) and further dilute 1 volume of this solution to 10
volumes with methanol (40%). Prepare solution (3) in the same manner as solution (1) using
meloxicam impurity standard BPCRS.
The chromatographic procedure may be carried out using a stainless steel column
(15 cm 4.6 mm) packed with stationary phase C (5 m) (Inertsil ODS-2 is suitable). Carry out a
linear gradient elution at 45 with a flow rate of 1 ml per minute using the following conditions.
Mobile phase A: A 0.1% w/v solution of potassium dihydrogen orthophosphate adjusted to pH 6.0 with
dilute sodium hydroxide solution.
Mobile phase B: Methanol.
Time
(minutes)
Mobile phase A
% v/v
Mobile phase B
% v/v
0
2.5
12
60
60
30
40
40
70
Use detection wavelengths of 260 and 350 nm. Allow the chromatography to proceed for 15
minutes.
Inject 10 l of each solution. The test is not valid unless, at each wavelength, the chromatogram
obtained with solution (3) closely resembles the corresponding chromatogram supplied with
meloxicam impurity standard BPCRS.
In the chromatogram obtained with solution (1) at 350 nm, the areas of any peaks corresponding
to impurity A and impurity C are not greater than half the area of the peak in the chromatogram
obtained with solution (2) at 350 nm (0.1% of impurity A assuming a relative response factor of 0.5,
and 0.05% for impurity C). In the chromatogram obtained with solution (1) at 260 nm, the area of
any peak corresponding to impurity B is not greater than the area of the peak in the chromatogram
obtained with solution (2) at 350 nm (0.1%). In both the chromatograms obtained with solution (1)
at 350 nm and at 260 nm, the area of any other secondary peak is not greater than the area of the peak
in the chromatogram obtained with solution (2) at 350 nm (0.1%). Calculate the percentage content
of impurities A and C at 350 nm, the percentage content of impurity B at 260 nm and the percentage
content of any other secondary peaks at the wavelength of higher response. The nominal total content
of any such impurities is not greater than 0.3%.
Heavy metals 2.0 g complies with limit test C for heavy metals, Appendix VII (10 ppm). Prepare the
standard using 2 ml of lead standard solution (10 ppm Pb).
25-71
Loss on drying When dried to a constant weight at 105, loses not more than 0.5% of its weight.
Use 3 g.
Sulphated ash Not more than 0.1%, Appendix IX A. Use 1 g.
Assay Dissolve 0.25 g in a mixture of 50 ml of anhydrous acetic acid and 5 ml of anhydrous formic acid
and carry out the method for non-aqueous titration, Appendix VIII A, determining the end-point
potentiometrically. Each ml of 0.1M perchloric acid VS is equivalent to 35.14 mg of C14H13N3O4S2.
Storage Meloxicam should be kept in a well-closed container.
IMPURITIES
O O
S
Me
N
COOEt
OH
A. ethyl 4-hydroxy-2-methyl-2H-1,2-benzothiazine-3-carboxylate 1,1-dioxide

2N
N
S
Me
B. 5-methylthiazol-2-ylamine
O O
S
Me
N
Et
N
OH
S
Me
C. 4-hydroxy-2-methyl-N-ethyl-N-(5-methyl-1,3-thiazol-2-yl)-2H-1,2-benzothiazine-3carboxamide 1,1-dioxide
O O
S
Me
N
COOMe
OH
D. methyl 4-hydroxy-2-methyl-2H-1,2-benzothiazine-3-carboxylate 1,1-dioxide,

O O
S
Me
N
Me
N
OH
S
Me
E. 4-hydroxy-2-methyl-N-methyl-N-(5-methyl-1,3-thiazol-2-yl)-2H-1,2-benzothiazine-3carboxamide 1,1-dioxide
25-72
Melphalan
Cl
N
Cl
NH 2
COOH
C13H18Cl2N2O2
305.2
148-82-3
Definition Melphalan is 4-bis(2-chloroethyl)amino-L-phenylalanine. It contains not less than

93.0% and not more than 100.5% of C13H18Cl2N2O2, calculated with reference to the dried
substance.
Characteristics A white or almost white powder; odourless or almost odourless.
Practically insoluble in water; slightly soluble in methanol; practically insoluble in chloroform and in
ether. It dissolves in dilute mineral acids.
Identification
melphalan (RS 212).
B. A 0.5% w/v solution in methanol is laevorotatory.
Ionisable chlorine Dissolve 0.4 g in a mixture of 75 ml of water and 2 ml of nitric acid, allow to
stand for 2 minutes and titrate with 0.1M silver nitrate VS determining the end point potentiometrically. Not more than 0.8 ml is required.
Loss on drying When dried at 100 at a pressure not exceeding 0.7 kPa for 2 hours, loses not more
than 7.0% of its weight. Use 1 g.
Assay To 0.4 g add 20 ml of a 20% w/v solution of potassium hydroxide, heat on a water bath for 2
hours, replacing water lost by evaporation, and cool. Add 75 ml of water and 4 ml of nitric acid, cool
and titrate with 0.1M silver nitrate VS determining the end point potentiometrically. Subtract the
volume of 0.1M silver nitrate VS used in the test for Ionisable chlorine. Each ml of 0.1M silver nitrate
VS represented by the difference is equivalent to 15.26 mg of C13H18Cl2N2O2.
Storage Melphalan should be kept in a well-closed container, protected from light and stored at a
temperature not exceeding 25.
Preparations
Melphalan Injection
Melphalan Tablets
25-73
Menadiol Sodium Phosphate

NaO
O
ONa
P
O
Me
O
NaO
O
ONa
C11H8Na4O8P2,6H2O
530.2
6700-42-1
Definition Menadiol Sodium Phosphate is tetrasodium 2-methylnaphthalene-1,4-diyl

di(orthophosphate) hexahydrate. It contains not less than 98.0% and not more than 100.5% of
C11H8Na4O8P2, calculated with reference to the anhydrous substance.
Characteristics A white to pink, crystalline powder; odour, characteristic; hygroscopic.
Very soluble in water; practically insoluble in ethanol (96%).
Identification
menadiol sodium phosphate (RS 213).
B. To 10 ml of a 2% w/v solution add 10 ml of 1M sulphuric acid, 10 ml of 0.1M cerium(IV) sulphate
and 1 ml of hydrogen peroxide solution (20 vol) and extract with two 10-ml quantities of chloroform.
Evaporate the combined chloroform extracts to dryness on a water bath and dry the residue at 40 at
a pressure not exceeding 0.7 kPa. The infrared absorption spectrum of the residue, Appendix II A, is
concordant with the reference spectrum of menadione.
C. Dissolve 40 mg in 2 ml of water, heat gently with 2 ml of sulphuric acid until white fumes are
evolved, add nitric acid dropwise until digestion is complete and cool. Add 2 ml of water, heat until
white fumes are evolved again, cool, add a further 10 ml of water and neutralise to litmus paper with
5M ammonia. The solution yields reaction A characteristic of sodium salts and reaction B characteristic
of phosphates, Appendix VI.
Inorganic phosphate Dissolve 25 mg in 10 ml of water, add 4 ml of 1M sulphuric acid, 1 ml of a
10% w/v solution of ammonium molybdate and 2 ml of methylaminophenylsulphite reagent and allow
to stand for 15 minutes. The absorbance of the solution at 730 nm, Appendix II B, is not more than
the absorbance of a solution prepared in the same manner but using 10 ml of a 0.0025% w/v solution
of potassium dihydrogen orthophosphate in place of the solution of the substance being examined
(0.7%).
Total phosphate Dissolve 85 mg in 50 ml of glacial acetic acid, add 5 ml of mercury(II) acetate solution and carry out Method I for non-aqueous titration, Appendix VIII A, determining the end point
potentiometrically. Not less than 7.7 ml and not more than 8.3 ml of 0.1M perchloric acid VS is
required.
Related substances Carry out in subdued light the method for thin-layer chromatography, Appendix
III A, using silica gel GF254 as the coating substance and a mixture of 50 volumes of propan-1-ol, 50
volumes of a 2% w/v solution of ammonium chloride, 5 volumes of butan-1-ol and 1.5 volumes of
diethylamine as the mobile phase. Apply separately to the plate 5 l of each of three solutions in
methanol (50%) containing (1) 4.0 w/v of the substance being examined, (2) 0.020% w/v of the
substance being examined and (3) 0.0080% w/v of 2-methyl-1,4-naphthaquinone. After removal of the
plate, allow it to dry in air and examine under ultraviolet light (254 nm). Any spot corresponding to
2-methyl-1,4-naphthaquinone (menadione) in the chromatogram obtained with solution (1) is not
more intense than the spot in the chromatogram obtained with solution (3) and any other secondary
spot is not more intense than the spot in the chromatogram obtained with solution (2).
Water 19.0 to 21.5% w/w, Appendix IX A. Use 0.25 g.
Assay Dissolve 0.1 g in 25 ml of water, add 25 ml of glacial acetic acid and 25 ml of 3M hydrochloric
acid and titrate with 0.02M cerium(IV) sulphate VS using platinum and calomel electrodes and determining the end point potentiometrically. Each ml of 0.02M cerium(IV) sulphate VS is equivalent to
4.221 mg of C11H8Na4O8P2.
Action and use Prevention of vitamin K deficiency.
Preparations
Menadiol Phosphate Injection
Menadiol Phosphate Tablets
25-74
Menadione
O
Me
O
C11H8O2
172.2
58-27-5
Menadione complies with the requirements of the 3rd edition of the European Pharmacopoeia [0507]. These
Action and use Synthetic vitamin K analogue.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Menadione contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent
of 2-methyl-1,4-naphthoquinone, calculated with reference to the dried substance.
CHARACTERS
A pale-yellow, crystalline powder, practically insoluble in water, freely soluble in toluene, soluble in
ether, sparingly soluble in alcohol and in methanol. It is unstable in light.
IDENTIFICATION
obtained with menadione CRS.
C. Dissolve about 1 mg in 5 ml of alcohol R, add 2 ml of ammonia R and 0.2 ml of ethyl
cyanoacetate R. An intense bluish-violet colour develops. Add 2 ml of hydrochloric acid R. The colour
disappears.
D. Dissolve about 10 mg in 1 ml of alcohol R, add 1 ml of hydrochloric acid R and heat in a waterbath. A red colour develops.
TESTS
Related substances Carry out the test protected from bright light. Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating substance.
Test solution. Dissolve 0.2 g of the substance to be examined in acetone R and dilute to 10 ml with the
same solvent.
Reference solution. Dilute 0.5 ml of the test solution to 100 ml with acetone R.
Apply separately to the plate 5 l of each solution. Develop over a path of 15 cm using a mixture of 1
volume of nitromethane R, 2 volumes of acetone R, 5 volumes of ethylene chloride R and 90 volumes of
cyclohexane R. Dry the plate in a current of hot air. Repeat the development and drying a further two
times. Examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with the test
solution, apart from the principal spot, is not more intense than the spot in the chromatogram
obtained with the reference solution (0.5 per cent).
Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying over
diphosphorus pentoxide R at a pressure of 2 kPa to 3 kPa for 4 h.
ASSAY
Dissolve 0.150 g in 15 ml of glacial acetic acid R in a flask with a stopper fitted with a valve. Add
15 ml of dilute hydrochloric acid R and 1 g of zinc powder R. Close the flask. Allow the mixture to stand
for 60 min, protected from light, with occasional shaking. Filter the solution over a cotton wad, wash
with three quantities, each of 10 ml, of carbon dioxide-free water R. Add 0.1 ml of ferroin R and
immediately titrate the combined filtrate and washings with 0.1M ammonium and cerium nitrate.
1 ml of 0.1M ammonium and cerium nitrate is equivalent to 8.61 mg of C11H8O2.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
25-75
Menotrophin
9002-68-0
Definition Menotrophin is a dry preparation containing glycoprotein gonadotrophins possessing
follicle-stimulating and luteinising activities. It contains not less than 40 IU of follicle-stimulating
hormone activity per mg. The ratio of IU of luteinising hormone (LH) activity to IU of folliclestimulating hormone (FSH) activity is approximately 1. The preparation is exclusively or
predominantly of pituitary origin and obtained from the urine of post-menopausal women but, where
necessary, chorionic gonadotrophin obtained from the urine of pregnant women may be added to
achieve the above ratio.
Production Menotrophin may be prepared by a suitable fractionation procedure followed by ionexchange chromatography. It is prepared in conditions designed to minimise microbial contamination. The manufacturing process must have been shown to eliminate any adventitious viral
contamination such as hepatitis virus or HIV by appropriate validated methods.
Characteristics An almost white or slightly yellow powder.
Soluble in water.
Identification Causes enlargement of the ovaries of immature female rats and increases the weight
of the seminal vesicles and prostate gland of immature male rats when administered as directed in the
Assay.
Hepatitis antigens Examined by a suitably sensitive immunochemical method, Appendix XIV B,
hepatitis antigens are not detected.
HIV antigens Examined by a suitably sensitive immunochemical method, Appendix XIV B, HIV
antigens are not detected.
Pyrogens Complies with the test for pyrogens, Appendix XIV D. Use per kg of the rabbits weight
1 ml of a solution in sodium chloride injection containing 5 IU of follicle stimulating hormone activity
per ml.
Water Not more than 5% w/w when determined by gas chromatography, Appendix III B, using
throughout dry glassware which may be siliconised and the following solutions. For solution (1)
dilute 15 l of anhydrous methanol (internal standard) with sufficient propan-2-ol R1 to produce
100 ml. For solution (2) dissolve 4 mg of the substance being examined in 0.5 ml of propan-2-ol R1.
For solution (3) dissolve 4 mg of the substance being examined in 0.5 ml of solution (1). For solution (4) add 10 l of water to 50 ml of solution (1).
The chromatographic procedure may be carried out using a stainless steel column (1 m 2 mm)
packed with porous polymer beads (60 to 80 mesh) (Chromosorb 102 is suitable), maintained at
120 with a thermal conductivity detector at 150 and using helium as the carrier gas.
From the chromatograms obtained and taking into account any water detectable in solution (1),
calculate the percentage content of water taking 0.9972 g as its weight per ml at 20.
Assay Carry out the biological assay of menotrophin described below. For each component the
estimated potency is not less than 80% and not more than 125% of the stated potency. The fiducial
limits of error are not less than 64% and not more than 156% of the stated potency.
Storage Menotrophin should be kept in an airtight, tamper-evident container and protected from
light. Under these conditions it may be expected to retain its potency for not less than 3 years.
Labelling The label states (1) the number of IU (Units) of follicle-stimulating hormone activity and
the number of IU (Units) of luteinising hormone activity in the container; (2) the number of
IU(Units) of follicle-stimulating hormone activity per mg and the number of IU (Units) of luteinising
hormone activity per mg; (3) where applicable, the number of IU (Units) of chorionic gonadotrophin
activity per mg; (4) the date after which the material is not intended to be used; (5) the conditions
under which it should be stored; (6) where applicable, that it is sterile.
Action and use Gonadotrophic hormone.
Preparation
Menotrophin Injection
Menotrophin intended for use in the manufacture of a parenteral dosage form without a further appropriate
sterilisation procedure complies with the following additional requirement.
Sterility Complies with the test for sterility, Appendix XVI A.
BIOLOGICAL ASSAY OF MENOTROPHIN
The potency of menotrophin with respect to its follicle-stimulating hormone activity is estimated by
comparing its effect in enlarging the ovaries of immature female rats with that of the Standard
Preparation of human urinary FSH and human urinary LH under the conditions of a suitable
25-76
method of assay. The potency of menotrophin with respect to its luteinising hormone activity is
estimated by comparing its effect in increasing the weight of the seminal vesicles or the prostate gland
of immature male rats with that of the Standard Preparation of human urinary FSH and human
urinary LH under the conditions of a suitable method of assay.
Standard Preparation
The Standard Preparation is the 2nd International Standard for Urinary FSH and LH, human,
established in 1988, consisting of a freeze-dried extract of urine from post-menopausal women
together with lactose (supplied in ampoules containing 54 IU of follicle stimulating hormone activity
and 46 IU of luteinising hormone activity), or another suitable preparation the potency of which has
been determined in relation to the International Standard.
Suggested method
Follicle-stimulating hormone activity Select female rats of the same strain, 19 to 28 days old and
differing in age by not more than 3 days, and having weights such that the difference between the
heaviest rat and the lightest rat is not more than 10 g. Assign the rats at random to six equal groups
of at least five animals. If sets of six litter mates are available, allot one litter mate from each set to
each group and mark according to litter.
Choose three doses of the Standard Preparation and three doses of the preparation being examined
such that the smallest dose produces a positive response in some of the rats and the largest dose does
not produce a maximum response in all of the rats. Use doses in geometric progression. As an initial
approximation, total doses of 1.5, 3.0 and 6.0 IU may be tried although the dose will depend on the
sensitivity of the animals used, which may vary widely.
Dissolve separately the total quantities of the preparation being examined and of the Standard
Preparation corresponding to the daily doses to be used in sufficient albuminphosphate buffer pH 7.2
containing not less than 70 IU of chorionic gonadotrophin per ml so that the daily dose is about 0.2 ml.
Add a suitable antimicrobial preservative such as 0.4% w/v of phenol or 0.002% w/v of thiomersal.
Store the solutions at a temperature of 2 to 8. Inject subcutaneously into each rat the daily dose
allocated to its group. Repeat the procedure after 24 and 48 hours. About 24 hours after the last
injection, kill the rats and remove the ovaries. Remove any extraneous fluid and tissue and immediately weigh the ovaries from each animal. Record the combined weight of both ovaries from each rat.
Calculate the result of the assay by standard statistical methods using the weight of the ovaries as the
response. (The precision of the assay may be improved by a suitable correction of the organ weight
with reference to the weight of the animal from which it was taken; an analysis of covariance may be
used).
Luteinising hormone activity Select male rats of the same strain, approximately 19 to 28 days old
and differing in age by not more than 3 days, and having weights such that the difference between the
heaviest rat and the lightest rat is not more than 10 g. Assign the rats at random to six equal groups
of at least five animals. If sets of six litter mates are available, allot one litter mate from each set to
each group and mark according to litter.
Choose three doses of the Standard Preparation and three doses of the preparation being examined
such that the smallest dose is sufficient to produce a positive response in some of the rats and the
largest dose does not produce a maximum response in all of the rats. Use doses in geometric
progression. As an initial approximation, total doses of 7, 14 and 28 IU may be tried although the
dose will depend on the sensitivity of the animals used, which may vary widely.
Dissolve separately the total quantities of the preparation being examined and of the Standard
Preparation corresponding to the daily doses to be used in sufficient albuminphosphate buffer pH 7.2
so that the daily dose is about 0.2 ml. Add a suitable antimicrobial preservative such as 0.4% w/v of
phenol or 0.002% w/v of thiomersal. Store the solutions at a temperature of 2 to 8. Inject
subcutaneously into each rat the daily dose allocated to its group on 4 consecutive days at the same
time each day. On the fifth day, about 24 hours after the last injection, kill the rats and remove the
seminal vesicles or the prostate gland. Remove any extraneous fluid and tissue and weigh immediately the seminal vesicles or the prostate gland. Calculate the result of the assay by standard statistical
methods, using the weight of the vesicles or the prostate gland as the response. (The precision of the
assay may be improved by a suitable correction of the organ weight with reference to the weight of
the animal from which it was taken; an analysis of covariance may be used.)
25-77
Mepivacaine Hydrochloride
Me
Me
O
,HCl
N
N
H H
Me
and enantiomer
C15H22N2O,HCl
282.8
1722-62-9
Mepivacaine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Local anaesthetic.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Mepivacaine hydrochloride contains not less than 98.5 per cent and not more than the equivalent of
101.0 per cent of (RS)-N-(2,6-dimethylphenyl)-1-methylpiperidine-2-carboxamide hydrochloride,
calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder, freely soluble in water and in alcohol, very slightly soluble in methylene
chloride.
It melts at about 260C with decomposition.
IDENTIFICATION
First identification: A, B, D.
obtained with mepivacaine hydrochloride CRS. Examine the substances prepared as discs.
B. Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable silica gel
Test solution. Dissolve 20 mg of the substance to be examined in alcohol R and dilute to 5 ml with the
same solvent.
Reference solution (a). Dissolve 20 mg of mepivacaine hydrochloride CRS in alcohol R and dilute to 5 ml
Reference solution (b). Dissolve 20 mg of mepivacaine hydrochloride CRS and 20 mg of lidocaine hydrochloride CRS in alcohol R and dilute to 5 ml with the same solvent.
of concentrated ammonia R, 5 volumes of methanol R and 100 volumes of ether R. Allow the plate to
dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram
obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained
with reference solution (b) shows two clearly separated principal spots.
C. To 5 ml of solution S (see Tests) add 1 ml of dilute sodium hydroxide solution R and shake with two
quantities, each of 10 ml, of ether R. Dry the combined upper layers over anhydrous sodium sulphate R.
Filter and evaporate the ether on a water-bath. Dry the residue at 100C to 105C for 2 h. The
melting point (2.2.14) is 151C to 155C.
D. It gives reaction (a) of chlorides (2.3.1).
TESTS
Solution S Dissolve 1.5 g in carbon dioxide-free water R and dilute to 30 ml with the same solvent.
solution B7 (Method II, 2.2.2).
pH (2.2.3). Dilute 2 ml of solution S to 5 ml with carbon dioxide-free water R. The pH of the solution
is 4.0 to 5.0.
Optical rotation (2.2.7). 0.10 to +0.10, determined on solution S.
25-78
Test solution. Dissolve 20.0 mg of the substance to be examined in the mobile phase and dilute to
Reference solution (a). Dissolve 20.0 mg of the substance to be examined and 30.0 mg of mepivacaine
impurity B CRS in the mobile phase and dilute to 100.0 ml with the mobile phase. Dilute 1.0 ml of
the solution to 100.0 ml with the mobile phase.
Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute
a stainless steel column 0.125 m long and 4.6 mm in internal diameter packed with basedeactivated octadecylsilyl silica gel for chromatography R (5 m),
as mobile phase at a flow rate of 1 ml per minute a mixture of 35 volumes of acetonitrile R and 65
volumes of 0.02M phosphoric acid (adjusted to pH 7.6 with strong sodium hydroxide solution R),
Inject 20 l of reference solution (a). Adjust the sensitivity of the system so that the heights of the
two principal peaks in the chromatogram obtained are at least 20 per cent of the full scale of the
recorder. The test is not valid unless the resolution between the peaks corresponding to mepivacaine
impurity B and mepivacaine is at least 2.5.
Inject 20 l of the test solution and 20 l of reference solution (b). Continue the chromatography
of the test solution for three times the retention time of mepivacaine. In the chromatogram obtained
with the test solution: the area of any peak, apart from the principal peak, is not greater than twice
cent) and the area of not more than one of these peaks is greater than the area of the principal peak in
the chromatogram obtained with reference solution (b) (0.1 per cent); the sum of the areas of all the
peaks, apart from the principal peak, is not greater than five times the area of the principal peak in the
chromatogram obtained with reference solution (b) (0.5 per cent). Disregard any peak with an area
less than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (b).
2,6-Dimethylaniline Dissolve 0.50 g in methanol R and dilute to 10 ml with the same solvent. To
2 ml of the solution add 1 ml of a freshly prepared 10 g/l solution of dimethylaminobenzaldehyde R in
methanol R and 2 ml of glacial acetic acid R and allow to stand for 10 min. Any yellow colour in the
manner using 2 ml of a 5 mg/l solution of 2,6-dimethylaniline R in methanol R (100 ppm).
Heavy metals (2.4.8). Dissolve 1.0 g in water R and dilute to 25 ml with the same solvent. Carry out
the prefiltration. 10 ml of the filtrate complies with limit test E for heavy metals (5 ppm). Prepare the
100C to 105C.
ASSAY
Dissolve 0.250 g in a mixture of 5.0 ml of 0.01M hydrochloric acid and 50 ml of alcohol R. Carry out a
1 ml of 0.1M sodium hydroxide is equivalent to 28.28 mg of C15H23ClN2O.
STORAGE
IMPURITIES
Me
H2N
Me
A. 2,6-dimethylaniline,
H
N
Me
O
H
and enantiomer
N
Me
B. (RS)-N-(2,6-dimethylphenyl)piperidine-2-carboxamide,
25-79
Me
O
N
N
Me
C. N-(2,6-dimethylphenyl)pyridine-2-carboxamide,
Me
Me
O
N
H
and enantiomer
N
Me
D. (RS)-N-(2,6-dimethylphenyl)-1-methyl-1,2,5,6-tetrahydropyridine-2-carboxamide,
Me
Me
O
N
H
Cl
and enantiomer
N
Me
E. (RS)-N-(4-chloro-2,6-dimethylphenyl)-1-methylpiperidine-2-carboxamide.
__________________________________________________________________________________________________________ Ph Eur
25-80
Meprobamate
Me
H2N
O
O
C9H18N2O4
Prn
NH2
O
O
218.3
57-53-4
Meprobamate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0407]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Meprobamate contains not less than 97.0 per cent and not more than the equivalent of 101.0 per
cent of 2-methyl-2-propylpropane-1,3-diyl dicarbamate, calculated with reference to the dried
substance.
CHARACTERS
A white or almost white, amorphous or crystalline powder, slightly soluble in water and in ether,
freely soluble in alcohol.
IDENTIFICATION
obtained with meprobamate CRS.
C. To 0.5 g add 1 ml of acetic anhydride R and 0.05 ml of sulphuric acid R, mix and allow to stand for
30 min, shaking frequently. Pour the solution dropwise into 50 ml of water R, mix and allow to stand.
Initiate crystallisation by scratching the wall of the tube with a glass rod. Collect the precipitate by
filtration, wash and dry at 60C. The precipitate melts (2.2.14) at 124C to 128C.
D. Dissolve 0.2 g in 15 ml of 0.5M alcoholic potassium hydroxide and boil under a reflux condenser for
15 min. Add 0.5 ml of glacial acetic acid R and 1 ml of a 50 g/l solution of cobalt nitrate R in ethanol R.
A deep-blue colour develops.
TESTS
Appearance of solution Dissolve 1.0 g in 20 ml of ethanol R. The solution is clear (2.2.1) and
colourless (Method II, 2.2.2).
coating substance.
Test solution. Dissolve 0.20 g of the substance to be examined in alcohol R and dilute to 10 ml with
the same solvent.
Reference solution. Dilute 0.1 ml of the test solution to 10 ml with alcohol R.
10 volumes of pyridine R, 30 volumes of acetone R and 70 volumes of hexane R. Dry the plate at
120C for 30 min, allow to cool and spray with a solution of 0.25 g of vanillin R in a cooled mixture
of 10 ml of alcohol R and 40 ml of sulphuric acid R and heat at 100C to 105C for 30 min. Any spot
in the chromatogram obtained with the test solution, apart from the principal spot, is not more
intense than the spot in the chromatogram obtained with the reference solution (1.0 per cent).
Heavy metals (2.4.8). Dissolve 2.0 g in a mixture of 15 volumes of water R and 85 volumes of
acetone R and dilute to 20 ml with the same mixture of solvents. 12 ml of the solution complies with
limit test B for heavy metals (10 ppm). Prepare the standard using lead standard solution (1 ppm Pb)
obtained by diluting lead standard solution (100 ppm Pb) R with the mixture of water R and acetone R.
Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in vacuo at
60C.
ASSAY
Dissolve 0.1000 g in 15 ml of a 25 per cent V/V solution of sulphuric acid R and boil under a reflux
25-81
condenser for 3 h. Cool, dissolve by cautiously adding 30 ml of water R, cool again and place in a
steam-distillation apparatus. Add 40 ml of strong sodium hydroxide solution R and distil immediately by
passing steam through the mixture. Collect the distillate into 40 ml of a 40 g/l solution of boric acid R
until the total volume in the receiver reaches about 200 ml. Add 0.25 ml of methyl red mixed solution R. Titrate with 0.1M hydrochloric acid until the colour changes from green to violet. Carry out a
blank titration.
1 ml of 0.1M hydrochloric acid is equivalent to 10.91 mg of C9H18N2O4.
__________________________________________________________________________________________________________ Ph Eur
25-82
Meptazinol Hydrochloride
Me
N
Et
,HCl
OH
C15H23NO,HCl
269.8
59263-76-2
Definition Meptazinol Hydrochloride is 3-(3-ethyl-1-methylperhydroazepin-3-yl)phenol hydrochloride. It contains not less than 99.0% and not more than 101.0% of C15H23NO,HCl, calculated
Characteristics A white or almost white powder.
Very soluble in water and in methanol; freely soluble in ethanol (96%); very slightly soluble in
acetone. It dissolves in dilute solutions of alkali hydroxides.
Identification
A. The light absorption, Appendix II B, in the range 230 to 350 nm of a 0.01% w/v solution in ethanol
(96%) exhibits a maximum only at 275 nm. The absorbance at 275 nm is about 0.80.
B. The infrared absorption spectrum, Appendix II A, is concordant with the reference spectrum of
meptazinol hydrochloride (RS 215).
Acidity or alkalinity To 10 ml of a 2% w/v solution in carbon dioxide-free water add 0.2 ml of 0.01M
sodium hydroxide VS and 0.1 ml of methyl red solution; the solution is yellow. Add 0.4 ml of 0.01M
hydrochloric acid VS; the solution is red.
Colour of solution A 10.0% w/v solution is not more intensely coloured than reference solution Y6,
Appendix IV B, Method II.
silica gel precoated plate (Merck silica gel 60 F254 plates are suitable) and a mixture of 1 volume of
18M ammonia, 30 volumes of chloroform and 70 volumes of ethyl acetate as the mobile phase. Apply
separately to the plate 10 l of each of three solutions of the substance being examined in methanol
containing (1) 1.0% w/v, (2) 0.01% w/v and (3) 0.005% w/v, respectively. After removal of the plate,
allow it to dry in air, examine under ultraviolet light (254 nm), expose to iodine vapour for 2 hours
and examine again. For each examination any secondary spot in the chromatogram obtained with
solution (1) is not more intense than the spot in the chromatogram obtained with solution (2) (1%)
and not more than one such spot is more intense than the spot in the chromatogram obtained with
solution (3) (0.5%).
Loss on drying When dried at 105 for 3 hours, loses not more than 0.5% of its weight. Use 1 g.
Assay Dissolve 0.15 g in 50 ml of anhydrous acetic acid and add 5 ml of mercury(II) acetate solution.
Carry out Method I for non-aqueous titration, Appendix VIII A, determining the end point potentiometrically. Each ml of 0.1M perchloric acid VS is equivalent to 26.98 mg of C15H23NO,HCl.
Storage Meptazinol Hydrochloride should kept in a well-closed container and stored at a temperature not exceeding 25.
Action and use Opioid analgesic.
Preparations
Meptazinol Injection
Meptazinol Tablets
IMPURITIES
Me
N
O
Et
OH
A. 3-ethyl-3-(3-hydroxyphenyl)-1-methylperhydroazepin-2-one
25-83
Me
N
Et
B. 3-ethyl-1-methyl-3-phenylperhydroazepine
Me
N
Et
N
Et
OH
Me
C. 4-(3-ethyl-1-methylperhydroazepin-3-yl)-2-[3-(3-ethyl-1-methylperhydroazepin-3yl)phenyl]phenol
Me
Me
N
Et
Et
O
D. 3,3oxybis(3-ethyl-1-methylperhydroazepin-3-ylbenzene)
Me
N
Et
OBun
E. 3-ethyl-3-(3-butoxyphenyl)-1-methylperhydroazepine
26-1
Mepyramine Maleate
OMe
Me2N
N
COOH
COOH
C17H23N3O,C4H4O4
401.5
59-33-6
Mepyramine Maleate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Mepyramine Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Mepyramine maleate contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of N-(4-methoxybenzyl)-NN-dimethyl-N-(2-pyridyl)ethylenediamine hydrogen
maleate, calculated with reference to the dried substance.
CHARACTERS
A white or slightly yellowish, crystalline powder, very soluble in water, freely soluble in alcohol, very
slightly soluble in ether.
IDENTIFICATION
obtained with mepyramine maleate CRS. Examine the substances as 50 g/l solutions in methylene
chloride R using a 0.1 mm cell.
C. Dissolve 0.100 g in 0.01M hydrochloric acid and dilute to 100.0 ml with the same acid. Dilute
350 nm (2.2.25), the solution shows two absorption maxima, at 239 nm and 316 nm. The specific
absorbances at the maxima are 431 to 477 and 196 to 220, respectively.
the chromatogram obtained with reference solution (b).
E. Triturate 0.1 g with 3 ml of water R and 1 ml of strong sodium hydroxide solution R. Shake with
three quantities, each of 5 ml, of ether R. To 0.1 ml of the aqueous layer add a solution of 10 mg of
resorcinol R in 3 ml of sulphuric acid R. Heat on a water-bath for 15 min; no colour develops. To the
rest of the aqueous layer add 1 ml of bromine water R. Heat on a water-bath for 15 min and then heat
to boiling and cool. To 0.2 ml of this solution add a solution of 10 mg of resorcinol R in 3 ml of
sulphuric acid R. Heat on a water-bath for 15 min. A violet-pink colour develops.
TESTS
Appearance of solution Dilute 5 ml of solution S to 25 ml with carbon dioxide-free water R. The
2.2.2).
pH (2.2.3). Dilute 1 ml of solution S to 10 ml with carbon dioxide-free water R. The pH of this
solution is 4.9 to 5.2.
Related substances Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the
coating substance.
with the same solvent. Prepare immediately before use.
26-2
Reference solution (a). Dissolve 0.4 g of mepyramine maleate CRS in chloroform R and dilute to 10 ml
Reference solution (b). Dilute 1 ml of reference solution (a) to 10 ml with chloroform R.
Reference solution (c). Dilute 0.1 ml of reference solution (a) to 50 ml with chloroform R.
Reference solution (d). To 2 ml of reference solution (c) add 2 ml of chloroform R.
volumes of diethylamine R and 100 volumes of ethyl acetate R. Allow the plate to dry in air and
examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with test solution (a),
apart from the principal spot, does not show more intense fluorescence quenching than the spot in
the chromatogram obtained with reference solution (c) (0.2 per cent). The test is not valid unless the
principal spots in the chromatograms obtained with test solution (a) and with reference solution (a)
have Rf values not less than 0.2 and the spot in the chromatogram obtained with reference solution
(d) is clearly visible. During evaluation, ignore the spot remaining at the starting point (maleic acid).
80C.
Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on the residue obtained in the test
for loss on drying.
ASSAY
Dissolve 0.150 g in 40 ml of anhydrous acetic acid R. Titrate with 0.1M perchloric acid determining the
STORAGE
__________________________________________________________________________________________________________ Ph Eur
26-3
Mercaptopurine
SH
H
N
N
N
C5H4N4S,H2O
170.2
6112-76-1
Mercaptopurine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0096].
Preparation
Mercaptopurine Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Mercaptopurine contains not less than 98.5 per cent and not more than the equivalent of 101.0 per
cent of purine-6-thiol, calculated with reference to the anhydrous substance.
CHARACTERS
A yellow, crystalline powder, practically insoluble in water and in ether, slightly soluble in alcohol. It
dissolves in solutions of the alkali hydroxides.
IDENTIFICATION
A. Dissolve 20 mg in 5 ml of dimethyl sulphoxide R and dilute to 100 ml with 0.1M hydrochloric acid.
Dilute 5 ml of the solution to 200 ml with 0.1M hydrochloric acid. Examined between 230 nm and
350 nm (2.2.25), the solution shows only one absorption maximum, at 325 nm.
B. Dissolve about 20 mg in 20 ml of alcohol R heated to 60C and add 1 ml of a saturated solution of
mercuric acetate R in alcohol R. A white precipitate is formed.
C. Dissolve about 20 mg in 20 ml of alcohol R heated to 60C and add 1 ml of a 10 g/l solution of
lead acetate R in alcohol R. A yellow precipitate is formed.
TESTS
Hypoxanthine Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the
coating substance.
Test solution. Dissolve 50 mg of the substance to be examined in 1 ml of dimethyl sulphoxide R and
dilute to 10 ml with methanol R.
Reference solution. Dissolve 10 mg of hypoxanthine R in 10 ml of dimethyl sulphoxide R and dilute to
100 ml with methanol R.
volumes of concentrated ammonia R, 7 volumes of water R and 90 volumes of acetone R. Allow the
plate to dry in air and examine in ultraviolet light at 254 nm. Any spot corresponding to
hypoxanthine in the chromatogram obtained with the test solution is not more intense than the spot
in the chromatogram obtained with the reference solution (2.0 per cent).
Water (2.5.12). 10.0 per cent to 12.0 per cent, determined on 0.250 g by the semi-micro determination of water.
ASSAY
Dissolve 0.100 g in 50 ml of dimethylformamide R. Titrate with 0.1M tetrabutylammonium hydroxide,
determining the end-point potentiometrically (2.2.20).
1 ml of 0.1M tetrabutylammonium hydroxide is equivalent to 15.22 mg of C5H4N4S.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
26-4
Mercuric Chloride
Mercuric Chloride complies with the requirements of the 3rd edition of the European Pharmacopoeia [0120].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Mercuric chloride contains not less than 99.5 per cent and not more than the equivalent of 100.5 per
cent of HgCl2, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder or colourless or white crystals or heavy crystalline masses, soluble in
water, in ether and in glycerol, freely soluble in alcohol.
IDENTIFICATION
A. It gives the reactions of chlorides (2.3.1).
B. Solution S (see Tests) gives the reactions of mercury (2.3.1).
TESTS
Appearance of solution Solution S is not more opalescent than reference suspension II (2.2.1) and
is colourless (Method II, 2.2.2).
Acidity or alkalinity To 10 ml of solution S add 0.1 ml of methyl red solution R. The solution is red.
Add 0.5 g of sodium chloride R. The solution becomes yellow. Not more than 0.5 ml of 0.01M hydrochloric acid is required to change the colour to red.
Mercurous chloride Dissolve 1.0 g in 30 ml of ether R. The solution shows no opalescence.
Loss on drying (2.2.32). Not more than 1.0 per cent, determined on 2.00 g by drying in vacuo for
24 h.
ASSAY
Dissolve 0.500 g in 100 ml of water R. Add 20.0 ml of 0.1M sodium edetate and 5 ml of buffer solution
pH 10.9 R. Allow to stand for 15 min. Add 0.1 g of mordant black 11 triturate R and titrate with 0.1M
zinc sulphate until the colour changes to purple. Add 3 g of potassium iodide R, allow to stand for
2 min, add a further 0.1 g of mordant black 11 triturate R and titrate with 0.1M zinc sulphate.
1 ml of 0.1M zinc sulphate used in the second titration is equivalent to 27.15 mg of HgCl2.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
26-5
Mestranol
Me
OH
C
CH
H
H
MeO
C21H26O2
310.4
72-33-3
Mestranol complies with the requirements of the 3rd edition of the European Pharmacopoeia [0509]. These
Action and use Oestrogen.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Mestranol contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent of
3-methoxy-19-nor-17-pregna-1,3,5(10)-trien-20-yn-17-ol, calculated with reference to the dried
substance.
CHARACTERS
A white or almost white, crystalline powder, practically insoluble in water, soluble in ether, sparingly
soluble in alcohol.
IDENTIFICATION
obtained with mestranol CRS.
C. Examine the chromatograms obtained in the test for related substances in daylight and in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with test solution (b) is
similar in position, colour, fluorescence and size to the principal spot in the chromatogram obtained
with reference solution (a).
D. Dissolve about 5 mg in 1 ml of sulphuric acid R. A red colour develops with a greenish-yellow
fluorescence in ultraviolet light at 365 nm. Add the solution to 10 ml of water R and mix. The
solution becomes pink and a pink to violet precipitate is formed on standing.
TESTS
Specific optical rotation (2.2.7). Dissolve 0.100 g in anhydrous pyridine R and dilute to 10.0 ml
with the same solvent. The specific optical rotation is 20 to 24, calculated with reference to the
dried substance.
Absorbance (2.2.25). Dissolve 25.0 mg in alcohol R and dilute to 25.0 ml with the same solvent.
Dilute 10.0 ml of this solution to 100.0 ml with alcohol R. Examined between 260 nm and 310 nm,
the solution shows two absorption maxima, at 279 nm and 288 nm, and a minimum at 286 nm. The
specific absorbances at the maxima are 62 to 68 and 59 to 64, respectively.
coating substance.
Reference solution (a). Dissolve 10 mg of mestranol CRS in chloroform R and dilute to 10 ml with the
same solvent.
Reference solution (b). Dilute 1 ml of test solution (b) to 10 ml with chloroform R.
Reference solution (c). Dilute 5 ml of reference solution (b) to 10 ml with chloroform R.
10 volumes of alcohol R and 90 volumes of toluene R. Allow the plate to dry in air until the odour of
26-6
the solvent is no longer perceptible. Heat at 110C for 10 min. Spray the hot plate with alcoholic
solution of sulphuric acid R. Heat again at 110C for 10 min. Examine in daylight and in ultraviolet
light at 365 nm. Any spot in the chromatogram obtained with test solution (a), apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution
(b) (1.0 per cent) and at most one such spot is more intense than the spot in the chromatogram
100C to 105C for 3 h.
ASSAY
Dissolve 0.200 g in 40 ml of tetrahydrofuran R and add 5 ml of a 100 g/l solution of silver nitrate R.
Titrate with 0.1M sodium hydroxide, determining the end-point potentiometrically (2.2.20). Carry out
a blank titration.
1 ml of 0.1M sodium hydroxide is equivalent to 31.04 mg of C21H26O2.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
26-7
Metaraminol Tartrate
H
OH
OH
CH3
COOH
, HOOC
H
NH2
OH
OH
C9H13NO2,C4H6O6
317.3
17171-57-2
Definition Metaraminol Tartrate is (1R,2S)-2-amino-1-(3-hydroxyphenyl)propan-1-ol hydrogen

(2R,3R)-tartrate. It contains not less than 99.0% and not more than 101.0% of C9H13NO2,C4H6O6,
Characteristics A white, crystalline powder; odourless or almost odourless.
Freely soluble in water; sparingly soluble in ethanol (96%); practically insoluble in chloroform and in
ether.
Identification
A. In the test for Related substances the principal spot in the chromatogram obtained with solution
(2) corresponds to that in the chromatogram obtained with solution (4).
B. To 0.5 ml of a 0.05% w/v solution add 0.5 ml of phosphomolybdotungstic reagent and 5 ml of dilute
sodium carbonate solution and allow to stand for 5 minutes. An intense blue colour is produced.
C. To 4 ml of a 0.05% w/v solution add 5 ml of borate buffer pH 9.6 and 1 ml of a freshly prepared
0.5% w/v solution of sodium 1,2-naphthaquinone-4-sulphonate and allow to stand for 1 minute. Add
0.2 ml of a 2% v/v solution of benzalkonium chloride solution and 5 ml of toluene and shake. A mauve
colour is immediately produced in the toluene layer (distinction from phenylephrine).
Phenones Absorbance of a 0.2% w/v solution at 310 nm, not more than 0.2, calculated with reference to the dried substance, Appendix II B.
III A, using a silica gel precoated plate (Merck silica gel 60 plates are suitable) and a mixture of 80
volumes of chloroform, 80 volumes of methanol and 10 volumes of 13.5M ammonia as the mobile
phase. Apply separately to the plate 10 l of each of the following solutions in methanol. Solutions (1),
(2) and (3) are solutions of the substance being examined containing (1) 1.0% w/v, (2) 0.050% w/v
and (3) 0.0050% w/v. Solution (4) contains 0.050% w/v of metaraminol tartrate BPCRS. After
removal of the plate, allow it to dry in air and spray with a solution prepared in the following manner.
Mix 25 ml of a 0.45% w/v solution of sulphanilic acid in 1M hydrochloric acid with 1.5 ml of a 5% w/v
solution of sodium nitrite, allow to stand for 5 minutes and mix cautiously with 25 ml of 2M sodium
carbonate. Any secondary spot in the chromatogram obtained with solution (1) is not more intense than
the spot in the chromatogram obtained with solution (3) (0.5%).
1 g.
Assay Carry out Method I for non-aqueous titration, Appendix VIII A, using 0.6 g and crystal violet
solution as indicator. Each ml of 0.1M perchloric acid VS is equivalent to 31.73 mg of
C9H13NO2,C4H6O6.
Storage Metaraminol Tartrate should be kept in a well-closed container.
Action and use Sympathomimetic.
Preparation
Metaraminol Injection
26-8
Metformin Hydrochloride
H
N
Me2N
NH2
,HCl
NH
C4H11N5,HCl
NH
165.6
1115-70-4
Metformin Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Hypoglycaemic.
Preparation
Metformin Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Metformin hydrochloride contains not less than 98.5 per cent and not more than the equivalent of
101.0 per cent of 1,1-dimethylbiguanide hydrochloride, calculated with reference to the dried
substance.
CHARACTERS
White crystals, freely soluble in water, slightly soluble in alcohol, practically insoluble in acetone and
in methylene chloride.
IDENTIFICATION
First identification: B, E.
obtained with metformin hydrochloride CRS. Examine the substances as discs prepared using potassium
chloride R.
C. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance.
same solvent.
Reference solution. Dissolve 20 mg of metformin hydrochloride CRS in water R and dilute to 5 ml with
the same solvent.
Apply separately to the plate 5 l of each solution. Develop over a path of 15 cm using the upper
layer of a mixture of 10 volumes of glacial acetic acid R, 40 volumes of butanol R and 50 volumes of
water R. Dry the plate at 100C to 105C for 15 min and spray with a mixture of equal volumes of a
100 g/l solution of sodium nitroprusside R, a 100 g/l solution of potassium ferricyanide R and a 100 g/l
solution of sodium hydroxide R, prepared 20 min before use. The principal spot in the chromatogram
obtained with the test solution is similar in position, colour and size to the principal spot in the
chromatogram obtained with the reference solution.
D. Dissolve about 5 mg in water R and dilute to 100 ml with the same solvent. To 2 ml of the
solution add 0.25 ml of strong sodium hydroxide solution R and 0.10 ml of -naphthol solution R. Mix
and allow to stand in iced water for 15 min. Add 0.5 ml of sodium hypobromite solution R and mix. A
pink colour develops.
E. It gives reaction (a) of chlorides (2.3.1).
TESTS
Reference solution (a). Dissolve 20.0 mg of cyanoguanidine R in water R and dilute to 100.0 ml with the
same solvent. Dilute 1.0 ml to 200.0 ml with the mobile phase.
1.0 ml to 20.0 ml with the mobile phase.
Reference solution (c). Dissolve 10.0 mg of melamine R in about 90 ml of water R. Add 5.0 ml of the
26-9
test solution and dilute to 100.0 ml with water R. Dilute 1.0 ml to 50.0 ml with the mobile phase.
a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with an irregular,
porous silica gel to which benzenesulphonic acid groups have been chemically bonded (10 m),
or a stainless steel column 0.11 m long and 4.7 mm in internal diameter packed with regular,
porous silica gel to which benzenesulphonic acid groups have been chemically bonded (5 m),
as mobile phase at a flow rate of 1 ml per minute a 17 g/l solution of ammonium dihydrogen
phosphate R adjusted to pH 3.0 with phosphoric acid R,
principal peak in the chromatogram obtained is at least 50 per cent of the full scale of the recorder.
Inject 20 l of reference solution (c). The test is not valid unless the resolution between the peaks
corresponding to melamine and metformin hydrochloride is at least ten.
Inject separately 20 l of the test solution, 20 l of reference solution (a) and 20 l of reference
solution (b). Continue the chromatography for twice the retention time of metformin hydrochloride.
In the chromatogram obtained with the test solution: the area of any peak corresponding to cyanoguanidine is not greater than the area of the peak in the chromatogram obtained with reference
solution (a) (0.02 per cent); the area of any peak, apart from the principal peak and any peak corresponding to cyanoguanidine, is not greater than the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.1 per cent).
100C to 105C for 5 h.
ASSAY
In order to avoid overheating in the reaction medium, mix thoroughly throughout and stop the
titration immediately after the end-point has been reached.
Dissolve 60.0 mg in 4 ml of anhydrous formic acid R. Add 50 ml of acetic anhydride R. Titrate with
1 ml of 0.1M perchloric acid is equivalent to 8.28 mg of C4H12ClN5.
STORAGE
IMPURITIES
NH
N
H
H2N
CN
A. cyanoguanidine,
NH2
N
H2N
N
N
NH
NH2
N
H
B. (4,6-diamino-1,3,5-triazin-2-yl)guanidine,
N(Me)2
H2N
N
N
NH2
C. N,N-dimethyl-1,3,5-triazine-2,4,6-triamine,
H2N
NH2
N
N
N
NH2
D. 1,3,5,-triazine-2,4,6-triamine (melamine),
NH
H2N
N
H
NH
N
H
Me
E. 1-methylbiguanide.
__________________________________________________________________________________________________________ Ph Eur
26-10
Methacrylic AcidEthyl Acrylate Copolymer (1:1)

corrected 1/01
Methacrylic AcidEthyl Acrylate Copolymer (1:1) complies with the requirements of the 3rd edition of the
European Pharmacopoeia [1128]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Methacrylic acidethyl acrylate copolymer (1:1) is a copolymer of methacrylic acid and ethyl acrylate
having a mean relative molecular mass of about 250,000. The ratio of carboxylic groups to ester
groups is about 1:1. It may contain suitable surface-active agents such as sodium dodecyl sulphate
and polysorbate 80. It contains not less than 46.0 per cent m/m and not more than 50.6 per cent m/m
of methacrylic acid units, calculated with reference to the dried substance.
CHARACTERS
A white, free-flowing powder, practically insoluble in water, freely soluble in ethanol and in
2-propanol, practically insoluble in ethyl acetate. It is freely soluble in a 40 g/l solution of sodium
hydroxide.
IDENTIFICATION
A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the Ph. Eur. reference
spectrum of methacrylic acidethyl acrylate copolymer (1:1).
B. It complies with the limits of the assay.
TESTS
Apparent viscosity Dissolve a quantity of substance to be examined corresponding to 37.5 g of the
dried substance in a mixture of 7.9 g of water R and 254.6 g of 2-propanol R. Determine the viscosity
(2.2.10) using a rotating viscometer. At a shear rate of 10 s1, the apparent viscosity is not less than
100 mPas and not more than 200 mPas.
Appearance of a film Place 1 ml of the solution prepared for the apparent viscosity test on a glass
plate and allow to dry. A clear brittle film is formed.
Ethyl acrylate and methacrylic acid Total content: not more than 0.1 per cent, determined by
liquid chromatography (2.2.29).
Blank solution. To 50.0 ml of methanol R add 25.0 ml of mobile phase.
Test solution. Dissolve 40 mg of the substance to be examined in 50.0 ml of methanol R and add
25.0 ml of mobile phase.
Reference solution. Dissolve 10 mg each of ethyl acrylate R and methacrylic acid R in methanol R and
dilute to 50.0 ml with the same solvent. Dilute 0.1 ml of this solution to 50.0 ml with methanol R and
add 25.0 ml of mobile phase.
as mobile phase at a flow rate of 2.5 ml/min a mixture of 30 volumes of methanol R and 70
volumes of phosphate buffer solution pH 2.0 R,
Inject 50 l of each solution. The test is not valid unless the resolution between the peaks corresponding to ethyl acrylate and methacrylic acid in the chromatogram obtained with the reference
solution is at least 2.0. The test is not valid if the chromatogram obtained with the blank solution
shows peaks with the same retention times as ethyl acrylate or methacrylic acid.
Calculate the percentage content of monomers from the area of the peaks in the chromatograms
obtained with the test solution and the reference solution and from the content of monomers in the
reference solution.
105C for 6 h.
ASSAY
Dissolve 1.000 g in a mixture of 40 ml of water R and 60 ml of 2-propanol R. Titrate slowly while
stirring with 0.5M sodium hydroxide, using phenolphthalein solution R as indicator.
1 ml of 0.5M sodium hydroxide is equivalent to 43.05 mg of C4H6O2 (methacrylic acid units).
26-11
LABELLING
The label states, where applicable, the name and concentration of any surface-active agents.
__________________________________________________________________________________________________________ Ph Eur
26-12
Methacrylic AcidEthyl Acrylate Copolymer (1:1) Dispersion 30 per

cent
corrected 1/01
Methacrylic AcidEthyl Acrylate Copolymer (1:1) Dispersion 30 per cent complies with the requirements of
the 3rd edition of the European Pharmacopoeia [1129]. These requirements are reproduced after the heading
Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Methacrylic acidethyl acrylate copolymer (1:1) dispersion 30 per cent is a dispersion in water of a
copolymer of methacrylic acid and ethyl acrylate having a mean relative molecular mass of about
250,000. The ratio of carboxylic groups to ester groups is about 1:1. It may contain suitable surfaceactive agents such as sodium dodecyl sulphate and polysorbate 80. It contains not less than 46.0 per
cent m/m and not more than 50.6 per cent m/m of methacrylic acid units, calculated with reference to
the residue on evaporation.
CHARACTERS
An opaque, white, slightly viscous liquid, miscible with water. On addition of solvents such as
acetone, ethanol or 2-propanol, a precipitate is formed which dissolves on addition of excess solvent.
It is miscible with a 40 g/l solution of sodium hydroxide.
It is sensitive to spoilage by microbial contaminants.
IDENTIFICATION
spectrum of methacrylic acidethyl acrylate copolymer (1:1) dispersion 30 per cent.
TESTS
Apparent viscosity Determine the viscosity (2.2.10) using a rotating viscometer. At a shear rate of
50 s1, the apparent viscosity is not more than 15 mPas.
Appearance of a film Place 1 ml on a glass plate and allow to dry. A clear, brittle film is formed.
Particulate matter Filter 100.0 g through a tared stainless steel sieve (90). Rinse with water R until
a clear filtrate is obtained and dry at 100C to 105C. The mass of the residue is not more than
1.00 g.
Ethyl acrylate and methacrylic acid Total content not more than 0.1 per cent, determined by
liquid chromatography (2.2.29) and calculated with reference to the dried substance.
Reference solution. Dissolve 10 mg each of ethyl acrylate R and methacrylic acid R in methanol R and
dilute to 50.0 ml with the same solvent. Dilute 0.1 ml of this solution to 50.0 ml with methanol R and
add 25.0 ml of mobile phase.
Inject 50 l of each solution. The test is not valid unless the resolution between the peaks corresponding to ethyl acrylate and methacrylic acid in the chromatogram obtained with the reference
solution is at least 2.0. The test is not valid if the chromatogram obtained with the blank solution
shows peaks with the same retention times as ethyl acrylate or methacrylic acid.
reference solution.
Residue on evaporation Dry 1.000 g at 110C for 5 h. The residue weighs not less than 0.285 g
26-13
and not more than 0.315 g.
per gram, determined by plate count.
ASSAY
STORAGE
Store protected from freezing. Handle the substance so as to minimise microbial contamination.
LABELLING
The label states, where applicable, the name and concentration of any surface-active agents.
__________________________________________________________________________________________________________ Ph Eur
26-14
Methacrylic AcidMethyl Methacrylate Copolymer (1:1)

corrected 1/01
Methacrylic AcidMethyl Methacrylate Copolymer (1:1) complies with the requirements of the 3rd edition of
the European Pharmacopoeia [1127]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Methacrylic acidmethyl methacrylate copolymer (1:1) is a copolymer of methacrylic acid and methyl
methacrylate having a mean relative molecular mass of about 135,000. The ratio of carboxylic groups
to ester groups is about 1:1. It contains not less than 46.0 per cent m/m and not more than 50.6 per
cent of methacrylic acid units, calculated with reference to the dried substance.
CHARACTERS
hydroxide.
IDENTIFICATION
spectrum of methacrylic acidmethyl methacrylate copolymer (1:1).
TESTS
Appearance of a film Place 1 ml of the solution prepared for the viscosity test on a glass plate and
allow to dry. A clear brittle film is formed.
Methyl methacrylate and methacrylic acid Total content: not more than 0.1 per cent, determined by liquid chromatography (2.2.29).
Reference solution. Dissolve 10 mg each of methyl methacrylate R and methacrylic acid R in methanol R
and dilute to 50.0 ml with the same solvent. Dilute 0.1 ml of this solution to 50.0 ml with methanol R
and add 25.0 ml of mobile phase.
Inject 50 l of each solution. The test is not valid unless the resolution between the peaks corresponding to methyl methacrylate and methacrylic acid in the chromatogram obtained with the
reference solution is at least 2.0. The test is not valid if the chromatogram obtained with the blank
solution shows peaks with the same retention times as methyl methacrylate or methacrylic acid.
reference solution.
105C for 6 h.
ASSAY
__________________________________________________________________________________________________________ Ph Eur
26-15
Methacrylic AcidMethyl Methacrylate Copolymer (1:2)

corrected 1/01
Methacrylic AcidMethyl Methacrylate Copolymer (1:2) complies with the requirements of the 3rd edition of
the European Pharmacopoeia [1130]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Methacrylic acidmethyl methacrylate copolymer (1:2) is a copolymer of methacrylic acid and methyl
methacrylate having a mean relative molecular mass of about 135,000. The ratio of carboxylic groups
to ester groups is about 1:2. It contains not less than 27.6 per cent m/m and not more than 30.7 per
cent m/m of methacrylic acid units, calculated with reference to the dried substance.
CHARACTERS
hydroxide.
IDENTIFICATION
spectrum of methacrylic acidmethyl methacrylate copolymer (1:2).
TESTS
Appearance of a film Place 1 ml of the solution prepared for the viscosity test on a glass plate and
allow to dry. A clear brittle film is formed.
Methyl methacrylate and methacrylic acid Total content: not more than 0.1 per cent, determined by liquid chromatography (2.2.29).
Reference solution. Dissolve 10 mg each of methyl methacrylate R and methacrylic acid R in methanol R
and dilute to 50.0 ml with the same solvent. Dilute 0.1 ml of this solution to 50.0 ml with methanol R
and add 25.0 ml of mobile phase.
Inject 50 l of each solution. The test is not valid unless the resolution between the peaks corresponding to methyl methacrylate and methacrylic acid in the chromatogram obtained with the
reference solution is at least 2.0. The test is not valid if the chromatogram obtained with the blank
solution shows peaks with the same retention times as methyl methacrylate or methacrylic acid.
reference solution.
100C to 105C for 6 h.
ASSAY
__________________________________________________________________________________________________________ Ph Eur
26-16
Methadone Hydrochloride
H
O
H3C
Me
NMe2
,HCl
and enantiomer
C21H27NO,HCl
345.9
1095-90-5
Methadone Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparations
Methadone Injection
Methadone Linctus
Methadone Oral Solution (1 mg per ml)
Methadone Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Methadone hydrochloride contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of (RS)-6-dimethylamino-4,4-diphenylheptan-3-one hydrochloride, calculated with
reference to the dried substance.
CHARACTERS
A white, crystalline powder, soluble in water, freely soluble in alcohol, practically insoluble in ether.
IDENTIFICATION
First identification: A, C, E.
A. The optical rotation (2.2.7) of solution S (see Tests) measured in a 2 dm tube is 0.05 to +0.05.
C. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the Ph. Eur. reference
spectrum of methadone hydrochloride.
D. To 2.0 ml of solution S add 1 ml of 0.1M hydrochloric acid and 6 ml of ammonium thiocyanate
solution R. A white precipitate is formed which becomes crystalline on stirring for a few minutes. The
crystalline precipitate, dried at 100C to 105C, melts (2.2.14) at 143C to 148C.
E. Dilute 1 ml of solution S to 5 ml with water R and add 1 ml of dilute ammonia R1. Mix, allow to
stand for 5 min and filter. The filtrate gives reaction (a) of chlorides (2.3.1).
TESTS
Acidity or alkalinity Dilute 10 ml of solution S to 25 ml with carbon dioxide-free water R. To 10 ml
of the solution add 0.2 ml of methyl red solution R and 0.2 ml of 0.01M sodium hydroxide. The solution
is yellow. Add 0.4 ml of 0.01M hydrochloric acid. The solution is red.
coating substance.
Test solution. Dissolve 0.5 g of the substance to be examined in alcohol R and dilute to 10 ml with the
same solvent.
Reference solution. Dilute 1 ml of the test solution to 10 ml with alcohol R. Dilute 1 ml of this solution
to 100 ml with alcohol R.
10 volumes of water R, 30 volumes of glacial acetic acid R and 60 volumes of alcohol R. Allow the plate
to dry in air and spray with dilute potassium iodobismuthate solution R. Any spot in the chromatogram
obtained with the test solution, apart from the principal spot, is not more intense than the spot in the
chromatogram obtained with the reference solution (0.1 per cent).
26-17
at 100C to 105C.
ASSAY
Dissolve 0.300 g in 50 ml of anhydrous acetic acid R. Add 5 ml of mercuric acetate solution R. Using
0.1 ml of crystal violet solution R as indicator, titrate with 0.1M perchloric acid until the colour changes
from violet-blue to green.
1 ml of 0.1M perchloric acid is equivalent to 34.59 mg of C21H28ClNO.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
26-18
Methanol
Methyl Alcohol
OH
3C
CH4O
32.04
67-56-1
Definition Methanol is methyl alcohol.

Characteristics A colourless, clear liquid, boiling at 65; hygroscopic.
Miscible with water, with absolute ethanol and with ether.
Identification
A. Complies with the test for Relative density.
B. Complies with the test for Refractive index.
C. The infrared absorption spectrum, Appendix II A, is concordant with the reference spectrum of
methanol (RS 387).
Acidity or alkalinity To 25 ml add 25 ml of water and 0.25 ml of phenolphthalein solution R1. The
solution remains colourless and not more than 0.9 ml of 0.01M sodium hydroxide VS is required to
change the colour of the solution to pink (30 ppm, calculated as acetic acid).
Clarity and colour Clear, Appendix IV A, and colourless, Appendix IV B, Method II.
Refractive index 1.328 to 1.330, Appendix V E.
Relative density 0.791 to 0.793, Appendix V G.
Acetone and aldehydes Dilute 1.25 ml to 5 ml with water and add 5 ml of alkaline potassium
mercuri-iodide solution. Any turbidity produced is not greater than that obtained by treating 5 ml of a
0.0006% w/v solution of acetone in the same manner (0.003%).
Reducing substances To 20 ml add 0.1 ml of 0.02M potassium permanganate VS; the pink colour is
not completely discharged within 5 minutes.
Non-volatile substances Evaporate 100 g to dryness on a water-bath and dry in an oven at 100 to
105. The residue weighs not more than 1 mg (10 ppm).
Water Not more than 0.1% w/w, Appendix IX C. Use 10 g.
Storage Methanol should be protected from moisture and stored at a temperature not exceeding
25.
IMPURITIES
A. Acetone
B. Aldehydes
26-19
Methaqualone
N
Me
Me
N
O
C16H14N2O
250.3
72-44-6
Methaqualone complies with the requirements of the 3rd edition of the European Pharmacopoeia [0510]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Methaqualone contains not less than 99.0 per cent and not more than the equivalent of 101.0 per
cent of 2-methyl-3-(2-methylphenyl)quinazolin-4(3H)-one, calculated with reference to the dried
substance.
CHARACTERS
A white or almost white, crystalline powder, very slightly soluble in water, soluble in alcohol,
sparingly soluble in ether. It dissolves in dilute sulphuric acid.
IDENTIFICATION
B. Dissolve 50.0 mg in 0.1M hydrochloric acid, warming if necessary, and dilute to 100.0 ml with the
same acid. Dilute 1.0 ml of this solution to 100.0 ml with 0.1M hydrochloric acid. Examined between
The specific absorbances at the maxima are 1270 to 1390 and 315 to 345, respectively.
spectrum of methaqualone.
D. Dissolve about 10 mg in 2 ml of alcohol R. Add 1 ml of dimethylaminobenzaldehyde solution R1 and
heat on a water-bath for 5 min. A reddish-orange colour develops.
TESTS
2.2.2).
Acidity Shake 0.6 g with 30 ml of carbon dioxide-free water R for 5 min and filter. To 10 ml of the
filtrate add 0.1 ml of phenolphthalein solution R. Not more than 0.2 ml of 0.01M sodium hydroxide is
required to change the colour of the indicator.
2-Aminobenzoic acid Dissolve 1.00 g in a mixture of 1 volume of water R and 3 volumes of
alcohol R and dilute to 50.0 ml with the same mixture of solvents (test solution). Prepare a reference
solution as follows: dissolve 40 mg of 2-aminobenzoic acid R in a mixture of 1 volume of water R and 3
volumes of alcohol R and dilute to 100.0 ml with the same mixture of solvents; dilute 10.0 ml of this
solution to 100.0 ml with a mixture of 1 volume of water R and 3 volumes of alcohol R; dilute 0.5 ml
of this solution to 50.0 ml with a mixture of 1 volume of water R and 3 volumes of alcohol R (reference solution). To the test solution and to the reference solution add 5 ml of dilute hydrochloric acid R
and cool in iced water for 5 min. Add 0.1 ml of sodium nitrite solution R and cool again in iced water
for 5 min. Add 1.5 ml of a freshly prepared 10 g/l solution of sulphamic acid R and shake until the
colour of the solution is discharged. Add 2.5 ml of a freshly prepared 2 g/l solution of naphthylethylenediamine dihydrochloride R and allow to stand for 2 h 30 min. Examine the solutions as
described for Method II, 2.2.2. The test solution is not more intensely coloured than the reference
solution (20 ppm).
o-Toluidine Dissolve 0.50 g in acetone R and dilute to 10.0 ml with the same solvent (test solution).
Prepare a reference solution as follows: dissolve 50 mg of freshly distilled o-toluidine R in 5 ml of
acetone R and dilute to 100.0 ml with dilute hydrochloric acid R ; dilute 10.0 ml of the solution to
100.0 ml with dilute hydrochloric acid R; dilute 0.1 ml of this solution to 10.0 ml with acetone R (refer-
26-20
ence solution). To the test solution and to the reference solution add 5 ml of water R and cool in
iced water for 5 min. Add 0.1 ml of sodium nitrite solution R and cool again in iced water for 5 min.
Add 10 ml of a freshly prepared 2 g/l solution of -naphthol R in dilute sodium hydroxide solution R.
Examine the solutions as described for Method II, 2.2.2. The test solution is not more intensely
coloured than the reference solution (10 ppm).
100C to 105C.
ASSAY
Dissolve 0.200 g in 30 ml of anhydrous acetic acid R. Titrate with 0.1M perchloric acid, determining the
1 ml of 0.1M perchloric acid is equivalent to 25.03 mg of C16H14N2O.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
26-21
Methenamine
1/01
N
N
N
C6H12N4
140.2
100-97-0
Methenamine complies with the requirements of the 3rd edition of the European Pharmacopoeia [1545]. These
Action and use Anti-infective.
When hexamine is prescribed or demanded, Methenamine shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Methenamine contains not less than 99.0 per cent and not more than the equivalent of 100.5 per
cent of 1,3,5,7-tetra-azotricyclo[3.3.1.13,7]decane, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder or colourless crystals, freely soluble in water, soluble in alcohol and in
methylene chloride.
IDENTIFICATION
spectrum of methenamine.
B. To 1 ml of solution S (see Tests) add 1 ml of sulphuric acid R and immediately heat to boiling.
Allow to cool. To 1 ml of the solution add 4 ml of water R and 5 ml of acetylacetone reagent R1. Heat
on a water-bath for 5 min. An intense yellow colour develops.
C. To 1 ml of solution S add 1 ml of dilute sulphuric acid R and immediately heat to boiling. The
solution gives the reaction of ammonium salts and salts of volatile bases (2.3.1).
D. Dissolve 10 mg in 5 ml of water R and acidify with dilute hydrochloric acid R. Add 1 ml of potassium
iodobismuthate solution R. An orange precipitate is formed immediately.
TESTS
indicator.
Free formaldehyde Dissolve 0.8 g in water R and dilute to 8 ml with the same solvent. Add 2 ml of
ammoniacal silver nitrate solution R. After 5 min, any grey colour in the solution is not more intense
than that in a standard prepared at the same time and in the same manner with a mixture of 8 ml of
freshly prepared formaldehyde standard solution (5 ppm CH2O) R and 2 ml of ammoniacal silver nitrate
solution R (50 ppm).
Ammonium (2.4.1). Dilute 2 ml of freshly prepared solution S to 13 ml with water R. Add 2 ml of
dilute sodium hydroxide solution R. The solution complies with the limit test for ammonium (50 ppm).
desiccator.
26-22
ASSAY
Dissolve 0.100 g in 30 ml of methanol R. Titrate with 0.1M perchloric acid, determining the end-point
potentiometrically (2.2.20).
1 ml of 0.1M perchloric acid is equivalent to 14.02 mg of C6H12N4.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
26-23
Methionine
H
H3C
C5H11NO2S
NH2
COOH
149.2
63-68-3
Methionine complies with the requirements of the 3rd edition of the European Pharmacopoeia [1027]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Methionine contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of (S)-2-amino-4-(methylthio)butanoic acid, calculated with reference to the dried substance.
PRODUCTION
When Methionine is produced by a process involving fermentation steps, it complies with the
requirements of the monograph on Products of fermentation (1468).
CHARACTERS
A white or almost white, crystalline powder or colourless crystals, soluble in water, very slightly
soluble in alcohol, practically insoluble in ether.
IDENTIFICATION
obtained with methionine CRS. Examine the substances prepared as discs.
D. Dissolve 0.1 g of the substance to be examined and 0.1 g of glycine R in 4.5 ml of dilute sodium
hydroxide solution R. Add 1 ml of a 25 g/l solution of sodium nitroprusside R. Heat to 40C for 10 min.
Allow to cool and add 2 ml of a mixture of 1 volume of phosphoric acid R and 9 volumes of hydrochloric acid R. A dark red colour develops.
TESTS
substance.
gel plate R.
Test solution (a). Dissolve 0.10 g of the substance to be examined in dilute hydrochloric acid R and
dilute to 10 ml with the same acid.
Reference solution (a). Dissolve 10 mg of methionine CRS in a 10 g/l solution of hydrochloric acid R and
dilute to 50 ml with the same acid solution.
Reference solution (c). Dissolve 10 mg of methionine CRS and 10 mg of serine CRS in a 10 g/l solution
of hydrochloric acid R and dilute to 25 ml with the same acid solution.
20 volumes of glacial acetic acid R, 20 volumes of water R and 60 volumes of butanol R. Allow the
plate to dry in air, spray with ninhydrin solution R and heat at 100C to 105C for 15 min. Any spot in
the chromatogram obtained with test solution (a), apart from the principal spot, is not more intense
than the spot in the chromatogram obtained with reference solution (b) (0.5 per cent). The test is not
26-24
valid unless the chromatogram obtained with reference solution (c) shows two clearly separated
spots.
Chlorides To 10 ml of solution S add 25 ml of water R, 5 ml of dilute nitric acid R and 10 ml of silver
nitrate solution R2. Allow to stand protected from light for 5 min. Any opalescence in the solution is
not more intense than that in a standard prepared at the same time and in the same manner using
10 ml of chloride standard solution (5 ppm Cl) R (200 ppm). Examine the tubes laterally against a black
background.
Sulphates (2.4.13). Dissolve 0.5 g in 3 ml of dilute hydrochloric acid R and dilute to 15 ml with
distilled water R. The solution complies with the limit test for sulphates (300 ppm).
Ammonium (2.4.1). 0.10 g complies with limit test B for ammonium (200 ppm). Prepare the
combined upper layers add 10 ml of water R and shake for 3 min. The lower layer complies with the
limit test for iron (10 ppm).
100C to 105C.
ASSAY
1 ml of 0.1M perchloric acid is equivalent to 14.92 mg of C5H11NO2S.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
26-25
DL-Methionine
H
H3C
NH2
COOH
and enantiomer
C5H11NO2S
149.2
59-51-8
DL-Methionine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0624].
Action and use Used in treatment of paracetamol overdose.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
DL-Methionine contains not less than 99.0 per cent and not more than the equivalent of 101.0 per
cent of (RS)-2-amino-4-(methylthio)butyric acid, calculated with reference to the dried substance.
CHARACTERS
Almost white, crystalline powder or small flakes, sparingly soluble in water, very slightly soluble in
alcohol, practically insoluble in ether. It dissolves in dilute acids and in dilute solutions of the alkali
hydroxides.
It melts at about 270C (instantaneous method).
IDENTIFICATION
obtained with DL-methionine CRS. Dry the substances at 105C.
B. Examine the chromatograms obtained in the test for related substances. The principal spot in the
C. Dissolve 2.50 g in 1M hydrochloric acid and dilute to 50.0 ml with the same acid. The angle of
optical rotation (2.2.7) is 0.05 to +0.05.
D. Dissolve 0.1 g of the substance to be examined and 0.1 g of glycine R in 4.5 ml of dilute sodium
hydroxide solution R. Add 1 ml of a 25 g/l solution of sodium nitroprusside R. Heat to 40C for 10 min.
Allow to cool and add 2 ml of a mixture of 1 volume of phosphoric acid R and 9 volumes of hydrochloric acid R. A deep-red colour develops.
TESTS
coating substance.
Test solution (a). Dissolve 0.2 g in water R and dilute to 10 ml with the same solvent.
Reference solution (a). Dissolve 20 mg of DL-methionine CRS in water R and dilute to 50 ml with the
same solvent.
Reference solution (b). Dilute 1 ml of reference solution (a) to 10 ml with water R.
plate to dry in air and spray with ninhydrin solution R. Heat the plate at 100C to 105C for 15 min.
Any spot in the chromatogram obtained with test solution (a), apart from the principal spot, is not
more intense than the spot in the chromatogram obtained with reference solution (b) (0.2 per cent).
Chlorides Dissolve 0.25 g in 35 ml of water R. Add 5 ml of dilute nitric acid R and 10 ml of silver
nitrate solution R2. Allow to stand protected from light for 5 min. Any opalescence in the solution is
not more intense than that in a standard prepared at the same time in the same manner using a
mixture of 10 ml of chloride standard solution (5 ppm Cl) R and 25 ml of water R (200 ppm). Examine
the tubes laterally against a black background.
26-26
Sulphates (2.4.13). Dissolve 1.0 g in 20 ml of distilled water R, heating to 60C. Cool to 10C and
filter. 15 ml of the solution complies with the limit test for sulphates (200 ppm).
100C to 105C.
ASSAY
Dissolve 0.140 g in 3 ml of anhydrous formic acid R. Add 30 ml of glacial acetic acid R. Immediately
after dissolution, titrate with 0.1M perchloric acid, determining the end-point potentiometrically
(2.2.20).
STORAGE
__________________________________________________________________________________________________________ Ph Eur
26-27
Methotrexate
H
HOOC
COOH
NH
O
NH2
N
N
N
Me
2N
C20H22N8O5
N
454.4
59-05-2
Methotrexate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0560]. These
Preparations
Methotrexate Injection
Methotrexate Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Methotrexate contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent
of (S)-2-[4-[[(2,4-diaminopteridin-6-yl)methyl]methylamino]benzoylamino]pentanedioic acid,
calculated with reference to the anhydrous substances.
CHARACTERS
A yellow or orange, crystalline, hygroscopic powder, practically insoluble in water, in alcohol and in
methylene chloride. It dissolves in dilute solutions of mineral acids and in dilute solutions of alkali
hydroxides and carbonates.
IDENTIFICATION
A. Dissolve 0.250 g in a 14 g/l solution of sodium carbonate R and dilute to 25.0 ml with the same
solution. The specific optical rotation (2.2.7) is +19 to +24 calculated with reference to the
B. Dissolve 10 mg in a 4 g/l solution of sodium hydroxide R and dilute to 100 ml with the same
solvent. Dilute 10 ml of this solution to 100 ml with a 4 g/l solution of sodium hydroxide R. Examined
between 230 nm and 380 nm (2.2.25), the solution shows three absorption maxima, at 258 nm,
302 nm and 371 nm, respectively. The ratio of the absorbance measured at the maximum at 302 nm
to that measured at the maximum at 371 nm is 2.8 to 3.3.
obtained with methotrexate CRS.
TESTS
Related substances Examine by liquid chromatography (2.2.29), as prescribed under Assay but
setting the spectrophotometer at 265 nm.
Inject 20 l of reference solution (c). The test is not valid unless in the chromatogram obtained, the
resolution between the peaks corresponding to impurity C and methotrexate respectively is at least
2.0.
for three times the retention time of methotrexate.
When the chromatograms are recorded in the prescribed conditions, the approximate relative
retention times are the following:
impurity A
0.4
impurity B
0.3
impurity C
0.8
impurity D 2.3
impurity E
0.2
In the chromatogram obtained with the test solution, the area of at most five peaks, apart from the
principal peak, is greater than 0.2 times the area of the principal peak in the chromatogram obtained
with reference solution (b) (0.1 per cent) and no such peak has an area greater than that of the
26-28
principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent). The sum
of the areas of all the peaks, apart from the principal peak, is not greater than 2.6 times the area of
the principal peak in the chromatogram obtained with reference solution (b) (1.3 per cent).
Disregard any peak with an area less than 0.05 times that of the principal peak in the chromatogram obtained with reference solution (b).
(R)-Methotrexate Not more than 3.0 per cent. Examine by liquid chromatography (2.2.29).
Reference solution (a). Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase.
Reference solution (b). Dissolve 4.0 mg of (RS)-methotrexate R in the mobile phase and dilute to
a stainless steel column 0.15 m long and 4.0 mm in internal diameter packed with bovine
albumin R bound to silica gel for chromatography R (film thickness 7 m; pore size 30 nm),
as mobile phase at a flow rate of 1.5 ml/min a solution prepared as follows: to 500 ml of a 7.1 g/l
solution of anhydrous disodium hydrogen phosphate R add 600ml of a 6.9 g/l solution of sodium
dihydrogen phosphate monohydrate R and mix. Adjust to pH 6.9 with dilute sodium hydroxide
solution R. To 920 ml of this mixture add 80 ml of propanol R,
Inject 20 l of each solution. The test is not valid unless in the chromatogram obtained with
reference solution (b); the resolution between the peaks corresponding to (S)-methotrexate and
(R)-methotrexate respectively is at least 3.0.
In the chromatogram obtained with the test solution the area of any peak corresponding to
(R)-methotrexate is not greater than the three times the area of the principal peak in the chromatogram obtained with reference solution (a) (3.0 per cent).
Heavy metals (2.4.8). 1.0 g complies with test C for heavy metals (50 ppm). Prepare the standard
using 5 ml of lead standard solution (10 ppm Pb) R.
ASSAY
Reference solution (a). Dissolve 25.0 mg of methotrexate CRS in the mobile phase and dilute to
Reference solution (b). Dilute 0.5 ml of reference solution (a) to 100.0 ml with the mobile phase.
Reference solution (c). Dilute 25.0 mg of the substance to be examined and 25.0 mg of methotrexate
impurity C CRS in the mobile phase and dilute to the 250.0 ml with the mobile phase.
as mobile phase at a flow rate of 1.2 ml/min a mixture of 7 volumes of acetonitrile R and 93
volumes of a solution obtained as follows: dissolve 7.8 g of citric acid R and 17.9 g of anhydrous
disodium hydrogen phosphate R in water R and dilute to 100 ml with the same solvent,
Inject reference solution (a) six times. The assay is not valid unless the relative standard deviation
of the area of the methotrexate peak is not greater than 2.0 per cent. Inject 20 l of the test solution
and 20 l of reference solution (a). Calculate the percentage content of methotrexate using the
chromatograms obtained with the test solution and reference solution (a).
STORAGE
IMPURITIES
NH2
N
N
2N
OH
A. (2,4-diaminopteridin-6-yl)methanol,
26-29
O
N
H
R1
N
N
2N
H COOH
COOH
N
R2
B. R1 = NH2, R2 = H: (S)-2-[4-[[(2,4-diaminopteridin-6-yl)methyl]amino]benzoylamino]pentanedioic acid (4-aminofolic acid, aminopterine),

C. R1 = OH, R2 = CH3: (S)-2-[4-[[(2-amino-4-hydroxypteridin-6-yl)methyl]methylamino]benzoylamino]pentanedioic acid (N-methylfolic acid, methopterine),
COOH
R
N
N
2N
N
Me
D. R = OH: 4-[[(2-amino-4-hydroxypteridin-6-yl)methyl]methylamino]benzoic acid

(N10-methylpteroic acid),
E. R = NH2: 4-[[(2,4-diaminopteridin-6-yl)methyl]methylamino]benzoic acid (2,4-diamino-N10methylpterioc acid, APA),
O
NH2
N
N
2N
H COOH
N
H
COOH
N
Me
F. (R)-2-[4-[[(2,4-diaminopteridin-6-yl)methyl]methylamino]benzoylamino]pentanedioic acid
((R)-methotrexate).
__________________________________________________________________________________________________________ Ph Eur
26-30
Methoxamine Hydrochloride
MeO
OH
CH3
,HCl
H
NH2
OMe
mixture of 4 stereoisomers
C11H17NO3,HCl
247.7
61-16-5
Definition Methoxamine Hydrochloride is all-rac-2-amino-1-(2,5-dimethoxyphenyl)propan-1-ol

hydrochloride. It contains not less than 99.0% and not more than 101.0% of C11H17NO3,HCl,
Characteristics Colourless crystals or white, plate-like crystals or a white, crystalline powder;
odourless or almost odourless.
Freely soluble in water; soluble in ethanol (96%); very slightly soluble in chloroform and in ether.
Identification
methoxamine hydrochloride (RS 220).
B. Yields the reactions characteristic of chlorides, Appendix VI.
Related substances Carry out the method for thin-layer chromatography, Appendix III A, using silica
gel GF254 as the coating substance and a mixture of 86 volumes of chloroform, 12 volumes of methanol
and 2 volumes of 13.5M ammonia as the mobile phase. Apply separately to the plate 5 l of each of
three solutions in methanol containing (1) 2.0% w/v of the substance being examined, (2) 0.010% w/v
of 2,5-dimethoxybenzaldehyde and (3) 0.020% w/v of the substance being examined. After removal of
the plate, allow it to dry in air and examine under ultraviolet light (365 nm). Any spot corresponding
to 2,5-dimethoxybenzaldehyde in the chromatogram obtained with solution (1) is not more intense
than the spot in the chromatogram obtained with solution (2). Spray the plate with a 0.3% w/v
solution of ninhydrin in butan-1-ol containing 3% v/v of glacial acetic acid and heat at 105 for 5
minutes. Any other secondary spot in the chromatogram obtained with solution (1) is not more intense
than the spot in the chromatogram obtained with solution (3).
Assay Carry out Method I for non-aqueous titration, Appendix VIII A, using 0.5 g and
1-naphtholbenzein solution as indicator. Each ml of 0.1M perchloric acid VS is equivalent to 24.77 mg of
C11H17NO3,HCl.
Preparation
Methoxamine Injection
26-31
Methyl Hydroxybenzoate
Methylparaben
COOMe
HO
C8H8O3
152.1
99-73-3
Methyl Hydroxybenzoate complies with the requirements of the 3rd edition of the European Pharmacopoeia
for Methyl Parahydroxybenzoate [0409]. These requirements are reproduced after the heading Definition
below.
Action and use Antimicrobial preservative.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Methyl parahydroxybenzoate contains not less than 99.0 per cent and not more than the equivalent
of 100.5 per cent of methyl 4-hydroxybenzoate.
CHARACTERS
A white, crystalline powder or colourless crystals, very slightly soluble in water, freely soluble in
alcohol and in methanol.
IDENTIFICATION
obtained with methyl parahydroxybenzoate CRS.
D. To about 10 mg in a test-tube add 1 ml of sodium carbonate solution R, boil for 30 s and cool
(solution a). To a further 10 mg in a similar test-tube add 1 ml of sodium carbonate solution R; the
substance partly dissolves (solution b). Add at the same time to solution (a) and solution (b) 5 ml of
aminopyrazolone solution R and 1 ml of potassium ferricyanide solution R and mix. Solution (b) is yellow
to orange-brown. Solution (a) is orange to red, the colour being clearly more intense than any similar
colour which may be obtained with solution (b).
TESTS
Solution S Dissolve 1.0 g in alcohol R and dilute to 10 ml with the same solvent.
Acidity To 2 ml of solution S add 3 ml of alcohol R, 5 ml of carbon dioxide-free water R and 0.1 ml of
bromocresol green solution R. Not more than 0.1 ml of 0.1M sodium hydroxide is required to change the
colour of the indicator to blue.
Related substances Examine by thin-layer chromatography (2.2.27), using as the coating substance
a suitable octadecylsilyl silica gel with a fluorescent indicator having an optimal intensity at 254 nm.
Reference solution (a). Dilute 0.5 ml of test solution (a) to 100 ml with acetone R.
Reference solution (b). Dissolve 10 mg of methyl parahydroxybenzoate CRS in acetone R and dilute to
Reference solution (c). Dissolve 10 mg of ethyl parahydroxybenzoate CRS in 1 ml of test solution (a) and
dilute to 10 ml with acetone R.
Apply to the plate 2 l of each solution. Develop over a path of 15 cm using a mixture of 1 volume of
glacial acetic acid R, 30 volumes of water R and 70 volumes of methanol R. Allow the plate to dry in air
and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with test solution
(a), apart from the principal spot, is not more intense than the spot in the chromatogram obtained
with reference solution (a) (0.5 per cent). The test is not valid unless the chromatogram obtained
with reference solution (c) shows two clearly separated principal spots.
26-32
ASSAY
Place 2.000 g in a ground-glass-stoppered flask and add 40.0 ml of 1M sodium hydroxide. Heat gently
under a reflux condenser for 1 h. Allow to cool and rinse the condenser with water R. Titrate the
excess sodium hydroxide with 0.5M sulphuric acid, continuing the titration until the second point of
inflexion and determining the end-point potentiometrically (2.2.20). Carry out a blank titration.
IMPURITIES
COOR
HO
A. R = H: 4-hydroxybenzoic acid,
B. R = CH2-CH3: ethyl 4-hydroxybenzoate,
C. R = CH2-CH2-CH3: propyl 4-hydroxybenzoate,
D. R = CH2-CH2-CH2-CH3: butyl 4-hydroxybenzoate.
__________________________________________________________________________________________________________ Ph Eur
26-33
Methyl Nicotinate
N
COOMe
C7H7NO2
137.1
93-60-7
Definition Methyl Nicotinate is methyl pyridine-3-carboxylate. It contains not less than 99.0%
and not more than 101.0% of C7H7NO2, calculated with reference to the anhydrous substance.
Characteristics White or almost white, crystals or crystalline powder; odour, characteristic.
Very soluble in water, in chloroform and in ethanol (96%); freely soluble in ether.
Identification
methyl nicotinate (RS 222).
B. The light absorption, Appendix II B, in the range 230 to 350 nm of a 0.002% w/v solution exhibits
a maximum only at 264 nm. The absorbance at 264 nm is about 0.46.
C. To 2 ml of a 0.1% w/v solution add 6 ml of cyanogen bromide solution and 1 ml of a 2.5% v/v
solution of aniline. A golden yellow colour is produced.
gel GF254 as the coating substance and a mixture of 48 volumes of chloroform, 45 volumes of ethanol
(96%) and 8 volumes of water as the mobile phase. Apply separately to the plate 10 l of each of two
solutions of the substance being examined in methanol containing (1) 5.0% w/v and (2) 0.025% w/v.
After removal of the plate, allow it to dry in air and examine under ultraviolet light (254 nm). Any
secondary spot in the chromatogram obtained with solution (1) is not more intense than the spot in the
chromatogram obtained with solution (2).
Assay Carry out Method I for non-aqueous titration, Appendix VIII A, using 0.3 g and determining
the end point potentiometrically. Each ml of 0.1M perchloric acid VS is equivalent to 13.71 mg of
C7H7NO2.
Storage Methyl Nicotinate should be kept in a well-closed container.
Action and use Vasodilator.
26-34
Methyl Salicylate
COOMe
OH
C8H8O3
152.1
119-36-860-7
Methyl Salicylate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0230].
Action and use Counter-irritant.
Preparations
Methyl Salicylate Liniment
Methyl Salicylate Ointment
When oil of wintergreen, wintergreen or wintergreen oil is prescribed or demanded, Methyl Salicylate
shall be dispensed or supplied unless it is ascertained that Methyl Salicylate Liniment is required.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Methyl salicylate contains not less than 99.0 per cent m/m and not more than the equivalent of
100.5 per cent m/m of methyl 2-hydroxybenzoate.
CHARACTERS
A colourless or slightly yellow liquid, very slightly soluble in water, miscible with alcohol and with
fatty and essential oils.
IDENTIFICATION
A. Heat 0.25 ml with 2 ml of dilute sodium hydroxide solution R on a water-bath for 5 min. Add 3 ml of
dilute sulphuric acid R. A crystalline precipitate is formed. Filter. The precipitate, washed with water R
and dried at 100C to 105C, melts (2.2.14) at 156C to 161C.
B. To 10 ml of a saturated solution add 0.05 ml of ferric chloride solution R1. A violet colour develops.
TESTS
Appearance of solution To 2 ml add 10 ml of alcohol R. The solution is clear (2.2.1) and not more
intensely coloured than reference solution Y7 (Method II, 2.2.2).
Acidity Dissolve 5.0 g in a mixture of 0.2 ml of bromocresol green solution R and 50 ml of alcohol R
previously neutralised to a blue colour by addition of 0.1M sodium hydroxide. Not more than 0.4 ml of
0.1M sodium hydroxide is required to restore the blue colour.
Refractive index (2.2.6): 1.535 to 1.538.
ASSAY
Dissolve 0.500 g in 25 ml of alcohol R. Add 0.05 ml of phenol red solution R and neutralise with 0.1M
sodium hydroxide. To the neutralised solution add 50.0 ml of 0.1M sodium hydroxide and heat under a
reflux condenser on a water-bath for 30 min. Cool and titrate with 0.1M hydrochloric acid. Calculate
the volume of 0.1M sodium hydroxide used in the saponification. Carry out a blank titration.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
26-35
Industrial Methylated Spirit

Industrial Methylated Spirits; IMS
Definition Industrial Methylated Spirit is a mixture of nineteen volumes of ethanol of an
appropriate strength with one volume of approved wood naphtha. Two strengths are available
containing 99% by volume and 95% by volume of alcohol (also known as 74 OP and 66 OP
respectively).
Characteristics A colourless, clear, mobile, volatile liquid, boiling at about 78; odour, spirituous
and of wood naphtha.
Identification Mix 0.1 ml with 0.05 ml of an 11% w/w solution of orthophosphoric acid and 0.25 ml
of dilute potassium permanganate solution. After 1 minute add a few mg of sodium metabisulphite and
shake until the mixture is decolorised. Add 1.5 ml of a 50% v/v solution of sulphuric acid and a few
mg of finely powdered chromotropic acid sodium salt, shake well and heat on a water bath for 5
minutes. A deep violet colour is produced.
Acidity or alkalinity 25 ml requires not more than 0.2 ml of 0.1M sodium hydroxide VS to produce a
pink colour with phenolphthalein solution R1 and not more than 1.0 ml of 0.1M hydrochloric acid VS to
produce a red colour with methyl red solution.
Clarity of solution Dilute 5.0 ml to 100 ml with water. The solution is clear, Appendix IV A.
Apparent density For 66 OP grade, not greater than 811.6 kg m3, and for 74 OP grade, not
greater than 792.8 kg m3, Appendix V G.
Aldehydes To 5.0 ml add 5 ml of water and 1 ml of decolorised fuchsin solution and allow to stand for
30 minutes. Any colour produced is not more intense than that obtained by treating in the same
manner 5 ml of a 0.005% w/v solution of redistilled acetaldehyde in aldehyde-free ethanol (96%)
(50 ppm).
Non-volatile matter When evaporated and dried at 105, leaves not more than 0.01% w/v of
residue.
26-36
Industrial Methylated Spirit (Ketone-free)

Definition Industrial Methylated Spirit (Ketone-free) is a mixture of nineteen volumes of ethanol
of an appropriate strength with one volume of approved wood naphtha substantially free from
ketones. Two strengths are available containing 99% by volume and 95% by volume of alcohol
(also known as 74 OP and 66 OP respectively).
Characteristics; Identification; Acidity or alkalinity; Clarity of solution; Apparent density;
Aldehydes; Non-volatile matter Complies with the requirements stated under Industrial
Methylated Spirit.
Ketones Dilute 5 ml to 10 ml with water, add 1 ml of a 1.0% w/v solution of 2-nitrobenzaldehyde in
ethanol (50%) followed by 1 ml of a 15% w/v solution of sodium hydroxide in water and allow to stand
for 15 minutes. Any colour produced is not more intense than that produced by treating in the same
manner 10 ml of a 0.025% v/v solution of acetone in ethanol (50%) (500 ppm).
26-37
Methylcellulose
9004-67-5
Methylcellulose complies with the requirements of the 3rd edition of the European Pharmacopoeia [0345].
Action and use Bulk-forming laxative; pharmaceutical aid.
Preparations
Methylcellulose Granules
Methylcellulose Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Methylcellulose is a partly O-methylated cellulose.
CHARACTERS
A white, yellowish-white or greyish-white powder or granules, hygroscopic after drying, practically
insoluble in hot water, in acetone, in ethanol, in ether and in toluene. It dissolves in cold water giving
a colloidal solution.
IDENTIFICATION
A. Heat 10 ml of solution S (see Tests) in a water-bath while stirring. At a temperature above 50C
the solution becomes cloudy or a flocculent precipitate is formed. The solution becomes clear again
on cooling.
B. To 10 ml of solution S add 0.3 ml of dilute acetic acid R and 2.5 ml of a 100 g/l solution of tannic
acid R. A yellowish-white, flocculent precipitate is formed which dissolves in dilute ammonia R1.
C. In a test-tube about 160 mm long, thoroughly mix 1 g with 2 g of finely powdered manganese
sulphate R. Introduce to a depth of 2 cm into the upper part of the tube a strip of filter paper
impregnated with a freshly prepared mixture of l volume of a 20 per cent V/V solution of
diethanolamine R and 11 volumes of a 50 g/l solution of sodium nitroprusside R, adjusted to about pH
9.8 with 1M hydrochloric acid. Insert the tube 8 cm into a silicone-oil bath at 190C to 200C. The
filter paper does not become blue within 10 min. Carry out a blank test.
D. Dissolve 0.2 g completely, without heating, in 15 ml of a 70 per cent m/m solution of sulphuric
acid R. Pour the solution with stirring into 100 ml of iced water and dilute to 250 ml with iced
water R. In a test-tube, mix thoroughly while cooling in iced water l ml of the solution with 8 ml of
sulphuric acid R, added dropwise. Heat in a water-bath for exactly 3 min and immediately cool in iced
water. While the mixture is cold, carefully add 0.6 ml of ninhydrin solution R2 and mix well. Allow to
stand at 25C. A pink colour is produced immediately and does not become violet within 100 min.
E. Place l ml of solution S on a glass plate. After evaporation of the water a thin film is formed.
F. 0.2 g does not dissolve in 10 ml of toluene R nor in 10 ml of ethanol R.
TESTS
Solution S While stirring, introduce a quantity of the substance to be examined equivalent to l.0 g of
the dried substance into 50 g of carbon dioxide-free water R heated to 90C. Allow to cool, adjust the
mass of the solution to 100 g with carbon dioxide-free water R and stir until dissolution is complete.
Allow to stand at 2C to 8C for 1 h before carrying out the test for appearance of solution.
Appearance of solution Solution S is not more opalescent than reference suspension III (2.2.1) and
not more intensely coloured than reference solution Y6 (Method II, 2.2.2).
Apparent viscosity While stirring, introduce a quantity of the substance to be examined equivalent
to 6.00 g of the dried substance into 150 g of water R heated to 90C. Stir with a propeller-type
stirrer for 10 min, place the flask in a bath of iced water, continue the stirring, and allow to remain in
the bath of iced water for 40 min to ensure that solution is complete. Adjust the mass of the solution
to 300 g, and centrifuge the solution to expel any entrapped air. Adjust the temperature of the
solution to 20 0.1C. Determine the viscosity (2.2.10) with a rotating viscometer at 20C and a
shear rate of 10 s1. The apparent viscosity is not less than 75 per cent and not more than 140 per
cent of the value stated on the label.
26-38
Loss on drying (2.2.32). Not more than 10.0 per cent, determined on 1.000 g by drying in an
oven at 100C to 105C.
STORAGE
LABELLING
The label states the apparent viscosity in millipascal seconds for a 2 per cent m/m solution.
__________________________________________________________________________________________________________ Ph Eur
26-39
Methyldopa
HO
COOH
Me
NH2
HO
C10H13NO4,1H2O
238.2
41372-08-1
Methyldopa complies with the requirements of the 3rd edition of the European Pharmacopoeia [0045]. These
Action and use Antihypertensive.
Preparation
Methyldopa Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Methyldopa contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent
of (S)-2-amino-3-(3,4-dihydroxyphenyl)-2-methylpropionic acid, calculated with reference to the
CHARACTERS
A white or yellowish white, crystalline powder or colourless or almost colourless crystals, slightly
soluble in water, very slightly soluble in alcohol, practically insoluble in ether. It is freely soluble in
IDENTIFICATION
obtained with methyldopa CRS.
B. Dissolve about 2 mg in 2 ml of water R and add 0.2 ml of ferric chloride solution R2. A green colour
develops which changes to bluish-violet on the addition of 0.1 g of hexamethylenetetramine R.
C. Dissolve about 5 mg in a mixture of 5 ml of 1M hydrochloric acid and 5 ml of water R. Add 0.1 ml
of sodium nitrite solution R containing 100 g/l of ammonium molybdate R. A yellow colour is produced
which becomes brownish-red on the addition of strong sodium hydroxide solution R.
D. To about 5 mg add 1 ml of water R, 1 ml of pyridine R and about 5 mg of nitrobenzoyl chloride R
and heat to boiling. Add, while shaking, 0.2 ml of sodium carbonate solution R. An orange or amber
colour develops.
TESTS
Appearance of solution Dissolve 1.0 g in 1M hydrochloric acid and dilute to 25 ml with the same
solvent. The solution is not more intensely coloured than reference solution BY6 or B6 (Method II,
2.2.2).
Acidity Dissolve 1.0 g with heating in 100 ml of carbon dioxide-free water R. Add 0.1 ml of methyl red
solution R. Not more than 0.5 ml of 0.1M sodium hydroxide is required to produce the pure yellow
colour of the indicator.
Optical rotation (2.2.7). Dissolve a quantity equivalent to 2.20 g of the anhydrous substance in
aluminium chloride solution R and dilute to 50.0 ml with the same solution. The angle of optical
rotation is 1.10 to 1.23.
Absorbance (2.2.25). Dissolve 40.0 mg in 0.1M hydrochloric acid and dilute to 100.0 ml with the
same acid. Dilute 10.0 ml of this solution to 100.0 ml with 0.1M hydrochloric acid. Examined between
230 nm and 350 nm, the solution shows a single absorption maximum, at 280 nm. The specific
absorbance at this maximum is 122 to 137, calculated with reference to the anhydrous substance.
Methoxymethyldopa and related substances Examine by thin-layer chromatography (2.2.27),
using cellulose for chromatography R as the coating substance.
Test solution. Dissolve 0.1 g of the substance to be examined in a mixture of 0.4 ml of hydrochloric
acid R1 and 9.6 ml of methanol R.
Reference solution (a). Dissolve 5 mg of 3-methoxymethyldopa CRS in 100 ml of methanol R.
Reference solution (b). To 1 ml of the test solution add 1 ml of reference solution (a).
26-40
Apply separately to the plate 10 l of the test solution, 10 l of reference solution (a) and 20 l of
reference solution (b). Develop over a path of 10 cm using a mixture of 15 volumes of glacial acetic
acid R, 25 volumes of water R and 65 volumes of butanol R. Dry the plate immediately in a current of
warm air and spray with a mixture of 5 volumes of a 50 g/l solution of sodium nitrite R and 45
volumes of a 3 g/l solution of nitroaniline R in a mixture of 20 volumes of water R and 80 volumes of
hydrochloric acid R. Dry the plate immediately in a current of warm air and spray with a 200 g/l
solution of sodium carbonate R. Examine the chromatograms immediately. Any spot in the chromatogram obtained with the test solution, apart from the principal spot, is not more intense than the spot
in the chromatogram obtained with reference solution (a) (0.5 per cent). The test is not valid unless
ASSAY
Dissolve 0.200 g in a mixture of 15 ml of anhydrous formic acid R 30 ml of anhydrous acetic acid R and
30 ml of dioxan R. Titrate with 0.1M perchloric acid until a green colour is obtained using 0.1 ml of
crystal violet solution R as indicator.
1 ml of 0.1M perchloric acid is equivalent to 21.12 mg of C10H13NO4.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
26-41
Methyldopate Hydrochloride
COOEt
HO
,HCl
Me
NH2
HO
C12H17NO4,HCl
275.7
2508-79-4
Definition Methyldopate Hydrochloride is ethyl 3-(3,4-dihydroxyphenyl)-2-methyl-L-alaninate

hydrochloride. It contains not less than 98.5% and not more than 101.0% of C12H17NO4,HCl,
Characteristics A white or almost white, crystalline powder; odourless or almost odourless.
Freely soluble in water, in ethanol (96%) and in methanol; slightly soluble in chloroform; practically
insoluble in ether.
Identification
methyldopate hydrochloride (RS 224).
B. The light absorption, Appendix II B, in the range 230 to 350 nm of a 0.008% w/v solution in 0.1M
hydrochloric acid exhibits a maximum only at 280 nm. The absorbance at the maximum at 280 nm is
about 0.80.
C. Yields reaction A characteristic of chlorides, Appendix VI.
Specific optical rotation In a 4% w/v solution in 0.1M hydrochloric acid and determined at 405 nm,
13.5 to 14.9, calculated with reference to the dried substance, Appendix V F.
gel GF254 as the coating substance and a mixture of equal volumes of acetone, butan-1-ol, glacial acetic
acid, toluene and water as the mobile phase, but allowing the solvent front to ascend 12 cm above the
line of application. Apply separately to the plate 2 l of each of three solutions in methanol containing
(1) 10.0% w/v of the substance being examined, (2) 0.25% w/v of methyldopa BPCRS and (3)
0.040% w/v of methyldopa BPCRS. After removal of the plate, allow it to dry in air, heat at 105 for
10 minutes and examine under ultraviolet light (254 nm). Expose the plate to iodine vapour for 10
minutes and examine again. By each method of visualisation any spot corresponding to methyldopa
in the chromatogram obtained with solution (1) is not more intense than the spot in the chromatogram obtained with solution (2) and any other secondary spot is not more intense than the spot in the
Loss on drying When dried to constant weight at 105 at a pressure not exceeding 0.7 kPa, loses
not more than 0.5% of its weight. Use 1 g.
C12H17NO4,HCl.
Storage Methyldopate Hydrochloride should be protected from light.
Preparation
Methyldopate Injection
26-42
Methylphenobarbital
Me
O
Et
O
NH
O
and enantiomer
C13H14N2O3
246.3
113-38-8
Methylphenobarbital complies with the requirements of the 3rd edition of the European Pharmacopoeia [0189].
Action and use Anticonvulsant.
Preparation
Methylphenobarbital Tablets
When methylphenobarbitone is prescribed or demanded, Methylphenobarbital shall be dispensed
or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Methylphenobarbital contains not less than 99.0 per cent and not more than the equivalent of
102.0 per cent of 5-ethyl-1-methyl-5-phenyl-1H,3H,5H-pyrimidine-2,4,6-trione, calculated with
CHARACTERS
A white, crystalline powder or colourless crystals, practically insoluble in water, slightly soluble in
ether, very slightly soluble in ethanol. It forms water-soluble compounds with alkali hydroxides and
carbonates and with ammonia.
IDENTIFICATION
A. Determine the melting point (2.2.14) of the substance to be examined. Mix equal parts of the
substance to be examined and methylphenobarbital CRS and determine the melting point of the
mixture. The difference between the two melting points (which are about 178C) is not greater than
2C.
obtained with methylphenobarbital CRS.
Test solution. Dissolve 0.1 g of the substance to be examined in chloroform R and dilute to 100 ml with
the same solvent.
Reference solution. Dissolve 0.1 g of methylphenobarbital CRS in chloroform R and dilute to 100 ml with
the same solvent.
Apply separately to the plate 10 l of each solution. Develop over a path of 18 cm using the lower
layer of a mixture of 5 volumes of concentrated ammonia R, 15 volumes of alcohol R and 80 volumes of
chloroform R. Examine immediately in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution.
D. To about 10 mg add 0.2 ml of sulphuric acid R and 0.1 ml of nitric acid R. Heat on a water-bath
for 10 min. Cool in iced water and add 5 ml of water R and 5 ml of strong sodium hydroxide solution R.
Add 5 ml of acetone R, shake and allow to stand. A dark-red colour develops in the upper layer.
TESTS
Appearance of solution Dissolve 1.0 g, with gentle heating, in a mixture of 4 ml of dilute sodium
hydroxide solution R and 6 ml of water R. The solution is clear (2.2.1) and not more intensely coloured
than reference solution Y6 (Method II, 2.2.2).
Acidity Boil 1.0 g with 50 ml of water R for 2 min, allow to cool and filter. To 10 ml of the filtrate
26-43
add 0.15 ml of methyl red solution R. The solution is orange-yellow. Not more than 0.1 ml of 0.1M
sodium hydroxide is required to produce a pure yellow colour.
coating substance.
the same solvent.
Reference solution. Dissolve 0.1 g of phenobarbital CRS in chloroform R and dilute to 100 ml with the
same solvent. Dilute 10 ml of the solution to 100 ml with chloroform R.
chloroform R. Examine immediately in ultraviolet light at 254 nm. Spray with diphenylcarbazone
mercuric reagent R. Allow the plate to dry in air and spray with freshly prepared alcoholic potassium
hydroxide solution R diluted 1 in 5 with aldehyde-free alcohol R. Heat at 100C to 105C for 5 min and
examine immediately. When examined in ultraviolet light and after spraying, any spot in the chromatogram obtained with the test solution, apart from the principal spot, is not more intense than the
spot in the chromatogram obtained with the reference solution (1.0 per cent).
100C to 105C.
ASSAY
Dissolve 0.200 g in 5 ml of pyridine R. Add 0.5 ml of thymolphthalein solution R and 10 ml of silver
nitrate solution in pyridine R. Titrate with 0.1M ethanolic sodium hydroxide until a pure blue colour is
obtained. Carry out a blank titration.
1 ml of 0.1M ethanolic sodium hydroxide is equivalent to 24.63 mg of C13H14N2O3.
__________________________________________________________________________________________________________ Ph Eur
26-44
Methylprednisolone
O
Me
H
HO
OH
OH
Me
O
H Me
C22H30O5
374.5
83-43-2
Methylprednisolone complies with the requirements of the 3rd edition of the European Pharmacopoeia [0561].
Action and use Corticosteroid.
Preparation
Methylprednisolone Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Methylprednisolone contains not less than 97.0 per cent and not more than the equivalent of
103.0 per cent of 11,17,21-trihydroxy-6-methylpregna-1,4-diene-3,20-dione, calculated with
CHARACTERS
alcohol, slightly soluble in acetone and in methylene chloride.
IDENTIFICATION
Second identification: C, D.
obtained with methylprednisolone CRS. If the spectra obtained in the solid state show differences,
dissolve the substance to be examined and the reference substance separately in the minimum
volume of acetone R, evaporate to dryness on a water-bath and record new spectra using the residues.
B. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel F254 plate R.
Test solution. Dissolve 10 mg of the substance to be examined in a mixture of 1 volume of methanol R
and 9 volumes of methylene chloride R and dilute to 10 ml with the same mixture of solvents.
Reference solution (a). Dissolve 20 mg of methylprednisolone CRS in a mixture of 1 volume of
solvents.
Reference solution (b). Dissolve 10 mg of hydrocortisone CRS in reference solution (a) and dilute to
10 ml with reference solution (a).
Apply to the plate 5 l of each solution. Prepare the mobile phase by adding a mixture of 1.2 volumes
of water R and 8 volumes of methanol R to a mixture of 15 volumes of ether R and 77 volumes of
methylene chloride R. Develop over a path of 15 cm. Carry out a second development over a path of
15 cm using a mixture of 5 volumes of butanol R saturated with water R, 15 volumes of toluene R and
80 volumes of ether R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The
principal spot in the chromatogram obtained with the test solution is similar in position and size to
the principal spot in the chromatogram obtained with reference solution (a). Spray with alcoholic
solution of sulphuric acid R. Heat at 120C for 10 min or until the spots appear. Allow to cool.
Examine the chromatograms in daylight and in ultraviolet light at 365 nm. The principal spot in the
chromatogram obtained with the test solution is similar in position, colour in daylight, fluorescence in
ultraviolet light at 365 nm and size to the principal spot in the chromatogram obtained with reference
C. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel F254 plate R.
Test solution (a). Dissolve 25 mg of the substance to be examined in methanol R and dilute to 5 ml
with the same solvent. This solution is also used to prepare test solution (b). Dilute 2 ml of the
solution to 10 ml with methylene chloride R.
26-45
Test solution (b). Transfer 0.4 ml of the solution obtained during the preparation of test solution (a)
to a glass tube 100 mm long and 20 mm in diameter and fitted with a ground-glass stopper or a
polytetrafluoroethylene cap and evaporate the solvent with gentle heating under a stream of
nitrogen R. Add 2 ml of a 15 per cent V/V solution of glacial acetic acid R and 50 mg of sodium
bismuthate R. Stopper the tube and shake the suspension in a mechanical shaker protected from light
for 1 h. Add 2 ml of a 15 per cent V/V solution of glacial acetic acid R and filter into a 50 ml
separating funnel, washing the filter with two quantities, each of 5 ml, of water R. Shake the clear
filtrate with 10 ml of methylene chloride R. Wash the organic layer with 5 ml of 1M sodium hydroxide
and two quantities, each of 5 ml, of water R. Dry over anhydrous sodium sulphate R.
Reference solution (a). Dissolve 25 mg of methylprednisolone CRS in methanol R and dilute to 5 ml with
the same solvent. This solution is also used to prepare reference solution (b). Dilute 2 ml of the
Reference solution (b). Transfer 0.4 ml of the solution obtained during preparation of reference
solution (a) to a glass tube 100 mm long and 20 mm in diameter and fitted with a ground-glass
stopper or a polytetrafluoroethylene cap and evaporate the solvent with gentle heating under a stream
of nitrogen R. Add 2 ml of a 15 per cent V/V solution of glacial acetic acid R and 50 mg of sodium
bismuthate R. Stopper the tube and shake the suspension in a mechanical shaker protected from light
for 1 h. Add 2 ml of a 15 per cent V/V solution of glacial acetic acid R and filter into a 50 ml
Apply to the plate 5 l of test solution (a), 5 l of reference solution (a), 10 l of test solution (b) and
10 l of reference solution (b), applying the latter two in small quantities in order to obtain small
spots. Develop over a path of 15 cm using a mixture of 5 volumes of butanol R saturated with
water R, 10 volumes of toluene R and 85 volumes of ether R. Allow the plate to dry in air and examine
in ultraviolet light at 254 nm. The principal spot in each of the chromatograms obtained with the test
solutions is similar in position and size to the principal spot in the chromatogram obtained with the
corresponding reference solution. Spray with alcoholic solution of sulphuric acid R and heat at 120C
for 15 min. Allow to cool. Examine the plate in daylight and in ultraviolet light at 365 nm. The
principal spot in each of the chromatograms obtained with the test solutions is similar in position,
colour in daylight, fluorescence in ultraviolet light at 365 nm and size to the principal spot in the
chromatogram obtained with the corresponding reference solution. The principal spots in the
chromatograms obtained with test solution (b) and reference solution (b) have an Rf value distinctly
higher than that of the principal spots in the chromatograms obtained with test solution (a) and
D. Add about 2 mg to 2 ml of sulphuric acid R and shake to dissolve. Within 5 min, an intense red
colour develops. When examined in ultraviolet light at 365 nm, brownish-red fluorescence is seen.
Add the solution to 10 ml of water R and mix. The colour fades and there is a yellowish-green
fluorescence in ultraviolet light at 365 nm.
TESTS
solvent. The specific optical rotation is +79.0 to +86.0, calculated with reference to the dried
substance.
Test solution. Dissolve 25.0 mg of the substance to be examined in a mixture of equal volumes of
acetonitrile R and methanol R and dilute to 10.0 ml with the same mixture of solvents.
Reference solution (a). Dissolve 2 mg of methylprednisolone CRS and 2 mg of betamethasone CRS in
mobile phase A and dilute to 100.0 ml with the same mobile phase.
as mobile phase at a flow rate of 2.5 ml/min a linear-gradient programme using the following
conditions:
Mobile phase A. In a 1000 ml volumetric flask mix 250 ml of acetonitrile R with 700 ml of water R
and allow to equilibrate; adjust the volume to 1000 ml with water R and mix again,
Mobile phase B. Acetonitrile R,
26-46
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B Comment

(per cent V/V)
0
15
40
100
100
0
0
0
100
41
100
46 = 0
100
isocratic
begin linear gradient
end chromatogram,
return to 100A
begin equilibration
with A
end equililbration,
begin next
chromatogram

Equilibrate the column with mobile phase B at a flow rate of 2.5 ml/min for at least 30 min and
then with mobile phase A for 5 min. For subsequent chromatograms, use the conditions described
from 40 min to 46 min. Adjust the sensitivity of the system so that the height of the principal peak in
the chromatogram obtained with 20 l of reference solution (b) is at least 50 per cent of the full scale
of the recorder.
Inject 20 l of reference solution (a). When the chromatograms are recorded in the conditions
described above, the retention times are: methylprednisolone, about 11.5 min and betamethasone,
about 12.5 min. The test is not valid unless the resolution between the peaks corresponding to
methylprednisolone and betamethasone is at least 1.5; if necessary, adjust the concentration of
acetonitrile in mobile phase A.
Inject separately 20 l of a mixture of equal volumes of acetonitrile R and methanol R as a blank,
20 l of the test solution and 20 l of reference solution (b). In the chromatogram obtained with the
test solution: the area of any peak, apart from the principal peak, is not greater than half the area of
the principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent); the sum
of the areas of all the peaks, apart from the principal peak, is not greater than twice the area of the
principal peak in the chromatogram obtained with reference solution (b) (2 per cent). Disregard any
peak due to the blank and any peak with an area less than 0.05 times the area of the principal peak in
100C to 105C.
ASSAY
Dissolve 0.100 g in alcohol R and dilute to 100.0 ml with the same solvent. Dilute 2.0 ml of the
STORAGE
IMPURITIES
O
Me
OH
OH
Me
H
H
O
H Me
A. 17,21-dihydroxy-6-methylpregna-1,4-diene-3,11,20-trione,
OH
HO
O
Me
OH
OH
Me
H
H
O
H Me
B. 11,17,21,21-tetrahydroxy-6-methylpregna-1,4-diene-3,20-dione,
26-47
Me O
HO
Me
H
H
O
H Me
C. 11-hydroxy-6-methylandrosta-1,4-diene-3,17-dione,
HO
HO
Me
Me
H
H
and Z-isomer at C*
O
H Me
D. (E)- and (Z)-11,20-dihydroxy-6-methylpregna-1,4,17(20)-triene-3,21-dione.

__________________________________________________________________________________________________________ Ph Eur
26-48
Methylprednisolone Acetate
O
H
Me
HO
OAc
OH
Me
H
H
O
H
C24H32O6
Me
416.5
53-36-1
Methylprednisolone Acetate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Methylprednisolone Acetate Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Methylprednisolone acetate contains not less than 97.0 per cent and not more than the equivalent of
103.0 per cent of 6-methyl-11,17,21-trihydroxypregna-1,4-diene-3,20-dione 21-acetate, calculated
CHARACTERS
acetone and in alcohol, slightly soluble in ether.
IDENTIFICATION
Second identification: C, D, E.
obtained with methylprednisolone acetate CRS. If the spectra obtained in the solid state show
differences, dissolve the substance to be examined and the reference substance separately in the
minimum volume of acetone R, evaporate to dryness and record new spectra using the residues.
B. Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating substance.
Reference solution (a). Dissolve 10 mg of methylprednisolone acetate CRS in a mixture of 1 volume of
solvents.
Reference solution (b). Dissolve 10 mg of prednisolone acetate CRS and 10 mg of methylprednisolone
acetate CRS in a mixture of 1 volume of methanol R and 9 volumes of methylene chloride R and dilute
to 10 ml with the same mixture of solvents.
volumes of butanol R, 10 volumes of toluene R and 85 volumes of ether R. Allow the plate to dry in air
and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with
with reference solution (a). Spray the plate with alcoholic solution of sulphuric acid R. Heat at 120C for
10 min or until the spots appear. Allow to cool. Examine in daylight and in ultraviolet light at
365 nm. The principal spot in the chromatogram obtained with the test solution is similar in
position, colour in daylight, fluorescence in ultraviolet light at 365 nm and size to the principal spot
in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows two spots which, when examined in ultraviolet light
at 365 nm, may not be completely separated.
26-49
Test solution (b). Transfer 2 ml of the solution obtained during preparation of test solution (a) to a
15 ml glass tube with a ground-glass stopper or a polytetrafluoroethylene cap. Add 10 ml of saturated
methanolic potassium hydrogen carbonate solution R and immediately pass a current of nitrogen R
through the solution for 5 min. Stopper the tube. Heat in a water-bath at 45C protected from light
for 1 h. Allow to cool.
Reference solution (a). Dissolve 25 mg of methylprednisolone acetate CRS in methanol R and dilute to
5 ml with the same solvent. This solution is also used to prepare reference solution (b). Dilute 2 ml
of the solution to 10 ml with methylene chloride R.
Reference solution (b). Transfer 2 ml of the solution obtained during preparation of reference solution
(a) to a 15 ml glass tube with a ground-glass stopper or a polytetrafluoroethylene cap. Add 10 ml of
saturated methanolic potassium hydrogen carbonate solution R and immediately pass a current of
nitrogen R through the solution for 5 min. Stopper the tube. Heat in a water-bath at 45C protected
from light for 1 h. Allow to cool.
Apply separately to the plate 5 l of each solution. Prepare the mobile phase by adding a mixture of
1.2 volumes of water R and 8 volumes of methanol R to a mixture of 15 volumes of ether R and 77
volumes of methylene chloride R. Develop over a path of 15 cm. Allow the plate to dry in air and
examine in ultraviolet light at 254 nm. The principal spot in each of the chromatograms obtained
with the test solutions is similar in position and size to the principal spot in the chromatogram
obtained with the corresponding reference solution. Spray with alcoholic solution of sulphuric acid R.
Heat at 120C for 10 min or until the spots appear. Allow to cool. Examine in daylight and in ultraviolet light at 365 nm. The principal spot in each of the chromatograms obtained with the test
solutions is similar in position, colour in daylight, fluorescence in ultraviolet light at 365 nm and size
to the principal spot in the chromatogram obtained with the corresponding reference solution. The
principal spots in the chromatograms obtained with test solution (b) and reference solution (b) have
an Rf value distinctly lower than that of the principal spots in the chromatograms obtained
respectively with test solution (a) and reference solution (a).
colour develops. When examined in ultraviolet light at 365 nm, a reddish-brown fluorescence is seen.
Add the solution to 10 ml of water R and mix. The colour fades and there is a greenish-yellow
fluorescence in ultraviolet light at 365 nm.
E. About 10 mg gives the reaction of acetyl (2.3.1).
TESTS
substance.
Reference solution (a). Dissolve 4 mg of methylprednisolone acetate CRS and 4 mg of dexamethasone
acetate CRS in the mobile phase and dilute to 20.0 ml with the mobile phase. Dilute 2.0 ml of this
solution to 10.0 ml with the mobile phase.
as mobile phase at a flow rate of 1 ml per minute a mixture prepared as follows: in a 1000 ml
volumetric flask mix 260 ml tetrahydrofuran R and 700 ml of water R and leave to equilibrate;
adjust the volume to 1000 ml with water R and mix again,
Equilibrate the column with the mobile phase at a flow rate of 1 ml per minute for about 45 min.
conditions, the retention times are: methylprednisolone acetate, about 43 min; dexamethasone
acetate, about 57 min. The test is not valid unless the resolution between the peaks corresponding to
methylprednisolone acetate and dexamethasone acetate is not less than 6.5. If necessary, adjust the
concentration of water R in the mobile phase.
Inject separately 20 l of the test solution and 20 l of reference solution (b). Continue the
chromatography for 1.5 times the retention time of the principal peak. In the chromatogram obtained
with the test solution, the sum of the areas of all the peaks, apart from the principal peak, is not
greater than half the area of the principal peak in the chromatogram obtained with reference solution
(b) (1.0 per cent). Disregard any peak due to the solvent and any peak with an area less than 0.025
times the area of the principal peak in the chromatogram obtained with reference solution (b).
26-50
at 100C to 105C.
ASSAY
Calculate the content of C24H32O6, taking the specific absorbance to be 355.
STORAGE
IMPURITIES
HO
Me
Me
OAc
OH
H
H
O
H Me
A. 20( + )-dihydro-6-methylprednisolone 21-acetate,
HO
O
Me
Me
OH
OH
H
H
O
H Me
B. 6-methylprednisolone,
HO
O
Me
O
OH
Me
H
H
O
H Me
C. 21-dehydromethylprednisolone,
HO
O
Me
O
H
Me
H
H
O
H Me
D. 21-dehydro-17-deoxymethylprednisolone,
HO
O
Me
OAc
OH
Me
H
H
E. prednisolone acetate,
O
Me
O
Me
OAc
H
H
O
H Me
F. 21-hydroxy-6-methylpregna-1,4-diene-3,11,20-trione 21-acetate,
26-51
HO
Me
O
Me
OAc
OH
H
H
O
H Me
G. 6-methylhydrocortisone acetate,
Me
Me
OAc
H
H
O
H Me
H. 11,21-dihydroxy-6-methylpregna-1,4,17(20)-trien-3-one 21-acetate.
__________________________________________________________________________________________________________ Ph Eur
26-52
Methylprednisolone Hydrogen Succinate

corrected 1/01
O
O
Me
H
HO
COOH
OH
Me
O
H Me
C26H34O8
474.6
2921-57-5
Methylprednisolone Hydrogen Succinate complies with the requirements of the 3rd edition of the European
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Methylprednisolone hydrogen succinate contains not less than 97.0 per cent and not more than the
equivalent of 4-[(11,17-dihydroxy-6-methyl-3,20-dioxopregna-1,4-dien-21-yl)oxy]-4-oxobutanoic
acid, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, hygroscopic powder, practically insoluble in water, slightly soluble in
acetone and in ethanol, practically insoluble in ether. It dissolves in dilute solutions of alkali
hydroxides.
IDENTIFICATION
obtained with methylprednisolone hydrogen succinate CRS.
B. Examine by thin layer chromatography (2.2.27), using a TLC silica gel F254 plate R.
Reference solution (a). Dissolve 20 mg of methylprednisolone hydrogen succinate CRS in a mixture of 1
volume of methanol R and 9 volumes of methylene chloride R and dilute to 20 ml with the same
Reference solution (b). Dissolve 10 mg of hydrocortisone hydrogen succinate CRS in reference solution (a)
and dilute to 10 ml with the same reference solution.
Apply to the plate 10 l of each solution. Develop over a path of 15 cm using a mixture of 0.1
volumes of anhydrous formic acid R, 1 volume of ethanol R and 15 volumes of methylene chloride R.
Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the
chromatogram obtained with the test solution is similar in position and size to the principal spot in
the chromatogram obtained with reference solution (a). Spray the plate with alcoholic solution of
sulphuric acid R. Heat at 120C for 10 min or until the spots appear. Allow to cool. Examine in
daylight and in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the
test solution is similar in position, colour in daylight, fluorescence in ultraviolet light at 365 nm and
size to the principal spot in the chromatogram obtained with reference solution (a). The test is not
valid unless the chromatogram obtained with reference solution (b) shows two spots which may,
however, not be completely separated.
C. Examine by thin layer chromatography (2.2.27), using a TLC silica gel F254 plate R.
Test solution (a). Dissolve 25 mg of the substance to be examined in methanol R with gentle heating
and dilute to 5 ml with the same solvent. This solution is also used to prepare test solution (b).
Dilute 2 ml of the solution to 10 ml with methylene chloride R.
Test solution (b). Transfer 2 ml of the solution obtained during the preparation of test solution (a) to a
15 ml glass tube with a ground-glass stopper or a polytetrafluoroethylene cap. Add 10 ml of a 0.8 g/l
26-53
solution of sodium hydroxide R in methanol R and immediately pass a stream of nitrogen R through the
solution for 5 min. Stopper the tube. Heat in a water-bath at 45C, protected from light, for 30 min.
Allow to cool.
Reference solution (a). Dissolve 25 mg of methylprednisolone hydrogen succinate CRS in methanol R with
gentle heating and dilute to 5 ml with the same solvent. This solution is also used to prepare
reference solution (b). Dilute 2 ml of the solution to 10 ml with methylene chloride R.
(a) to a 15 ml glass tube with a ground-glass stopper or a polytetrafluoroethylene cap. Add 10 ml of a
0.8 g/l solution of sodium hydroxide R in methanol R and immediately pass a stream of nitrogen R
through the solution for 5 min. Stopper the tube. Heat in a water-bath at 45C, protected from light,
for 30 min. Allow to cool.
Apply to the plate 5 l of each solution. Prepare the mobile phase by adding a mixture of 1.2 volumes
of water R and 8 volumes of methanol R to a mixture of 15 volumes of ether R and 77 volumes of
methylene chloride R. Develop over a path of 15 cm. Allow the plate to dry in air and examine in
ultraviolet light at 254 nm. The principal spot in each of the chromatograms obtained with the test
solutions is similar in position and size to the principal spot in the chromatogram obtained with the
corresponding reference solution.
Spray the plate with alcoholic solution of sulphuric acid R. Heat at 120C for 10 min or until the spots
appear. Allow to cool. Examine in daylight and in ultraviolet light at 365 nm. The principal spot in
each of the chromatograms obtained with the test solutions is similar in position, colour in daylight,
fluorescence in ultraviolet light at 365 nm and size to the principal spot in the chromatogram
obtained with the corresponding reference solution. The principal spot in each of the chromatograms
obtained with test solution (b) and reference solution (b) has an Rf value distinctly higher than that of
the principal spot in each of the chromatograms obtained with test solution (a) and reference solution
(a).
D. Add about 2 mg to 2 ml of sulphuric acid R and shake to dissolve. Within 5 min a reddish-brown
colour develops. Add the solution to 10 ml of water R and mix. The colour fades and a precipitate is
formed.
TESTS
Appearance of solution Dissolve 0.100 g in 5 ml of sodium hydrogen carbonate solution R. The
solution is clear (2.2.1).
substance.
Reference solution (a). Dissolve 25 mg of methylprednisolone hydrogen succinate for performance test CRS
in the mobile phase and dilute to 10.0 ml with the mobile phase.
volumes of a 3 per cent V/V solution of glacial acetic acid R,
conditions, the retention times are: methylprednisolone hydrogen succinate about 22 min and
methylhydrocortisone 21-(hydrogen succinate) (the impurity eluting immediately after the main peak
and appearing as a shoulder) about 24 min. Measure the height (A) above the base-line of the peak
due to methylhydrocortisone 21-(hydrogen succinate) and the height (B) above the base-line of the
lowest point of the curve separating this peak from the peak due to methylprednisolone hydrogen
succinate. The test is not valid unless A is greater than four times B. If necessary, adjust the concentration of acetonitrile in the mobile phase.
chromatography for twice the retention time of the principal peak. In the chromatogram obtained
with the test solution: the area of any peak, apart from the principal peak, is not greater than 0.5
times the area of the principal peak in the chromatogram obtained with reference solution (b)
(0.5 per cent); the sum of the areas of all the peaks, apart from the principal peak, is not greater than
26-54
the area of the principal peak in the chromatogram obtained with reference solution (b) (1 per
cent). Disregard any peak due to the solvent and any peak with an area less than 0.05 times the
area of the principal peak in the chromatogram obtained with reference solution (b).
at 100C to 105C.
ASSAY
STORAGE
IMPURITIES
O
Me
HO
OR1
OR2
Me
H
H
Me
A. R1 = R2 = H: methylprednisolone,
B. R1 = H, R2 = CO-CH2-CH2-CO2H: 4-[(11,21-dihydroxy-6-methyl-3,20-dioxopregna1,4-dien-17-yl)oxy]-4-oxobutanoic acid (methylprednisolone 17-(hydrogen succinate)),
C. R1 = CO-CH3, R2 = H: 11,17-dihydroxy-6-methyl-3,20-dioxopregna-1,4-dien-21-yl
acetate (methylprednisolone acetate),
O
O
Me
HO
COOH
OH
Me
H
H
O
Me
D. 4-[(11,17-dihydroxy-6-methyl-3,20-dioxopregn-4-en-21-yl)oxy]-4-oxobutanoic acid
(methylhydrocortisone 17-(hydrogen succinate)).
__________________________________________________________________________________________________________ Ph Eur
26-55
Methyltestosterone
Me Me
OH
Me
H
H
O
C20H30O2
302.5
58-18-4
Methyltestosterone complies with the requirements of the 3rd edition of the European Pharmacopoeia [0410].
Action and use Androgen; anabolic steroid.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Methyltestosterone contains not less than 97.0 per cent and not more than the equivalent of
103.0 per cent of 17-hydroxy-17-methylandrost-4-en-3-one, calculated with reference to the dried
substance.
CHARACTERS
A white or slightly yellowish-white, crystalline powder, practically insoluble in water, freely soluble in
alcohol, slightly soluble in ether.
IDENTIFICATION
obtained with methyltestosterone CRS.
C. After examination of the chromatograms obtained in the test for related substances, spray the
plate with a saturated solution of potassium dichromate R in a mixture of 30 volumes of water R and 70
volumes of sulphuric acid R and examine immediately in daylight. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the
chromatogram obtained with reference solution (a).
TESTS
substance.
a suitable silica gel containing a fluorescent indicator having an optimal intensity at 254 nm.
Test solution. Dissolve 0.2 g of the substance to be examined in a mixture of 1 volume of methanol R
and 9 volumes of chloroform R and dilute to 10 ml with the same mixture of solvents.
Reference solution (a). Dissolve 20 mg of methyltestosterone CRS in 1 ml of a mixture of 1 volume of
methanol R and 9 volumes of chloroform R.
Reference solution (b). Dilute 1 ml of the test solution to 100 ml with a mixture of 1 volume of
Reference solution (c). Dilute 5 ml of reference solution (b) to 10 ml with a mixture of 1 volume of
Reference solution (d). Dissolve 10 mg of testosterone CRS in 0.5 ml of reference solution (a) and dilute
to 10 ml with a mixture of 1 volume of methanol R and 9 volumes of chloroform R.
volume of anhydrous acetic acid R, 30 volumes of light petroleum R and 70 volumes of butyl acetate R.
Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with the test solution, apart from the principal spot, is not more intense than the spot
in the chromatogram obtained with reference solution (b) (1.0 per cent) and at most one such spot is
26-56
more intense than the spot in the chromatogram obtained with reference solution (c) (0.5 per
cent). The test is not valid unless the chromatogram obtained with reference solution (d) shows
two clearly separated spots.
at 100C to 105C for 2 h.
ASSAY
solution to 100.0 ml with alcohol R. Dilute 10.0 ml of this solution to 100.0 ml with alcohol R.
Measure the absorbance (2.2.25) at the maximum at 241 nm.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
26-57
Methylthioninium Chloride / Methylene Blue

For the purposes of product labelling in the United Kingdom, the pair of names given above shall be used
together when the substance is stated as an active ingredient in a formulated preparation (see Supplementary
Chapter II A, Changes in title).
N
Cl
Me2N
C16H18ClN3S,xH2O
NMe2
319.9
(anhydrous)
61-73-4
(anhydrous)
Methylthioninium Chloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Methylthioninium chloride (methylene blue) contains not less than 95.0 per cent and not more than
the equivalent of 101.0 per cent of 3,7-bis(dimethylamino)phenothiazin-5-ylium chloride, calculated
CHARACTERS
A dark blue, crystalline powder with a copper-coloured sheen, or green crystals with a bronzecoloured sheen, soluble in water, slightly soluble in alcohol.
IDENTIFICATION
A. Dissolve 10 mg in dilute hydrochloric acid R and dilute to 100 ml with the same acid. Dilute 5 ml of
the solution to 100 ml with dilute hydrochloric acid R. Examined between 240 nm and 800 nm
(2.2.25), the solution shows four absorption maxima, at 255 nm to 260 nm, 285 nm to 290 nm,
675 nm to 685 nm and 740 nm to 750 nm.
B. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel plate R.
the same solvent. Dilute 1 ml to 10 ml with methanol R.
Reference solution. Dissolve 10 mg of methylthioninium chloride CRS in methanol R and dilute to 10 ml
with the same solvent. Dilute 1 ml to 10 ml with methanol R.
of anhydrous formic acid R and 80 volumes of propanol R. Allow the plate to dry in air protected from
light. Examine in daylight. The principal spot in the chromatogram obtained with the test solution is
similar in position and size to the principal spot in the chromatogram obtained with the reference
solution. A secondary spot may appear above the principal spot in both chromatograms.
C. Dissolve about 1 mg in 10 ml of water R. Add 1 ml of glacial acetic acid R and 0.1 g of zinc
powder R. Heat to boiling. The solution becomes colourless. Filter and shake the filtrate. It becomes
blue in contact with air.
D. Ignite 50 mg with 0.5 g of anhydrous sodium carbonate R. Cool and dissolve the residue in 10 ml of
dilute nitric acid R. Filter. The filtrate, without further addition of dilute nitric acid R, gives reaction (a)
of chlorides (2.3.1).
TESTS
Methanol-insoluble substances To 1.0 g add 20 ml of methanol R and boil under a reflux
condenser for 5 min. Filter through a tared sintered-glass filter (40) and wash the filter with
methanol R until a colourless filtrate is obtained. Dry the filter at 100C and weigh. The residue
weighs not more than 10.0 mg (1.0 per cent).
Reference solution (a). Dissolve 15.0 mg of methylthioninium impurity A CRS in the mobile phase and
dilute to 100.0 ml with the mobile phase. To 1.0 ml of this solution, add 1.0 ml of the test solution
and dilute to 10.0 ml with the mobile phase.
26-58
volumes of a mixture of 3.4 ml of phosphoric acid R and 1000 ml of water R,
Inject 20 l of each solution. Adjust the sensitivity of the detector so that the height of the peak due
to the substance to be examined (retention time about 11 min) in the chromatogram obtained with
reference solution (a) is at least 80 per cent of the full scale of the recorder. The test is not valid
unless in the chromatogram obtained with reference solution (a), the resolution between the peaks
due to impurity A and methylthioninium is at least 1.5. If necessary, adjust the concentration of
acetonitrile in the mobile phase. Continue the chromatography of the test solution for twice the
retention time of the principal peak. In the chromatogram obtained with the test solution the area of
any peak corresponding to impurity A is not greater than five times the area of the principal peak in
the chromatogram obtained with reference solution (b) (5.0 per cent); the area of any peak, apart
from the principal peak and the peak due to impurity A, is not greater than half the area of the
principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent) and the sum
of the areas of any such peaks is not greater than the area of the principal peak in the chromatogram
obtained with reference solution (b) (1.0 per cent). Disregard any peak with an area less than 0.1
times that of the principal peak in the chromatogram obtained with reference solution (b) (0.1 per
cent).
Metals Examine by atomic emission spectrometry (2.2.22) in argon plasma, using as detector a
conventional optical system or a mass spectrometer; in the case of a mass spectrometer, use indium
as internal standard.
Test solution. In a 10 ml volumetric flask, dissolve with stirring 100 mg in 9 ml of water R, add
100.0 l of a 10 g/ml solution of indium prepared from indium elementary standard solution for atomic
spectrometry (1.000 g/l) R in nitric acid R which has been diluted fifty-fold with water R. Dilute to
Reference solutions. Into a 100 ml volumetric flask, introduce 10.0 ml of a standard solution containing
1.00 g/ml of each of the metals to be determined and prepared by dilution, with water R, of each
elementary standard solution for atomic spectrometry (1.000 g/l) R for the corresponding elements. Add
1.00 ml of a 10 g/ml solution of indium prepared from indium elementary standard solution for atomic
spectrometry (1.000 g/l) R in nitric acid R which has been diluted fifty-fold with water R. Dilute to
Blank solution. Dilute one hundred-fold with water R the 10 g/ml solution of indium used for the test
and reference solutions.
Element
Maximum content in ppm
Aluminium
Cadmium
Chromium
Copper
Tin
Iron
Manganese
Mercury
Molybdenum
Nickel
Lead
Zinc
100
1
10
100
10
100
10
1
10
10
10
100
26-59
Element
Optical detection
Aluminium
Cadmium
Chromium
Copper
Tin
Background 1
(nm)
Background 2
(nm)
Isotope
396.15
214.44
283.56
396.05
214.37
283.49
396.25
214.51
283.64
27
114
327.40
327.31
189.90
327.48
190.10
238.27
238.14
260.50
253.60
260.64
253.80
*1
55
200
202.02
231.54
216.90
202.04
231.66
217.10
95
60
208
213.80
213.91
66
115
190.00*2
238.20
Iron
Manganese
Mercury
260.57
253.70*3
202.03
231.60
Molybdenum
Nickel
Lead
217.00*2
213.86
Zinc
Indium
Mass detection
Signal
(nm)
*1
65
118
*1 Element difficult, if not impossible, to be determined with a mass spectrometer

as detector.
*2 Borderline sensitivity with conventional optical spectrometry.
*3 Mercury is often impossible to determine using conventional optical
spectrometry; it may be quantified using a device for the determination of
hydrides.
Loss on drying (2.2.32). 8.0 per cent to 22.0 per cent, determined on 1.000 g by drying in an
oven at 100C to 105C.
ASSAY
Dissolve 0.300 g in 30 ml of water R with heating. Cool, add 50.0 ml of potassium dichromate
solution R1 and dilute to 100.0 ml with water R. Allow to stand for 10 min. Filter and discard the first
20 ml of filtrate. Introduce 50.0 ml of the filtrate into a flask with a ground-glass neck, add 50 ml of
dilute sulphuric acid R and 8.0 ml of potassium iodide solution R. Allow to stand protected from light for
5 min, then add 80 ml of water R. Titrate with 0.1M sodium thiosulphate using 2 ml of starch solution R,
added towards the end of the titration, as indicator. Carry out a blank titration.
1 ml of 0.1M sodium thiosulphate is equivalent to 10.66 mg of C16H18ClN3S.
STORAGE
IMPURITIES
N
Cl
Me2N
NHMe
A. 3-(dimethylamino)-7-(methylamino)phenothiazin-5-ylium chloride.
__________________________________________________________________________________________________________ Ph Eur
26-60
Methysergide Maleate
O
CH2OH
H
N
H
NMe
CH3
H
COOH
COOH
MeN
,
and epimer at C*
C21H27N3O2,C4H4O4
469.5
129-49-7
Definition Methysergide Maleate is (1RS)-N-[1-(hydroxymethyl)propyl]-1-methyl-D-lysergamide

hydrogen maleate. It contains not less than 98.0% and not more than 101.0% of
C21H27N3O2,C4H4O4, calculated with reference to the dried substance.
Characteristics A white or almost white, crystalline powder which may have a yellow or pink tinge;
Slightly soluble in water and in methanol; practically insoluble in chloroform and in ether.
Identification
methysergide maleate (RS 227).
B. In the test for Related substances, the principal spot in the chromatogram obtained with solution
C. Dissolve 1 mg in 1 ml of ethanol (96%) and add 1 ml of dimethylaminobenzaldehyde solution R6. A
brownish red to violet colour is produced.
Acidity pH of a 0.2% w/v solution, 3.7 to 4.7, Appendix V L.
Specific optical rotation In a 0.25% w/v solution, +35.0 to +45.0, calculated with reference to
the dried substance, Appendix V F.
Related substances Carry out the method for thin-layer chromatography, Appendix III A, protected
from light using a suspension of silica gel G in 0.1M sodium hydroxide to prepare the plate and a
mixture of 90 volumes of chloroform and 10 volumes of methanol as the mobile phase. Apply
separately to the plate 5 l of each of the following solutions. Solution (1) contains 2.0% w/v of the
substance being examined in a mixture of 1 volume of 13.5M ammonia and 100 volumes of methanol.
Solutions (2) to (8) are solutions of methysergide maleate BPCRS in a mixture of 1 volume of 13.5M
ammonia and 100 volumes of methanol containing (2) 0.0050% w/v, (3) 0.010% w/v, (4) 0.015%
w/v, (5) 0.020% w/v, (6) 0.030% w/v, (7) 0.040% w/v and (8) 2.0% w/v respectively. After removal
of the plate, allow it to dry in air and examine under ultraviolet light (365 nm). Assess the intensities of
any secondary spots in the chromatogram obtained with solution (1) by reference to the spots in the
chromatograms obtained with solutions (2) to (7), making allowance for area in assessing the
intensities of spots of different Rf values and disregarding any spots less intense than the spot in the
chromatogram obtained with solution (2). The total of the intensities so assessed does not exceed
2%.
Assay Carry out Method I for non-aqueous titration, Appendix VIII A, using 0.4 g and 1-naphtholbenzein solution as indicator. Each ml of 0.1M perchloric acid VS is equivalent to 46.95 mg of
C21H27N3O2,C4H4O4.
Storage Methysergide Maleate should be kept in a well-closed container, protected from light and
stored at a temperature of 2 to 8.
Action and use Prophylaxis of migraine.
Preparation
Methysergide Tablets
26-61
Metipranolol
Me
OH
NHPr i
Me
AcO
Me
and enantiomer
C17H27NO4
309.4
22664-55-7
Definition Metipranolol is (RS)-4-(2-hydroxy-3-isopropylaminopropoxy)-2,3,6-trimethylphenyl

acetate. It contains not less than 99.0% and not more than 101.0% of C17H27NO4, calculated with
Characteristics A white, crystalline powder; melting point, about 108.
Practically insoluble in water; soluble in ethanol (96%), in acetone and in methanol. It dissolves in
Identification The infrared absorption spectrum, Appendix II A, is concordant with the reference
spectrum of metipranolol (RS 375).
Alkalinity Shake 0.5 g with 20 ml of water for 5 minutes and filter. The pH of the filtrate is 9.0 to
10.0, Appendix V L.
Colour of solution A 5.0% w/v solution in 1M hydrochloric acid is not more intensely coloured than
reference solution Y6, Appendix IV B.
Heavy metals 2 g complies with limit test C for heavy metals, Appendix VII. Use 2 ml of lead standard
solution (10 ppm Pb) to prepare the standard (10 ppm).
Iron Dissolve the residue obtained in the test for Sulphated ash in 1 ml of hydrochloric acid, evaporate
to dryness, dissolve the residue in a mixture of 0.5 ml of 6M acetic acid and 7 ml of water and add
sufficient water to produce 20 ml. 10 ml of this solution complies with the limit test for iron, Appendix
VII (10 ppm).
following solutions in methanol. Solution (1) contains 0.10% w/v of the substance being examined.
Solution (2) contains 0.0005% w/v of the substance being examined. Solution (3) contains 0.001%
w/v of desacetylmetipranolol BPCRS. Solution (4) contains 0.02% w/v of the substance being examined
and 0.02% w/v of desacetylmetipranolol BPCRS.
The chromatographic procedure may be carried out using (a) a stainless steel column (15
cm 4.6 mm) packed with stationary phase C (5 m) (Hypersil ODS is suitable), (b) as the mobile
phase with a flow rate of 2 ml per minute a mixture of 1 volume of perchloric acid, 45 volumes of
methanol and 54 volumes of water, the pH of the mixture being adjusted to 3.0 with 13.5M ammonia
and (c) a detection wavelength of 275 nm. For solution (1) allow the chromatography to proceed for
at least 3 times the retention time of the principal peak.
between the two principal peaks is at least 5.
In the chromatogram obtained with solution (1) the area of any peak corresponding to
desacetylmetipranolol is not greater than the area of the principal peak in the chromatogram obtained
with solution (3) (1%) and the sum of the areas of any secondary peaks is not greater than the area of
the principal peak in the chromatogram obtained with solution (2) (0.5%).
C17H27NO4.
Storage Metipranolol should be kept in a well-closed container and protected from light.
Action and use Beta-adrenoceptor antagonist.
Preparation
Metipranolol Eye Drops
26-62
IMPURITIES
Me
Me
OH
O
HO
Me
A. desacetylmetipranolol
NHPri
26-63
Metixene Hydrochloride
H
N
Me
,HCl
S
and enantiomer
C20H23NS,HCl,H2O
363.9
7081-40-5
Metixene Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Anticholinergic.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Metixene hydrochloride contains not less than 98.0 per cent and not more than the equivalent of
102.0 per cent of (RS)-1-methyl-3-[(9H-thioxanthen-9-yl)methyl]piperidine hydrochloride,
CHARACTERS
A white or almost white, crystalline or fine crystalline powder, soluble in water, soluble in alcohol and
in methylene chloride, practically insoluble in ether.
IDENTIFICATION
obtained with metixene hydrochloride CRS.
TESTS
Appearance of solution Dissolve 0.40 g in methanol R and dilute to 20.0 ml with the same solvent.
The solution is clear (2.2.1) and not more intensely coloured than reference solution Y6 (Method I,
2.2.2).
pH (2.2.3). Dissolve 0.18 g in carbon dioxide-free water R heating if necessary at about 50C, cool and
dilute to 10.0 ml with the same solvent. The pH of the solution, measured immediately, is 4.4 to 5.8.
Related substances Examine by thin layer chromatography (2.2.27), using a TLC silica gel plate R.
Carry out the test protected from light.
Test solution. Dissolve 50 mg of the substance to be examined in methylene chloride R and dilute to
Reference solution (a). Dissolve 5 mg of metixene hydrochloride CRS in methylene chloride R and dilute to
Reference solution (b). Dissolve 20 mg of thioxanthene CRS in 50 ml of methylene chloride R. Dilute
1.0 ml of this solution to 20.0 ml with methylene chloride R.
Reference solution (c). Dissolve 5 mg of thioxanthone CRS in 50 ml of methylene chloride R. Dilute
1.0 ml of this solution to 20.0 ml with methylene chloride R.
Reference solution (d). Dilute 4 ml of reference solution (a) to 10.0 ml with methylene chloride R.
Apply to the plate as narrow bands 5 l of each solution. Develop over a path of 10 cm using a
mixture of 10 volumes of glacial acetic acid R, 10 volumes of methanol R and 80 volumes of methylene
chloride R. Dry the plate in a stream of cold air. Spray with a mixture of 1 volume of sulphuric acid R
and 9 volumes of alcohol R and heat at 100C for 10 min. Allow the plate to cool and examine in
ultraviolet light at 365 nm. Any band corresponding to thioxanthene in the chromatogram obtained
with the test solution is not more intense than the band in the chromatogram obtained with reference
solution (b) (0.2 per cent); any band corresponding to thioxanthone in the chromatogram obtained
with the test solution is not more intense than the band in the chromatogram obtained with reference
solution (c) (0.05 per cent); any band apart from the principal band and the bands corresponding to
thioxanthene and thioxanthone is not more intense than the band in the chromatogram obtained with
reference solution (a) (0.5 per cent) and at most one such band is more intense than the band in the
26-64
chromatogram obtained with reference solution (d) (0.2 per cent). The test is not valid unless the
bands in the chromatogram obtained with reference solutions (b) and (c) are clearly visible and
separated.
Loss on drying (2.2.32). Not less then 4.0 per cent and not more than 6.0 per cent, determined on
0.500 g by drying in an oven at 138C to 142C.
ASSAY
Dissolve 0.250 g of the substance to be examined in a mixture of 5.0 ml of 0.01M hydrochloric acid
and 50 ml of alcohol R. Carry out a potentiometric titration (2.2.20), using 0.1M sodium
hydroxide. Read the volume added between the two points of inflexion.
1 ml of 0.1M sodium hydroxide is equivalent to 34.59 mg of C20H24ClNS.
STORAGE
IMPURITIES
A. 9H-thioxanthene,
O
B. 9H-thioxanthen-9-one (thioxanthone).
__________________________________________________________________________________________________________ Ph Eur
26-65
Metoclopramide
revised 1/01
O
Cl
H2N
C14H22ClN3O2
NEt2
N
H
OMe
299.8
364-62-5
Metoclopramide complies with the requirements of the 3rd edition of the European Pharmacopoeia [1348].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Metoclopramide contains not less than 99.0 per cent and not more than the equivalent of 101.0 per
cent of 4-amino-5-chloro-N-(2-diethylaminoethyl)-2-methoxybenzamide, calculated with reference
CHARACTERS
A white or almost white, fine powder, practically insoluble in water, sparingly soluble in methylene
chloride, sparingly soluble to slightly soluble in alcohol.
IDENTIFICATION
obtained with metoclopramide CRS. Examine the substances prepared as discs.
C. Examine the chromatograms obtained in test A for related substances (see Tests) in ultraviolet
light at 254 nm before spraying with dimethylaminobenzaldehyde solution R1. The principal spot in the
chromatogram obtained with test solution (a) is similar in position and size to the principal spot in
TESTS
Appearance of solution Dissolve 2.5 g in 25 ml of 1M hydrochloric acid. The freshly prepared
2.2.2).
Related substances
A. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel F254 plate R.
Test solution (b). Dissolve 0.160 g of the substance to be examined in methanol R and dilute to 10 ml
Reference solution (a). Dissolve 20 mg of metoclopramide CRS and 10 mg of sulpiride CRS in
methanol R and dilute to 5 ml with the same solvent.
Reference solution (b). Dissolve 20 mg of N,N-diethylethane-1,2-diamine R in methanol R and dilute to
50 ml with the same solvent. Dilute 2 ml of the solution to 25 ml with methanol R.
of concentrated ammonia R, 10 volumes of dioxan R, 14 volumes of methanol R and 90 volumes of
methylene chloride R. Allow the plate to dry in air. Examine in ultraviolet light at 254 nm
(identification C). Spray with dimethylaminobenzaldehyde solution R1. Allow the plate to dry in air.
Any spot corresponding to impurity E (not visualised in ultraviolet light at 254 nm) in the chromatogram obtained with test solution (b) is not more intense than the spot in the chromatogram obtained
with reference solution (b) (0.2 per cent). The test is not valid unless the chromatogram obtained
with reference solution (a) shows two clearly separated spots.
26-66
Reference solution (b). Dissolve 10.0 mg of metoclopramide impurity A CRS in the mobile phase and
dilute to 100.0 ml with the mobile phase. Mix 1.0 ml of this solution with 0.1 ml of the test solution
as mobile phase at a flow rate of 1.5 ml/min a mixture prepared as follows: dissolve 6.8 g of
potassium dihydrogen phosphate R in 700 ml of water R; add 0.2 ml of N,N-dimethyloctylamine R
and adjust to pH 4.0 with dilute phosphoric acid R; dilute to 1000 ml with water R, add 250 ml of
acetonitrile R and mix,
Inject 10 l of each solution. Adjust the sensitivity of the system so that the heights of the principal
peaks in the chromatogram obtained with reference solution (b) are at least 50 per cent of the full
scale of the recorder. The test is not valid unless, in the chromatogram obtained with reference
solution (b), the resolution between the two principal peaks is at least 2.0. Continue the chromatography of the test solution for eight times the retention time of metoclopramide. In the chromatogram obtained with the test solution: the area of any peak, apart from the principal peak, is not
greater than the area of the principal peak in the chromatogram obtained with reference solution (a)
(0.2 per cent) and the sum of the areas of any such peaks is not greater than three times the area of
the principal peak in the chromatogram obtained with reference solution (a) (0.6 per cent). Disregard
any peak with an area less than 0.1 times that of the principal peak in the chromatogram obtained
100C to 105C.
ASSAY
Dissolve 0.250 g in 50 ml of anhydrous acetic acid R and add 5 ml of acetic anhydride R. Titrate with
IMPURITIES
O
O
H3C
Cl
N
H
OMe
N
H
NEt 2
A. 4-(acetylamino)-5-chloro-N-(2-diethylaminoethyl)-2-methoxybenzamide,
O
H3C
Cl
COOMe
N
H
OMe
B. methyl 4-(acetylamino)-5-chloro-2-methoxybenzoate,
Cl
COOH
H2N
OMe
C. 4-amino-5-chloro-2-methoxybenzoic acid,
COOMe
O
H3C
N
H
OMe
D. methyl 4-(acetylamino)-2-methoxybenzoate,
26-67
NEt 2
H2N
E. N,N-diethylethane-1,2-diamine,
O
Cl
NEt 2
N
H
OH
H2N
F. 4-amino-5-chloro-N-(2-diethylaminoethyl)-2-hydroxybenzamide,
O
O
Cl
NEt 2
N
H
OMe
H2N
G. 4-amino-5-chloro-N-(2-diethylaminoethyl)-2-methoxybenzamide N-oxide,
COOH
O
H3C
N
H
OH
H. 4-(acetylamino)-2-hydroxybenzoic acid.
__________________________________________________________________________________________________________ Ph Eur
26-68
Metoclopramide Hydrochloride
O
Cl
H2N
N
H
NEt2
,HCl
OMe
C14H22ClN3O2,HCl,H2O 354.3
54143-57-6
Metoclopramide Hydrochloride complies with the requirements of the 3rd edition of the European
Preparations
Metoclopramide Injection
Metoclopramide Oral Solution
Metoclopramide Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Metoclopramide hydrochloride contains not less than 99.0 per cent and not more than the equivalent
of 101.0 per cent of 4-amino-5-chloro-N-(2-diethylaminoethyl)-2-methoxybenzamide hydrochloride,
calculated with reference to the anhydrous substance.
CHARACTERS
White or almost white, crystalline powder or crystals, very soluble in water, freely soluble in alcohol,
sparingly soluble in methylene chloride, practically insoluble in ether.
IDENTIFICATION
A. The pH (2.2.3) of solution S (see Tests) is 4.5 to 6.0.
obtained with metoclopramide hydrochloride CRS. Examine the substances as discs prepared using
potassium chloride R.
C. Examine the chromatograms obtained in the test for related substances in ultraviolet light before
spraying with dimethylaminobenzaldehyde solution R1. The principal spot in the chromatogram
obtained with test solution (b) is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a).
D. Dilute 1 ml of solution S to 2 ml with water R. The solution gives reaction (a) of chlorides (2.3.1).
E. Dissolve about 2 mg in 2 ml of water R. The solution gives the reaction of primary aromatic
amines (2.3.1).
TESTS
coating substance.
Reference solution (a). Dissolve 20 mg of metoclopramide hydrochloride CRS in methanol R and dilute to
Reference solution (b). Dilute 5 ml of test solution (a) to 100 ml with methanol R. Dilute 1 ml of this
solution to 10 ml with methanol R.
Reference solution (c). Dissolve 10 mg of N,N-diethylethylenediamine R in methanol R and dilute to
26-69
volumes of concentrated ammonia R, 10 volumes of dioxan R, 14 volumes of methanol R and 90
volumes of methylene chloride R. Allow the plate to dry in air. Examine in ultraviolet light at
254 nm. Any spot in the chromatogram obtained with test solution (a), apart from the principal spot,
is not more intense than the spot in the chromatogram obtained with reference solution (b) (0.5 per
cent). Spray with dimethylaminobenzaldehyde solution R1. Allow the plate to dry in air. Any spot in the
chromatogram obtained with test solution (a) that has not been visualised in ultraviolet light
at 254 nm is not more intense than the spot in the chromatogram obtained with reference solution
(c) (0.5 per cent).
ASSAY
Dissolve 0.2500 g in a mixture of 5.0 ml of 0.01M hydrochloric acid and 50 ml of alcohol R. Carry out
a potentiometric titration (2.2.20), using 0.1M sodium hydroxide. Read the volume of 0.1M sodium
hydroxide added between the two points of inflexion.
1 ml of 0.1M sodium hydroxide is equivalent to 33.63 mg of C14H23Cl2N3O2.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
27-1
Metoprolol Succinate
H
OH
NHPri
MeO
,HOOC
COOH
and enantiomer
C34H56N2O10
653
98418-47-4
Metoprolol Succinate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1448].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Metoprolol succinate contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of bis[(2RS)-1-[4-(2-methoxyethyl)phenoxy]-3-[(1-methylethyl)amino]propan-2-ol]
butanedioate, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder, freely soluble in water, soluble in methanol, sparingly soluble in alcohol,
very slightly soluble in ethyl acetate.
IDENTIFICATION
obtained with metoprolol succinate CRS. Examine the spectra as discs.
C. Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable
octadecylsilyl silica gel with a fluorescent indicator having an optimal intensity at 254 nm.
Test solution. Dissolve 15 mg of the substance to be examined in 2 ml of methanol R.
Reference solution (a). Dissolve 15 mg of metoprolol succinate CRS in 2 ml of methanol R.
Reference solution (b). Dissolve 15 mg of oxprenolol hydrochloride CRS and 15 mg of metoprolol succinate
CRS in 2 ml of methanol R.
Apply separately to the plate 10 l of each solution. Develop in an unsaturated tank over a path of
10 cm using a mixture of 0.5 volumes of perchloric acid R, 50 volumes of methanol R and 50 volumes
of water R. Dry the plate in a current of warm air and examine in ultraviolet light at 254 nm. The
the principal spot in the chromatogram obtained with reference solution (a). The test is not valid
TESTS
Solution S Dissolve 0.500 g in carbon dioxide-free water R and dilute to 25.0 ml with the same
solvent.
it is colourless (Method II, 2.2.2).
pH(2.2.3). The pH of solution S is 7.0 to 7.6.
Optical rotation (2.2.7). The angle of optical rotation, determined on solution S is 0.10 to
+0.10.
Related substances
A. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel plate R.
the same solvent.
27-2
Reference solution. Dilute 1 ml of the test solution to 50 ml with methanol R. Dilute 5 ml of this
Apply separately to the plate 10 l of each solution. Place two beakers each containing 30 volumes of
concentrated ammonia R at the bottom of a chromatographic tank containing a mixture of 20 volumes
of methanol R and 80 volumes of ethyl acetate R and allow to saturate for at least 1 h before use.
Develop over a path of 12 cm. Allow the plate to dry in air for at least 3 h. Expose the plate to iodine
vapour for at least 15 h. Any spot in the chromatogram obtained with the test solution, apart from
the principal spot, is not more intense than the spot in the chromatogram obtained with the reference
solution (0.2 per cent).
Reference solution (a). Dissolve 5.0 mg of metoprolol succinate CRS and 3.0 mg of metoprolol impurity D
Reference solution (c). If this solution is required (see below), it is to be prepared in a fume cupboard. This
solution is used only to determine the retention time of impurity C. Dissolve 10 mg of metoprolol succinate
CRS in 10 ml of 0.1M hydrochloric acid. Transfer this solution to an evaporating dish 10 cm in
diameter. Place the dish so that the surface of the solution is 5 cm from a lamp emitting ultraviolet
light (2.1.3) at 254 nm for 6 h. Dilute 0.5 ml of this solution to 25 ml with the mobile phase.
a stainless steel column 0.25m long and 4.0 mm in internal diameter packed with octadecylsilyl
as mobile phase at a flow rate of 1 ml/min a mixture prepared as follows: dissolve 3.9 g of
ammonium acetate R in 810 ml of water R, add 2.0 ml of triethylamine R, 10.0 ml of glacial acetic
acid R, 3.0 ml of phosphoric acid R and 190 ml of acetonitrile R and mix,
obtained with reference solution (b) is at least 50 per cent of the full scale of the recorder.
conditions, the retention times are: metoprolol about 9 min and impurity D about 12 min. The test is
not valid unless in the chromatogram obtained with reference solution (a), the resolution between the
peaks due to metoprolol and impurity D is at least 4.0. If necessary, adjust slightly the acetonitrile
composition of the mobile phase.
for three times the retention time of the principal peak. In the chromatogram obtained with the test
solution: the area of any peak, apart from the principal peak, is not greater than the area of the
principal peak in the chromatogram obtained with reference solution (b) (0.1 per cent); the sum of
the areas of all the peaks, apart from the principal peaks, is not greater than five times the area of the
principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent). If any of the
above limits are exceeded and if a peak occurs with a retention time of about 4.5 min (impurity C),
prepare reference solution (c). Inject 20 l of the test solution and 20 l of reference solution (c). In
the chromatogram obtained with the test solution: divide the area of the peak corresponding to the
principal peak in the chromatogram obtained with reference solution (c) (impurity C) by ten: this
calculated area is not greater than the area of the principal peak in the chromatogram obtained with
reference solution (b) (0.1 per cent); the sum of the areas of all the peaks, apart from the principal
peak, is not greater than five times the area of the principal peak in the chromatogram obtained
reference solution (b) (0.5 per cent). Disregard any peak with an area less than 0.3 times the area of
the principal peak in the chromatogram obtained with reference solution (b).
Heavy metals (2.4.8). Dissolve 2.0 g in 20 ml of water R. 12 ml of the solution complies with limit
test A for heavy metals (10 ppm). Prepare the standard using lead standard solution (1 ppm Pb) R.
100C to 105C.
ASSAY
STORAGE
27-3
IMPURITIES
by liquid chromatography:
H OH
O
NHEt
and enantiomer
eO
A. (2RS)-1-(ethylamino)-3-[4-(2-methoxyethyl)phenoxy]propan-2-ol,
OH
eO
B. 4-(2-methoxyethyl)phenol,
H OH
NHPr i
and enantiomer
OHC
C. 4-[(2RS)-2-hydroxy-3-[(1-methylethyl)amino]propoxy]benzaldehyde,
H OH
O
OH
and enantiomer
eO
D. (2RS)-3-[4-(2-methoxyethyl)phenoxy]propane-1,2-diol,
H OH
NHPr i
and enantiomer
E. (2RS)-1-[4-(2-hydroxyethyl)phenoxy]-3-[(1-methylethyl)amino]propan-2-ol.
by thin-layer chromatography:
OH
H
N
H
N
Me
Me
Me
F. 1,3-bis[(1-methylethyl)amino]propan-2-ol,
H OH
HO
H
N
Me
and enantiomer
Me
G. (2RS)-3-[(1-methylethyl)amino]propane-1,2-diol,
OH
O
OMe
Pr i
N
OH
*
MeO
H. 1,1-bis[(1-methylethyl)imino]bis[3-[4-(2-methoxyethyl)phenoxy]propan-2-ol].
__________________________________________________________________________________________________________ Ph Eur
27-4
Metoprolol Tartrate
H
OH
NHPri
MeO
OH
COOH
, HOOC
H
OH
and enantiomer
(C15H25NO3)2,C4H6O6
685
56392-17-7
Metoprolol Tartrate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1028].
Preparations
Metoprolol Injection
Metoprolol Tartrate Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Metoprolol tartrate contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of di[(RS)-3-[4-(2-methoxyethyl)phenoxy]-1-(isopropylamino)propan-2-ol] tartrate,
CHARACTERS
A white, crystalline powder or colourless crystals, very soluble in water, freely soluble in alcohol.
IDENTIFICATION
obtained with metoprolol tartrate CRS. If the spectra obtained in the solid state show differences,
record further spectra using discs prepared by placing 25 l of a 100 g/l solution in methylene
chloride R on a disc of potassium bromide R and evaporating the solvent. Examine immediately.
D. Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable
octadecylsilyl silica gel with a fluorescent indicator having an optimal intensity at 254 nm.
Reference solution (a). Dissolve 15 mg of metoprolol tartrate CRS in 2 ml of methanol R.
Reference solution (b). Dissolve 15 mg of oxprenolol hydrochloride CRS and 15 mg of metoprolol tartrate
CRS in 2 ml of methanol R.
10 cm using a mixture of 0.5 volumes of perchloric acid R, 50 volumes of methanol R and 50 volumes
of water R. Dry the plate in a current of warm air and examine in ultraviolet light at 254 nm. The
the principal spot in the chromatogram obtained with reference solution (a). The test is not valid
E. It gives reaction (b) of tartrates (2.3.1).
TESTS
solvent.
27-5
Specific optical rotation (2.2.7). +7.0 to +10.0, determined on solution S and calculated with
Related substances
A. Examine by thin-layer chromatography (2.2.27), using a suitable silica gel as the coating
substance.
the same solvent.
Reference solution (a). Dilute 1 ml of the test solution to 20 ml with methanol R. Dilute 5 ml of this
Reference solution (b). Dilute 4 ml of reference solution (a) to 10 ml with methanol R.
Apply separately to the plate 5 l of each solution. Place two beakers each containing 30 volumes of
concentrated ammonia R at the bottom of a chromatographic tank containing a mixture of 20 volumes
of methanol R and 80 volumes of ethyl acetate R and allow to saturate for at least 1 h before use.
Develop over a path of 12 cm. Allow the plate to dry in air for at least 3 h. Expose the plate to iodine
vapour for at least 15 h. Any spot in the chromatogram obtained with the test solution, apart from
the principal spot, is not more intense than the spot in the chromatogram obtained with reference
solution (a) (0.5 per cent) and at most one such spot is more intense than the spot in the chromatogram obtained with reference solution (b) (0.2 per cent). Disregard any spot that remains on the
starting-line.
Reference solution (a). Dissolve 5.0 mg of metoprolol tartrate CRS and 3.0 mg of metoprolol impurity D
CRS in the mobile phase and dilute to 100.0 ml with the mobile phase. Dilute 5.0 ml of the solution
to 10.0 ml with the mobile phase.
Reference solution (c). If this solution is required (see below), it is to be prepared in a fume cupboard. This
solution is used only to determine the retention time of metoprolol impurity C. Dissolve 10 mg of metoprolol
tartrate CRS in 10 ml of 0.1M hydrochloric acid. Transfer this solution to an evaporating dish 10 cm in
diameter. Place the dish so that the surface of the solution is 5 cm from a lamp emitting ultraviolet
light (2.1.3) at 254 nm for 6 h. Dilute 0.5 ml of this solution to 25 ml with the mobile phase.
a stainless steel column 0.3 m long and 3.9 mm in internal diameter packed with a suitable
octadecylsilyl silica gel for chromatography R (10 m),
as mobile phase at a flow rate of 1 ml per minute a mixture prepared as follows: dissolve 3.9 g of
ammonium acetate R in 810 ml of water R, add 2.0 ml of triethylamine R, 10.0 ml of glacial acetic
acid R, 3.0 ml of phosphoric acid R and 190 ml of acetonitrile R and mix,
maintaining the temperature of the column at 40C. Equilibrate the column with the mobile phase
at a flow rate of 1 ml per minute until a stable base-line is obtained (at least 20 min).
obtained with 10 l of reference solution (b) is not less than 50 per cent of the full scale of the
recorder.
conditions, the retention times are: metoprolol, about 10 min and metoprolol impurity D, about
13.5 min. The test is not valid unless in the chromatogram obtained with reference solution (a), the
resolution between the peaks due to metoprolol impurity D and metoprolol is at least 4.0. If
necessary, adjust slightly the acetonitrile composition of the mobile phase.
chromatography for three times the retention time of the principal peak. In the chromatogram
cent); the sum of the areas of all the peaks, apart from the principal peak, is not greater than 1.7
(0.5 per cent). If any of the above limits are exceeded and if a peak occurs with a retention time of
about 5 min (metoprolol impurity C), prepare reference solution (c). Inject separately 10 l of the
test solution and 10 l of reference solution (c). In the chromatogram obtained with the test solution:
divide the area of the peak corresponding to the principal peak in the chromatogram obtained with
27-6
reference solution (c) (metoprolol impurity C) by 10: this divided area is not greater than the area
of the principal peak in the chromatogram obtained with reference solution (b) (0.3 per cent); the
sum of the areas of all the peaks, apart from the principal peak, is not greater than 1.7 times the
Disregard any peak due to tartaric acid and any peak with an area less than 0.2 times the area of the
principal peak in the chromatogram obtained with reference solution (b).
Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying in vacuo
over anhydrous calcium chloride R for 4 h.
ASSAY
STORAGE
Store in a well-closed container protected from light.
IMPURITIES
by liquid chromatography:
OH
O
NHEt
MeO
A. 1-(ethylamino)-3-[4-(2-methoxyethyl)phenoxy]propan-2-ol,
OH
MeO
B. 4-(2-methoxyethyl)phenol,
OH
NHPri
O
OHC
C. 4-(2-hydroxy-3-isopropylaminopropoxy)benzaldehyde,
OH
O
OH
MeO
D. 3-[4-(2-methoxyethyl)phenoxy]propane-1,2-diol,
OH
NHPri
O
OMe
E. 1-isopropylamino-3-[2-(2-methoxyethyl)phenoxy]propan-2-ol,
OH
O
NHPri
F. 1-isopropylamino-3-phenoxypropan-2-ol,
OH
HO
G. 4-(2-hydroxyethyl)phenol,
27-7
OH
NHPri
O
HO
H. 1-isopropylamino-3-[4-(2-hydroxyethyl)phenoxy]propan-2-ol,
OH
NHPri
O
H2C
I. 1-isopropylamino-3-(4-vinylphenoxy)propan-2-ol,
OH
O
OH
MeO
i PrHN
J. 3-[2-hydroxy-3-[4-(2-methoxyethyl)phenoxy]propoxy]-1-isopropylaminopropan-2-ol,
OH
NHPri
MeO
H
H
and E-isomer
K. (E)- and (Z)-1-isopropylamino-3-[4-(2-methoxyvinyl)phenoxy]propan-2-ol,

by thin-layer chromatography:
OH
OH
iPrHN
NHPri
L. 1,1-oxybis[(3-isopropylamino)propan-2-ol],
OH
i PrHN
NHPri
M. 1,3-di-isopropylaminopropan-2-ol,
OH
HO
NHPri
N. 3-isopropylaminopropane-1,2-diol,
OH
O
MeO
NPri
O. isopropylamino-1,1-bis[3-[4-(2-methoxyethyl)phenoxy]propan-2-ol.
__________________________________________________________________________________________________________ Ph Eur
27-8
Metrifonate
O
P
Cl3C
H
OMe
OMe
OH
and enantiomer
C4H8Cl3O4P
257.4
52-68-6
Metrifonate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1133]. These
Action and use Insecticide; anthelmintic.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Metrifonate contains not less than 98.0 per cent and not more than the equivalent of 100.5 per cent
of dimethyl (RS)-(2,2,2-trichloro-1-hydroxyethyl)phosphonate, calculated with reference to the
CHARACTERS
A white, crystalline powder, freely soluble in water, very soluble in methylene chloride, freely soluble
in acetone and in alcohol.
It melts between 76C and 81C.
IDENTIFICATION
obtained with metrifonate CRS. Examine the substances prepared as discs.
the same solvent.
Reference solution. Dissolve 10 mg of metrifonate CRS in methanol R and dilute to 10 ml with the same
solvent.
Apply to the plate 10 l of each solution. Develop in an unsaturated tank over a path of 15 cm using
a mixture of 5 volumes of glacial acetic acid R, 25 volumes of dioxan R and 70 volumes of toluene R.
Allow the plate to dry in air. Spray with a 50 g/l solution of 4-(4-nitrobenzyl)pyridine R in acetone R
and heat at 120C for 15 min. Before the plate cools, spray with a 100 g/l solution of tetraethylene
pentamine R in acetone R. Examine immediately. The principal spot in the chromatogram obtained
with the test solution is similar in position, colour and size to the principal spot in the chromatogram
C. Dissolve about 20 mg in 1 ml of dilute sodium hydroxide solution R. Add 1 ml of pyridine R. Shake
and heat on a water-bath for 2 min. A red colour develops in the upper layer.
D. To 0.1 g add 0.5 ml of nitric acid R, 0.5 ml of a 500 g/l solution of ammonium nitrate R1 and
0.1 ml of concentrated hydrogen peroxide solution R. Heat on a water-bath for 10 min. Heat to boiling
and add 1 ml of ammonium molybdate solution R. A yellow colour is produced or a yellow precipitate is
formed.
TESTS
Appearance of solution Dissolve 5.0 g in 20 ml of methanol R. The solution is clear (2.2.1) and not
more intensely coloured than reference solution Y7 (Method II, 2.2.2).
Acidity Dissolve 2.5 g in carbon dioxide-free water R and dilute to 50 ml with the same solvent. Add
0.1 ml of methyl red solution R. Not more than 1.0 ml of 0.1M sodium hydroxide is required to change
the colour of the indicator to yellow.
Optical rotation (2.2.7). Dissolve 0.1 g in alcohol R and dilute to 10.0 ml with the same solvent.
Solvent mixture. Prepare a mixture of 10 volumes of mobile phase B and 90 volumes of mobile phase
A.
27-9
Test solution. Dissolve 0.20 g of the substance to be examined in the solvent mixture and dilute to
10.0 ml with the solvent mixture.
Reference solution (a). Use a freshly prepared solution. Dissolve 10.0 mg of desmethylmetrifonate CRS in
the solvent mixture and dilute to 20.0 ml with the solvent mixture. Dilute 1.0 ml of this solution to
Reference solution (b). Dissolve 0.10 g of dichlorvos R in the solvent mixture and dilute to 50.0 ml with
the solvent mixture. Dilute 1.0 ml of this solution to 50.0 ml with the solvent mixture.
Reference solution (c). Dilute 1.0 ml of the test solution to 10.0 ml with the solvent mixture. Dilute
5.0 ml of this solution to 100.0 ml with the solvent mixture.
Reference solution (d). Use a freshly prepared solution. Mix 1.0 ml of reference solution (a), 1.0 ml of
reference solution (b) and 0.025 ml of the test solution.
Reference solution (e). Dilute 4.0 ml of the test solution to 100.0 ml with the solvent mixture. Dilute
1.0 ml of this solution to 10.0 ml with the solvent mixture.
octadecylsilyl silica gel for chromatography (10 m),
as mobile phase at a flow rate of 1 ml/min:
Mobile phase A. A 1.36 g/l solution of potassium dihydrogen phosphate R, previously adjusted to
pH 2.9 with phosphoric acid R,
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
05
525
25end
90
9085
8545
10
1015
1555

equilibrating the column for 5 min with the same mixture of mobile phases used for the first 5 min
and maintaining the temperature of the column at 40C.
When the chromatograms are recorded in the prescribed conditions the peaks elute in the following
order: desmethylmetrifonate, metrifonate and dichlorvos. Inject 10 l of reference solution (e).
obtained is 50 per cent to 70 per cent of the full scale of the recorder. Inject 50 l of reference
solution (d). The test is not valid unless the resolution between the peaks corresponding to
desmethylmetrifonate and metrifonate is at least 3.0 and the resolution between the peaks corresponding to metrifonate and dichlorvos is at least 4.5.
Inject 50 l of the test solution and 50 l each of reference solutions (a), (b) and (c). Continue the
chromatography of the test solution for three times the retention time of metrifonate. In the chromatogram obtained with the test solution: the area of any peak corresponding to desmethylmetrifonate is
not greater than the area of the principal peak in the chromatogram obtained with reference solution
(a) (0.5 per cent); the area of any peak corresponding to dichlorvos is not greater than the area of the
principal peak in the chromatogram obtained with reference solution (b) (0.2 per cent); the area of
any other peak, apart from the principal peak and the peaks corresponding to desmethylmetrifonate
and dichlorvos respectively, is not greater than the area of the principal peak in the chromatogram
obtained with reference solution (c) (0.5 per cent) and the sum of the areas of all these peaks is not
greater than twice the area of the principal peak in the chromatogram obtained with reference
solution (c) (1 per cent). Disregard any peak with an area less than 0.1 times the area of the principal
peak in the chromatogram obtained with reference solution (e).
Chlorides Not more than 500 ppm. Dissolve 5.00 g in 30 ml of alcohol R and add a mixture of
15 ml of nitric acid R and 100 ml of water R. Using a silver electrode, titrate with 0.01M silver nitrate,
1 ml of 0.01M silver nitrate is equivalent to 0.3546 mg of Cl.
ASSAY
Dissolve 0.300 g in 30 ml of alcohol R. Add 10 ml of ethanolamine R and allow to stand for 1 h at
20C to 22C. Add a chilled mixture of 15 ml of nitric acid R and 100 ml of water R maintaining the
temperature of the mixture at 20C to 22C. Maintain at that temperature and titrate with 0.1M silver
nitrate, using a silver electrode and determining the end-point potentiometrically (2.2.20).
Calculate the percentage content of C4H8Cl3O4P, taking into account the content of chloride and
using the following expression:
27-10
Vp VCl 0.1
25.74 0.1
M Cl
M p
VP = volume of silver nitrate used in the assay in millilitres,
MP = mass of substance used in the assay in grams,
VCl = volume of silver nitrate used in the test for chlorides in millilitres,
MCl = mass of substance used in the test for chlorides in grams.
STORAGE
IMPURITIES
O
Cl3C
OH
OMe
and enantiomer
H OH
A. methyl (RS)-(2,2,2-trichloro-1-hydroxyethyl)phosphonate acid (desmethylmetrifonate),

O
Cl2C
OMe
OMe
B. 2,2-dichloroethenyl dimethyl phosphate (dichlorvos).

__________________________________________________________________________________________________________ Ph Eur
27-11
Metronidazole
CH2CH2OH
O2N
Me
N
C6H9N3O3
171.2
443-48-1
Metronidazole complies with the requirements of the 3rd edition of the European Pharmacopoeia [0675]. These
Preparations
Metronidazole Gel
Metronidazole Intravenous Infusion
Metronidazole Suppositories
Metronidazole Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Metronidazole contains not less than 99.0 per cent and not more than the equivalent of 101.0 per
cent of 2-(5-nitro-2-methylimidazol-1-yl)ethanol, calculated with reference to the dried substance.
CHARACTERS
A white or yellowish, crystalline powder, slightly soluble in water, in acetone, in alcohol and in
methylene chloride, very slightly soluble in ether.
IDENTIFICATION
B. Dissolve 40.0 mg in 0.1M hydrochloric acid and dilute to 100.0 ml with the same acid. Dilute
5.0 ml of the solution to 100.0 ml with 0.1M hydrochloric acid. Examined between 230 nm and
350 nm (2.2.25), the solution shows an absorption maximum at 277 nm and a minimum at 240 nm.
The specific absorbance at the maximum is 365 to 395.
obtained with metronidazole CRS. Examine the substances prepared as discs.
D. To about 10 mg add about 10 mg of zinc powder R, 1 ml of water R and 0.25 ml of dilute hydrochloric acid R. Place in a water-bath for 5 min. Cool. The solution gives the reaction of primary
aromatic amines (2.3.1).
TESTS
acid. The solution is not more opalescent than reference suspension II (2.2.1) and not more intensely
coloured than reference solution GY6 (Method II, 2.2.2).
coating substance.
Test solution. Dissolve 0.10 g in acetone R and dilute to 10 ml with the same solvent.
1 volume of water R, 10 volumes of alcohol R, 10 volumes of diethylamine R and 80 volumes of chloroform R. Allow the plate to dry in air. Examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with the test solution, apart from the principal spot, is not more intense than the
100C to 105C for 3 h.
27-12
ASSAY
Dissolve 0.1500 g in 50 ml of anhydrous acetic acid R. Titrate with 0.1M perchloric acid, determining
the end-point potentiometrically (2.2.20).
STORAGE
__________________________________________________________________________________________________________ Ph Eur
27-13
Metronidazole Benzoate
O
O
O2N
Me
N
C13H13N3O4
275.3
13182-89-3
Metronidazole Benzoate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Antiprotozoal; antibacterial.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Metronidazole benzoate contains not less than 98.5 per cent and not more than the equivalent of
101.0 per cent of 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl benzoate, calculated with reference to
the dried substance.
CHARACTERS
White or slightly yellowish, crystalline powder or flakes, practically insoluble in water, freely soluble
in methylene chloride, soluble in acetone, slightly soluble in alcohol, very slightly soluble in ether.
IDENTIFICATION
B. Dissolve 0.100 g in 1M hydrochloric acid and dilute to 100.0 ml with the same acid. Dilute 1.0 ml
of the solution to 100.0 ml with 1M hydrochloric acid. Examined between 220 nm and 350 nm
(2.2.25), the solution shows two absorption maxima, at 232 nm and 275 nm. The specific
absorbance at the maximum at 232 nm is 525 to 575.
obtained with metronidazole benzoate CRS.
D. Examine the chromatograms obtained in the test for related substances in ultraviolet light at
254 nm. The principal spot in the chromatogram obtained with test solution (b) is similar in position
and size to the principal spot in the chromatogram obtained with reference solution (a).
E. To about 10 mg add about 10 mg of zinc powder R, 1 ml of water R and 0.3 ml of hydrochloric
acid R. Heat on a water-bath for 5 min and cool. The solution gives the reaction of primary aromatic
amines (2.3.1).
TESTS
Appearance of solution Dissolve 1.0 g in dimethylformamide R and dilute to 10 ml with the same
solvent. The solution is not more opalescent than reference suspension II (2.2.1) and not more
intensely coloured than reference solution GY3 (Method II, 2.2.2).
Acidity Dissolve 2.0 g in a mixture of 20 ml of dimethylformamide R and 20 ml of water R, previously
neutralised with 0.02M hydrochloric acid or 0.02M sodium hydroxide using 0.2 ml of methyl red
solution R. Not more than 0.25 ml of 0.02M sodium hydroxide is required to change the colour of the
indicator.
coating substance. Heat the plate at 110C for 1 h and allow to cool before use.
Test solution (b). Dilute 1.0 ml of test solution (a) to 10 ml with acetone R.
Reference solution (a). Dissolve 20 mg of metronidazole benzoate CRS in acetone R and dilute to 10 ml
Reference solution (c). Dilute 2 ml of test solution (b) to 100 ml with acetone R.
27-14
Reference solution (d). Dissolve 10 mg of metronidazole CRS in acetone R and dilute to 100 ml with the
same solvent.
Reference solution (e). Dissolve 10 mg of 2-methyl-5-nitroimidazole R in acetone R and dilute to 100 ml
Reference solution (f). Dissolve 10 mg of metronidazole CRS and 10 mg of 2-methyl-5-nitroimidazole R
in acetone R and dilute to 50 ml with the same solvent.
Apply separately to the plate 10 l of each solution. Develop over a path of 15 cm using ethyl
acetate R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. In the chromatogram obtained with test solution (a): any spot corresponding to metronidazole or 2-methyl-5nitroimidazole is not more intense than the corresponding spot in the chromatograms obtained with
reference solutions (d) and (e) (0.5 per cent); any spot, apart from the principal spot and any spots
corresponding to metronidazole and 2-methyl-5-nitroimidazole, is not more intense than the spot in
the chromatogram obtained with reference solution (b) (0.5 per cent) and at most one such spot is
more intense than the spot in the chromatogram obtained with reference solution (c) (0.2 per cent).
The test is not valid unless the chromatogram obtained with reference solution (f) shows two clearly
separated principal spots.
80C for 3 h.
ASSAY
STORAGE
IMPURITIES
A. metronidazole,
O2N
H
N
Me
N
B. 2-methyl-5-nitroimidazole.
_______________________________________________________________________________________________________________________ Ph Eur
27-15
Metyrapone
N
O
Me
Me
N
C14H14N2O
226.3
54-36-4
Definition Metyrapone is 2-methyl-1,2-di(3-pyridyl)propan-1-one. It contains not less than

97.0% and not more than 103.0% of C14H14N2O, calculated with reference to the dried substance.
Characteristics A white to light amber, crystalline powder; odour, characteristic.
Sparingly soluble in water; freely soluble in chloroform and in ethanol (96%). It dissolves in dilute
mineral acids.
Identification
A. The infrared absorption spectrum, Appendix II A, is concordant with reference spectrum 1 of
metyrapone (RS 230).
hydrochloric acid exhibits a maximum only at 260 nm. The absorbance at 260 nm is about 1.0.
C. To 5 ml of a 1% w/v solution in 1M sulphuric acid add 0.2 ml of potassium tetraiodomercurate
solution. A cream precipitate is produced.
III A, using silica gel GF254 as the coating substance and a mixture of 90 volumes of propan-2-ol, 5
volumes of 13.5M ammonia and 5 volumes of water as the mobile phase. Apply separately to the plate
10 l of each of two solutions of the substance being examined in chloroform containing (1) 5.0% w/v
and (2) 0.010% w/v. After removal of the plate, allow it to dry in air and examine under ultraviolet
light (254 nm). Any secondary spot in the chromatogram obtained with solution (1) is not more intense
than the spot in the chromatogram obtained with solution (2).
Loss on drying When dried over phosphorus pentoxide at a pressure not exceeding 0.7 kPa for 6
hours, loses not more than 0.5% of its weight. Use 1 g.
Assay Carry out the following procedure protected from light. Dissolve 0.1 g in sufficient 0.1M
hydrochloric acid to produce 100 ml. Dilute 5 ml to 50 ml with 0.1M hydrochloric acid and dilute 5 ml
of this solution to 50 ml with 0.1M hydrochloric acid. Measure the absorbance of the resulting solution
at the maximum at 260 nm, Appendix II B. Calculate the content of C14H14N2O taking 500 as the
value of A(1%, 1 cm) at the maximum at 260 nm.
Storage Metyrapone should be protected from light.
Action and use Diagnostic agent for pituitary function.
Preparation
Metyrapone Capsules
27-16
Mexenone
O
OH
OMe
Me
C15H14O3
242.3
1641-17-4
Definition Mexenone is 2-hydroxy-4-methoxy-4-methylbenzophenone. It contains not less than

97.0% and not more than 103.0% of C15H14O3, calculated with reference to the dried substance.
Characteristics A pale yellow, crystalline powder; odourless or almost odourless.
Practically insoluble in water; freely soluble in acetone; sparingly soluble in ethanol (96%).
Identification
mexenone (RS 232).
methanol exhibits three maxima, at 243, 287 and 325 nm. The absorbances at the maxima are about
0.53, about 0.90 and about 0.66, respectively.
C. In the test for Related substances, the principal spot in the chromatogram obtained with solution
(2) corresponds to the spot in the chromatogram obtained with solution (4).
Iron Ignite 1.0 g with 1 g of anhydrous sodium carbonate, cool, dissolve the residue in 5 ml of hydrochloric acid and dilute to 40 ml with water. 10 ml of the solution complies with the limit test for iron,
gel GF254 as the coating substance and a mixture of 100 volumes of toluene and 10 volumes of butan2-one as the mobile phase. Apply separately to the plate 5 l of each of four solutions in butan-2-one
containing (1) 10% w/v of the substance being examined, (2) 0.20% w/v of the substance being
examined, (3) 0.10% w/v of the substance being examined and (4) 0.20% w/v of mexenone BPCRS.
After removal of the plate, allow it to dry in air and examine under ultraviolet light (254 nm). Any
secondary spot in the chromatogram obtained with solution (1) is not more intense than the spot in the
Loss on drying When dried to constant weight at 60 at a pressure not exceeding 3.5 kPa, loses not
Assay Dissolve 80 mg in sufficient methanol to produce 100 ml. Dilute 10 ml of the solution to
100 ml with methanol and further dilute 10 ml of this solution to 100 ml with methanol. Measure the
absorbance of the resulting solution at the maximum at 287 nm, Appendix II B, and calculate the
content of C15H14O3 taking 640 as the value of A(1%, 1 cm) at the maximum at 287 nm.
Action and use Topical sun-screening substance.
Preparation
Mexenone Cream
27-17
Mexiletine Hydrochloride
Me
NH2
O
CH3
,HCl
Me
and enantiomer
C11H17NO,HCl
215.7
5370-01-4
Mexiletine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Anti-arrhythmic.
Preparations
Mexiletine Capsules
Mexiletine Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Mexiletine hydrochloride contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of (RS)-[2-(2,6-dimethylphenoxy)-1-methylethyl]amine hydrochloride, calculated
with reference to the anhydrous substance.
CHARACTERS
A white or almost white, crystalline powder, freely soluble in water and in methanol, sparingly soluble
in methylene chloride, practically insoluble in ether.
IDENTIFICATION
A. The angle of optical rotation (2.2.7) is 0.1 to +0.1, determined on solution S (see Tests).
B. Dissolve 40.0 mg in a 0.5 per cent V/V solution of dilute hydrochloric acid R and dilute to 50.0 ml
with the same acid solution. Examined between 230 nm and 350 nm (2.2.25), the solution shows an
absorption maximum at 260 nm. The specific absorbance at the maximum is 10.9 to 12.1.
obtained with mexiletine hydrochloride CRS. If the spectra obtained in the solid state show differences,
dissolve the substance to be examined and the reference substance separately in methanol R, evaporate to dryness and record new spectra using the residues.
D. Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating substance.
the same solvent. Dilute 2 ml of this solution to 50 ml with methanol R.
Reference solution (a). Dissolve 20 mg of mexiletine hydrochloride CRS in methanol R and dilute to
Reference solution (b). Dissolve 10 mg of 2,6-dimethylphenol R in reference solution (a) and dilute to
5 ml with the same solution.
1 volume of concentrated ammonia R1, 14 volumes of methanol R and 85 volumes of methylene
chloride R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Spray with
ninhydrin solution R and heat at 110C for 15 min. For both methods of visualisation, the principal
principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless,
in ultraviolet light at 254 nm, the chromatogram obtained with reference solution (b) shows two
clearly separated spots.
E. Dilute 1.5 ml of solution S to 15 ml with water R. The solution gives reaction (a) of chlorides
(2.3.1).
TESTS
27-18
Appearance of solution Dilute 5 ml of solution S to 10 ml with water R. The solution is clear
(2.2.1) and colourless (Method II, 2.2.2).
Reference solution (a). Dissolve 20.0 mg of 2,6-dimethylphenol R in the mobile phase and dilute to
50.0 ml with the mobile phase. Dilute 1.0 ml of the solution to 100.0 ml with the mobile phase.
Reference solution (c). Dissolve 10.0 mg of 2,6-dimethylphenoxyacetone CRS in the mobile phase and
dilute to 10.0 ml with the mobile phase. To 1.0 ml of the solution add 2.5 ml of the test solution and
as mobile phase at a flow rate of 1 ml per minute a mixture of 40 volumes of water R and 60
volumes of acetonitrile R, containing 6 g/l of potassium dihydrogen phosphate R and 2.9 g/l of
sodium lauryl sulphate R,
Inject 20 l of reference solution (c). Adjust the sensitivity of the system so that the heights of the
two principal peaks are not less than 50 per cent of the full scale of the recorder. The test is not valid
unless the resolution between the first peak (2,6-dimethylphenoxyacetone) and the second peak
(mexiletine) is at least 2.0. Inject 20 l of the test solution, 20 l of reference solution (a) and 20 l of
reference solution (b). Continue the chromatography for twice the retention time of mexiletine
(about 8 min). In the chromatogram obtained with the test solution: the area of any peak corresponding to 2,6-dimethylphenol is not greater than the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.2 per cent); the area of any other peak, apart from the principal peak, is not greater than the area of the principal peak in the chromatogram obtained with
reference solution (b) (0.5 per cent). Disregard any peak with an area less than 0.1 times that of the
of water.
ASSAY
Dissolve 0.150 g in 50 ml of a mixture of equal volumes of acetic acid R and acetic anhydride R.
Titrate with 0.1M perchloric acid, determining the end-point potentiometrically (2.2.20).
1 ml of 0.1M perchloric acid is equivalent to 21.57 mg of C11H18ClNO.
STORAGE
IMPURITIES
Me
OH
Me
A. 2,6-dimethylphenol,
O
Me
O
CH3
Me
B. (2,6-dimethylphenoxy)acetone.
_______________________________________________________________________________________________________________________ Ph Eur
27-19
Mianserin Hydrochloride
Me
N
N
C18H20N2,HCl
,HCl
300.8
21535-47-7
Mianserin Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Mianserin Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Mianserin hydrochloride contains not less than 98.5 per cent and not more than the equivalent of
101.0 per cent of (RS)-2-methyl-1,2,3,4,10,14b-hexahydrodibenzo[c,f]pyrazino[1,2-a]azepine hydrochloride, calculated with reference to the dried substance.
CHARACTERS
White or almost white, crystalline powder or crystals, sparingly soluble in water, soluble in methylene
chloride, slightly soluble in alcohol.
IDENTIFICATION
A. Dissolve 50.0 mg in water R and dilute to 50.0 ml with the same solvent. Dilute 5.0 ml of the
solution to 50.0 ml with water R. Examined between 230 nm and 350 nm (2.2.25), the solution
shows an absorption maximum at 279 nm. The specific absorption at the maximum is 64 to 72.
obtained with mianserin hydrochloride CRS. Examine the substances as discs prepared using potassium
chloride R. If the spectra obtained show differences, dissolve the substance to be examined and the
reference substance separately in methanol R, evaporate to dryness and record new spectra using the
residues.
Reference solution (a). Dissolve 10 mg of mianserin hydrochloride CRS in methylene chloride R and dilute
Reference solution (b). Dissolve 10 mg of mianserin hydrochloride CRS and 10 mg of cyproheptadine
hydrochloride CRS in methylene chloride R and dilute to 5 ml with the same solvent.
volumes of diethylamine R, 20 volumes of ether R and 75 volumes of cyclohexane R. Examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is
similar in position and size to the principal spot in the chromatogram obtained with reference
TESTS
pH (2.2.3). Dissolve 0.10 g in carbon dioxide-free water R and dilute to 10 ml with the same solvent.
The pH of the solution is 4.0 to 5.5.
coating substance.
27-20
Test solution. Dissolve 0.20 g of the substance to be examined in a mixture of 1 volume of ammonia R
and 4 volumes of methanol R and dilute to 10 ml with the same mixture of solvents.
ammonia R and 4 volumes of methanol R.
10 volumes of methanol R and 90 volumes of methylene chloride R. Dry the plate in a current of cold
air. Expose the plate to iodine vapour for 20 min. Any spot in the chromatogram obtained with the
test solution, apart from the principal spot, is not more intense than the spot in the chromatogram
obtained with reference solution (a) (0.5 per cent) and at most one such spot is more intense than the
spot in the chromatogram obtained with reference solution (b) (0.1 per cent).
diphosphorus pentoxide R at 65C at a pressure not exceeding 700 Pa for 3 h.
ASSAY
1 ml of 0.1M sodium hydroxide is equivalent to 30.08 mg of C18H21ClN2.
STORAGE
IMPURITIES
Me
N
N
OH
A. 1-[(2-hydroxymethyl)phenyl]-4-methyl-2-phenylpiperazine.
_______________________________________________________________________________________________________________________ Ph Eur
27-21
Miconazole
N
N
H
O
Cl
Cl
Cl
Cl
and enantiomer
C18H14Cl4N2O
416.1
22916-47-8
Miconazole complies with the requirements of the 3rd edition of the European Pharmacopoeia [0935]. These
Action and use Antifungal.
Preparation
Miconazole Oromucosal Gel
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Miconazole contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of (RS)-1-[2-(2,4-dichlorobenzyloxy)-2-(2,4-dichlorophenyl)-ethyl]-1H-imidazole, calculated with
CHARACTERS
A white or almost white powder, very slightly soluble in water, freely soluble in methanol, soluble in
alcohol.
IDENTIFICATION
obtained with miconazole CRS. Examine the substances as discs prepared using potassium bromide R.
C. Examine by thin-layer chromatography (2.2.27), using a suitable octadecylsilyl silica gel as the
coating substance.
Reference solution (a). Dissolve 30 mg of miconazole CRS in the mobile phase and dilute to 5 ml with
the mobile phase.
Reference solution (b). Dissolve 30 mg of miconazole CRS and 30 mg of econazole nitrate CRS in the
mobile phase and dilute to 5 ml with the mobile phase.
Dry the plate in a current of warm air for 15 min and expose it to iodine vapour until the spots
appear. Examine in daylight. The principal spot in the chromatogram obtained with the test solution
is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b)
D. To 30 mg in a porcelain crucible add 0.3 g of anhydrous sodium carbonate R. Heat over an open
flame for 10 min. Allow to cool. Take up the residue with 5 ml of dilute nitric acid R and filter. To
1 ml of the filtrate add 1 ml of water R. The solution gives reaction (a) of chlorides (2.3.1).
TESTS
27-22
Appearance of solution Solution S is clear (2.2.1) and is not more intensely coloured than reference solution Y6 (Method II, 2.2.2).
Optical rotation (2.2.7). The angle of optical rotation of solution S is 0.10 to +0.10.
Reference solution (a). Dissolve 2.5 mg of miconazole CRS and 2.5 mg of econazole nitrate CRS in the
mobile phase and dilute to 100.0 ml with the mobile phase.
5.0 ml of this solution to 20.0 ml with the mobile phase.
as mobile phase at a flow rate of 2 ml per minute a solution of 6.0 g of ammonium acetate R in a
mixture of 300 ml of acetonitrile R, 320 ml of methanol R and 380 ml of water R,
recorder.
conditions, the retention times are: econazole nitrate, about 10 min; miconazole, about 20 min. The
test is not valid unless the resolution between the peaks corresponding to econazole nitrate and
miconazole is not less than ten; if necessary, adjust the composition of the mobile phase.
with the test solution: the area of any peak, apart from the principal peak, is not greater than the area
sum of the areas of all the peaks, apart from the principal peak, is not greater than twice that of the
principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent). Disregard
any peak due to the solvent and any peak with an area less than 0.2 times the area of the principal
peak in the chromatogram obtained with reference solution (b).
60C for 4 h.
ASSAY
Dissolve 0.300 g in 50 ml of a mixture of 1 volume of glacial acetic acid R and 7 volumes of methyl
ethyl ketone R. Using 0.2 ml of naphtholbenzein solution R as indicator, titrate with 0.1M perchloric acid
until the colour changes from orange-yellow to green.
1 ml of 0.1M perchloric acid is equivalent to 41.61 mg of C18H14Cl4N2O.
STORAGE
IMPURITIES
A. miconazole nitrate,
H
HO
Cl
Cl
and enantiomer
B. (RS)-1-(2,4-dichlorophenyl)-2-imidazol-1-ylethanol,
H
O
Cl
Cl
Cl
and enantiomer
C. (RS)-1-[2-(4-chlorobenzyloxy)-2-(2,4-dichlorophenyl)ethyl]imidazole,
27-23
NH2
H
O
Cl
Cl
Cl
Cl
and enantiomer
D. (RS)-2-(2,4-dichlorobenzyloxy)-2-(2,4-dichlorophenyl)ethylamine,
Cl
H
O
Cl
Cl
Cl
and enantiomer
E. (RS)-1-[2-(2,6-dichlorobenzyloxy)-2-(2,4-dichlorophenyl)ethyl]imidazole,
Me Ne
_
COO
H
O
Cl
Cl
Cl
Cl
and enantiomer
F. (RS)-1-(1-carboxylato-1-methylethyl)-3-[2-(2,4-dichlorobenzyloxy)-2-(2,4-dichlorophenyl)ethyl]imidazolium,
H
O
Cl
Cl
Cl
Cl
and enantiomer
G. (RS)-1-[2-(3,4-dichlorobenzyloxy)-2-(2,4-dichlorophenyl)ethyl]imidazole,
Cl
O
Cl
Cl
Cl
and enantiomer
H. (RS)-1-[2-(2,5-dichlorobenzyloxy)-2-(2,4-dichlorophenyl)ethyl]imidazole.
_______________________________________________________________________________________________________________________ Ph Eur
27-24
Miconazole Nitrate
N
N
H
,HNO3
O
Cl
Cl
Cl
Cl
and enantiomer
C18H14Cl4N2O,HNO3
479.1
22832-87-7
Miconazole Nitrate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0513].
Preparation
Miconazole Cream
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Miconazole nitrate contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of (RS)-1-[2-(2,4-dichlorobenzyloxy)-2-(2,4-dichlorophenyl)ethyl]-1H-imidazole
nitrate, calculated with reference to the dried substance.
CHARACTERS
A white or almost white powder, very slightly soluble in water, sparingly soluble in methanol, slightly
IDENTIFICATION
obtained with miconazole nitrate CRS. Examine the substances prepared as discs using potassium
bromide R.
C. Examine by thin-layer chromatography (2.2.27), using a suitable octadecylsilyl silica gel as the
coating substance.
Reference solution (a). Dissolve 30 mg of miconazole nitrate CRS in the mobile phase and dilute to 5 ml
Reference solution (b). Dissolve 30 mg of miconazole nitrate CRS and 30 mg of econazole nitrate CRS in
the mobile phase and dilute to 5 ml with the mobile phase.
Dry the plate in a current of warm air for 15 min and expose it to iodine vapour until the spots
appear. Examine in daylight. The principal spot in the chromatogram obtained with the test solution
is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b)
D. It gives the reaction of nitrates (2.3.1).
TESTS
Appearance of solution Solution S is clear (2.2.1) and is not more intensely coloured than reference solution Y7 (Method II, 2.2.2).
27-25
Optical rotation (2.2.7). The angle of optical rotation of solution S is 0.10 to +0.10.
Reference solution (a). Dissolve 2.5 mg of miconazole nitrate CRS and 2.5 mg of econazole nitrate CRS
as mobile phase at a flow rate of 2 ml per minute a solution of 6.0 g of ammonium acetate R in a
mixture of 300 ml of acetonitrile R, 320 ml of methanol R and 380 ml of water R,
recorder.
Inject 10 l of reference solution (a). When the chromatogram is recorded in the prescribed
conditions, the retention times are: econazole nitrate, about 10 min; miconazole nitrate, about
20 min. The test is not valid unless the resolution between the peaks corresponding to econazole
nitrate and miconazole nitrate is at least ten; if necessary, adjust the composition of the mobile phase.
with the test solution: the area of any peak apart from the principal peak is not greater than the area
sum of the areas of the peaks apart from the principal peak is not greater than twice the area of the
any peak due to the nitrate ion and any peak with an area less than 0.2 times the area of the principal
peak in the chromatogram obtained with reference solution (b).
100C to 105C for 2 h.
ASSAY
Dissolve 0.350 g in 75 ml of anhydrous acetic acid R, with slight heating if necessary. Titrate with
0.1M perchloric acid determining the end-point potentiometrically (2.2.20). Carry out a blank titration.
1 ml of 0.1M perchloric acid is equivalent to 47.91 mg of C18H15Cl4N3O4.
STORAGE
IMPURITIES
H
HO
Cl
Cl
and enantiomer
A. (RS)-1-(2,4-dichlorophenyl)-2-imidazol-1-ylethanol,
H
O
Cl
Cl
Cl
and enantiomer
B. (RS)-1-[2-(4-chlorobenzyloxy)-2-(2,4-dichlorophenyl)ethyl]imidazole,
27-26
NH2
H
O
Cl
Cl
Cl
Cl
and enantiomer
C. (RS)-2-(2,4-dichlorobenzyloxy)-2-(2,4-dichlorophenyl)ethylamine,
Cl
H
O
Cl
Cl
Cl
and enantiomer
D. (RS)-1-[2-(2,6-dichlorobenzyloxy)-2-(2,4-dichlorophenyl)ethyl]imidazole,
Me Ne
_
COO
H
O
Cl
Cl
Cl
Cl
and enantiomer
E. (RS)-1-(1-carboxylato-1-methylethyl)-3-[2-(2,4-dichlorobenzyloxy)-2-(2,4-dichlorophenyl)ethyl]imidazolium,
H
O
Cl
Cl
Cl
Cl
and enantiomer
F. (RS)-1-[2-(3,4-dichlorobenzyloxy)-2-(2,4-dichlorophenyl)ethyl]imidazole,
Cl
O
Cl
Cl
Cl
and enantiomer
G. (RS)-1-[2-(2,5-dichlorobenzyloxy)-2-(2,4-dichlorophenyl)ethyl]imidazole.
_______________________________________________________________________________________________________________________ Ph Eur
27-27
Midazolam
Me
N
N
Cl
N
F
C18H13ClFN3
325.8
59467-70-8
Midazolam complies with the requirements of the 3rd edition of the European Pharmacopoeia [0936]. These
Action and use Premedicament sedative.
Preparation
Midazolam Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Midazolam contains not less than 98.5 per cent and not more than the equivalent of 101.5 per cent
of 8-chloro-6-(2-fluorophenyl)-1-methyl-4H-imidazo[1,5-a][1,4]benzodiazepine, calculated with
CHARACTERS
A white or yellowish, crystalline powder, practically insoluble in water, freely soluble in acetone and
in alcohol, soluble in methanol.
IDENTIFICATION
obtained with midazolam CRS.
and size to the principal spot in the chromatogram obtained with reference solution (b).
D. Mix 90 mg with 0.30 g of anhydrous sodium carbonate R and ignite in a crucible until an almost
white residue is obtained (normally in less than 5 min). Allow to cool and dissolve the residue in 5 ml
of dilute nitric acid R. Filter (the filtrate is also used in identification test E). Add 1.0 ml of the filtrate
to a freshly prepared mixture of 0.1 ml of alizarin S solution R and 0.1 ml of zirconyl nitrate solution R.
Mix, allow to stand for 5 min and compare the colour of the solution with that of a blank prepared in
the same manner. The colour of the test solution is yellow and that of the blank is red.
E. To 1 ml of the filtrate obtained in identification test D add 1 ml of water R. The solution gives
reaction (a) of chlorides (2.3.1).
TESTS
Appearance of solution Dissolve 0.1 g in 0.1M hydrochloric acid and dilute to 10 ml with the same
acid. The solution is clear (2.2.1) and not more intensely coloured than reference solution Y6
(Method II, 2.2.2).
coating substance.
Test solution (a). Dissolve 0.2 g of the substance to be examined in alcohol R and dilute to 5 ml with
the same solvent.
Test solution (b). Dilute 1 ml of test solution (a) to 50 ml with alcohol R.
Reference solution (a). Dilute 1 ml of test solution (a) to 10 ml with alcohol R. Dilute 2 ml of the
solution to 100 ml with alcohol R.
27-28
Reference solution (b). Dissolve 8 mg of midazolam CRS in alcohol R and dilute to 10 ml with the same
solvent.
Reference solution (c). Dissolve 8 mg of midazolam CRS and 8 mg of chlordiazepoxide CRS in alcohol R
volumes of glacial acetic acid R, 15 volumes of water R, 20 volumes of methanol R and 80 volumes of
ethyl acetate R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Any spot in
than the spot in the chromatogram obtained with reference solution (a) (0.2 per cent). The test is not
spots.
100C to 105C for 2 h.
Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g in a platinum crucible.
ASSAY
0.1M perchloric acid determining the end-point potentiometrically (2.2.20). Titrate to the second point
of inflexion.
1 ml of 0.1M perchloric acid is equivalent to 16.29 mg of C18H13ClFN3.
STORAGE
IMPURITIES
Me
N
N
NH
Cl
A. 8-chloro-6-(2-fluorophenyl)-5,6-dihydro-1-methyl-4H-imidazo[1,5-a][1,4]benzodiazepine,
Me
N
N
N
Cl
B. 8-chloro-6-(2-fluorophenyl)-1-methyl-6H-imidazo[1,5-a][1,4]benzodiazepine,
Me
N
COOH
N
Cl
N
F
C. 8-chloro-6-(2-fluorophenyl)-1-methyl-4H-imdazo[1,5-a][1,4]benzodiazepine-3-carboxylic
acid.
__________________________________________________________________________________________________________ Ph Eur
27-29
Minocycline Hydrochloride
corrected 1/01
OH
OH
OH
O
CONH2
,HCl
NMe2
C23H28ClN3O7
H
H
OH
NMe2
494.9
13614-98-7
Minocycline Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Minocycline Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Minocycline hydrochloride contains not less than 96.0 per cent and not more than the equivalent of
102.5 per cent of (4S,4aS,5aR,12aS)-4,7-bis(dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo1,4,4a,5,5a,6,11,12a-octahydronaphthacene-2-carboxamide hydrochloride, calculated with reference
CHARACTERS
A yellow, crystalline powder, hygroscopic, sparingly soluble in water, slightly soluble in alcohol,
practically insoluble in ether. It dissolves in solutions of alkali hydroxides and carbonates.
IDENTIFICATION
A. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel GF254 plate R. Adjust the pH
of a 100 g/l solution of sodium edetate R to 9.0 with strong sodium hydroxide solution R and spray the
solution evenly onto the plate (about 10 ml for a plate 100 mm 200 mm). Allow the plate to dry in
a horizontal position for at least 1 h. At the time of use, dry the plate in an oven at 110C for 1 h.
the same solvent.
Reference solution (a). Dissolve 5 mg of minocycline hydrochloride CRS in methanol R and dilute to
Reference solution (b). Dissolve 5 mg of minocycline hydrochloride CRS and 5 mg of doxycycline hyclate
CRS in methanol R and dilute to 10 ml with the same solvent.
6 volumes of water R, 35 volumes of methanol R and 59 volumes of methylene chloride R. Dry the plate
in a current of air and examine in ultraviolet light at 254 nm. The spot in the chromatogram obtained
with the test solution is similar in position and size to the spot in the chromatogram obtained with
reference solution (a). The test is not valid unless the chromatogram obtained with reference solution
(b) shows two clearly separated principal spots.
B. To about 2 mg add 5 ml of sulphuric acid R. A bright yellow colour develops. Add 2.5 ml of
water R to the solution. The colour becomes pale yellow.
C. It gives reaction (a) of chlorides (2.3.1).
TESTS
solvent.
Appearance of solution Dilute 1.0 ml of solution S to 50.0 ml with water R. The solution is clear
(2.2.1) and not more intensely coloured than intensity 4 of the range of reference solutions of the
most appropriate colour (Method II, 2.2.2).
Light-absorbing impurities Carry out the measurement within 1 h of preparation of solution S. The
absorbance (2.2.25) of solution S measured at 560 nm is not greater than 0.06.
27-30
Inject separately reference solutions (b) and (c). Inject test solution (a) and continue the
chromatography for at least one and a half times the retention time of the principal peak. In the
chromatogram obtained with test solution (a): the area of any peak corresponding to 4-epiminocycline is not greater than the area of the principal peak in the chromatogram obtained with
reference solution (c) (1.2 per cent); the area of any peak appearing between the solvent peak and
the peak corresponding to 4-epiminocycline or of any peak appearing on the tail of the principal
peak is not greater than the area of the principal peak in the chromatogram obtained with reference
solution (c) (1.2 per cent) and the sum of the areas of such peaks is not greater than the area of the
principal peak in the chromatogram obtained with reference solution (b) (2 per cent).
standard using 2.5 ml of lead standard solution (10 ppm Pb) R.
Sterility (2.6.1). If intended for use in the manufacture of parenteral dosage forms without a further
appropriate sterilisation procedure, it complies with the test for sterility.
without a further appropriate procedure for the removal of bacterial endotoxins, not more than
1.25 I.U. of endotoxin per milligram.
ASSAY
Test solution (a). Dissolve 25.0 mg of the substance to be examined in the mobile phase and dilute to
Test solution (b). Dilute 10.0 ml of test solution (a) to 20.0 ml with the mobile phase.
Reference solution (a). Dissolve 12.5 mg of minocycline hydrochloride CRS in the mobile phase and
Reference solution (b). Dilute 2.0 ml of test solution (a) to 100.0 ml with the mobile phase.
Reference solution (c). Dilute 1.2 ml of test solution (a) to 100.0 ml with the mobile phase.
Reference solution (d). Dissolve 10 mg of minocycline hydrochloride CRS in water R and dilute to 5 ml
with the same solvent. Heat 5 ml of the solution on a water-bath for 60 min. Evaporate to dryness.
Take up the residue with 25 ml of the mobile phase.
Carry out the test protected from bright light. Store the solutions at a temperature of 2C to 8C and use them
within 3 h of preparation.
a column 0.20 m long and 4.6 mm in internal diameter packed with octylsilyl silica gel for chromatography R (5 m),
as mobile phase at a flow rate of 1 ml/min a mixture of 25 volumes of a 4 g/l solution of sodium
edetate R, 27 volumes of dimethylformamide R and 50 volumes of a 28 g/l solution of ammonium
oxalate R, adjusted to pH 7.0 using tetrabutylammonium hydroxide solution (104 g/l) R,
Inject reference solution (d). The assay is not valid unless the resolution between the two principal
peaks is at least 2. Inject reference solution (a) six times. The assay is not valid unless the relative
standard deviation of the peak area for minocycline is at most 1.5 per cent and the number of
theoretical plates calculated from the minocycline peak is at least 15,000 per metre. Inject alternately
test solution (b) and reference solution (a).
Calculate the percentage content of C23H28ClN3O7.
STORAGE
Store in an airtight container, protected from light. If the substance is sterile, store in a sterile,
airtight, tamper-proof container.
LABELLING
The label states:
where applicable, that the substance is sterile,
where applicable, that the substance is free from bacterial endotoxins.
27-31
IMPURITIES
OH
OH O
OH
H
R1
CONH2
OH
R2
R3
A. R1 = N(CH3)2, R2 = H, R 3 = N(CH3)2: (4R,4aS,5aR,12aS)-4,7-bis(dimethylamino)-3,10,12,12atetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydronaphthacene-2-carboxamide (4epiminocycline),

B. R1 = H, R2 = N(CH3)2, R3 = H: (4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydronaphthacene-2-carboxamide (sancycline),
C. R1 = NHCH3, R2 = N(CH3)2, R3 = H: (4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12atetrahydroxy-7-methylamino-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydronaphthacene-2carboxamide (7-monodemethylminocycline).

__________________________________________________________________________________________________________ Ph Eur
27-32
Minoxidil
NH2
N
N
O
NH2
C9H15N5O
209.3
38304-91-5
Minoxidil complies with the requirements of the 3rd edition of the European Pharmacopoeia [0937]. These
Preparation
Minoxidil Scalp Application
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Minoxidil contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of
6-(piperidin-1-yl)pyrimidine-2,4-diamine 3-oxide, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, slightly soluble in water, soluble in methanol and in
propylene glycol, very slightly soluble in ether.
IDENTIFICATION
A. Dissolve 20.0 mg in 0.1M hydrochloric acid and dilute to 100.0 ml with the same solvent (solution
a). Dilute 2.0 ml of solution (a) to 100.0 ml with 0.1M hydrochloric acid (solution b) and dilute 2.0 ml
of solution (a) to 100.0 ml with 0.1M sodium hydroxide (solution c). Examine solutions (b) and (c)
between 200 nm and 350 nm (2.2.25). Solution (b) shows two absorption maxima, at 230 nm and
281 nm. The specific absorbance at the maximum at 230 nm is 1015 to 1120 and that at the
maximum at 281 nm is 1060 to 1170. Solution (c) shows three absorption maxima, at 230 nm,
262 nm and 288 nm. The specific absorbance at the maximum at 230 nm is 1525 to 1685, that at
the maximum at 262 nm is 485 to 535 and that at the maximum at 288 nm is 555 to 605.
obtained with minoxidil CRS.
the same solvent.
Reference solution. Dissolve 10 mg of minoxidil CRS in methanol R and dilute to 10 ml with the same
solvent.
1.5 volumes of concentrated ammonia R and 100 volumes of methanol R. Allow the plate to dry in air
and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with
with the reference solution.
D. Dissolve about 10 mg in 1 ml of methanol R. Add 0.1 ml of copper sulphate solution R. A green
colour develops. The solution becomes greenish-yellow on the addition of 0.1 ml of dilute hydrochloric
acid R.
TESTS
Appearance of solution Dissolve 0.5 g in 12.5 ml of methanol R and dilute to 25 ml with water R.
The solution is clear (2.2.1) and not more intensely coloured than reference solution Y6 (Method II,
2.2.2).
27-33
Reference solution (b). Dissolve 5 mg of deoxyminoxidil (minoxidil impurity E) CRS in the mobile phase
and dilute to 20 ml with the mobile phase. To 2 ml of this solution, add 2 ml of the test solution and
dilute to 10 ml with the mobile phase.
as mobile phase at a flow rate of 1 ml per minute a mixture prepared as follows: dissolve 3.0 g of
docusate sodium R in a mixture of 10 ml of glacial acetic acid R and 300 ml of water R, adjust to
pH 3.0 with perchloric acid R and add 700 ml of methanol R,
Inject 10 l of each solution and continue the chromatography for twice the retention time of the
principal peak. In the chromatogram obtained with the test solution, the sum of the areas of any
peaks, apart from the principal peak, is not greater than 1.5 times the area of the principal peak in the
chromatogram obtained with reference solution (a) (1.5 per cent). The test is not valid unless, in the
chromatogram obtained with reference solution (b), the resolution between the peaks corresponding
to minoxidil and deoxyminoxidil is at least 2.0. Disregard any peak with an area less than 0.1 per cent
of that of the peak in the chromatogram obtained with reference solution (a).
100C to 105C.
ASSAY
end-point potentiometrically (2.2.20). Carry out a blank titration.
STORAGE
IMPURITIES
Cl
NH2
N
O
NH2
A. 6-chloropyrimidine-2,4-diamine 3-oxide,
Cl
NH2
N
NH2
B. 6-chloropyrimidine-2,4-diamine,
N
NH2
C. 3-(cyanoimino)-3-piperidinopropanoamide,
Me
S
O
O
O
NH2
N
O
NH2
D. 6-[[(4-methylphenyl)sulphonyl]oxy]pyrimidine-2,4-diamine 3-oxide,
27-34
NH2
N
NH2
E. 6-(piperidin-1-yl)pyrimidine-2,4-diamine (desoxyminoxidil).
_______________________________________________________________________________________________________________________ Ph Eur
27-35
Dementholised Mint Oil

Definition Dementholised Mint Oil is obtained by steam distillation, followed by partial
dementholisation and processing, from the flowering tops of Mentha arvensis L. var. piperascens
Holmes.
Characteristics A clear, colourless to pale yellow liquid, visibly free from water; odour,
characteristic.
Identification Carry out the method for thin-layer chromatography, Appendix III A, using silica gel G
as the coating substance and a mixture of 5 volumes of ethyl acetate and 95 volumes of toluene as the
mobile phase. Apply separately to the plate, as bands 20 mm long and not more than 3 mm wide,
20 l of each of three solutions in toluene containing (1) 1% v/v of the oil being examined, (2) 0.5%
w/v of menthol, 0.2% v/v of cineole, 0.1% w/v of thymol and 0.1% v/v of menthyl acetate and (3) 0.1%
v/v of cineole, 0.1% w/v of thymol and 0.1% w/v of carvone. After removal of the plate, allow it to dry
in air, spray with anisaldehyde solution, using 10 ml for a plate 200 mm 200 mm in size, and heat at
105 for 5 minutes. The chromatogram obtained with solution (2) shows, in order of increasing Rf
values, a blue band (menthol), a brown band (cineole), a pink band (thymol) and a blue band
(menthyl acetate). In the chromatogram obtained with solution (1) the bands corresponding to
menthol and menthyl acetate are prominent. There is no pink band corresponding to thymol but a
pink band corresponding to carvone may be present. In addition a blue band with an Rf value slightly
higher than that of menthol and an orange band with an Rf value slightly lower than that of menthyl
acetate may be present. In the chromatogram obtained with solution (3) three distinct bands are
obtained.
Acidity To 2 g add 0.25 ml of phenolphthalein solution R1 and titrate with 0.1M ethanolic potassium
hydroxide VS. Not more than 1.2 ml is required to change the colour of the solution.
Optical rotation Brazilian-type oil, 22 to 29; Chinese-type oil, 17 to 24, Appendix V F.
Refractive index Brazilian-type oil, 1.456 to 1.463; Chinese-type oil, 1.458 to 1.466, Appendix
V E.
Solubility in ethanol Soluble, at 20, in 4 volumes of ethanol (70%), Appendix X M; the solution
may become cloudy when diluted.
Weight per ml 0.889 to 0.900 g, Appendix V G.
Chromatographic profile Carry out the method for gas chromatography, Appendix III B, using the
following solutions. Solution (1) is the substance being examined. For solution (2) add to 1 g of
hexane 0.1 g of limonene, 0.2 g of cineole, 0.4 g of menthone, 0.1 g of menthofuran, 0.1 g of
(+)-isomenthone, 0.4 g of menthyl acetate, 0.2 g of pulegone, 0.6 g of menthol and 0.1 g of carvone and
mix.
The chromatographic procedure may be carried out using (a) a glass capillary column (25 m to
60 m about 0.25 mm) coated with polyethylene glycol 20,000 as bonded phase (Carbowax 20 M is
suitable) and (b) helium as the carrier gas at a flow rate of 1.5 ml per minute. Maintain the temperature of the column at 55 for 6 minutes then increase it at the rate of 4 per minute to 180; keep the
injection port temperature at 220 and the detector at 230.
Inject 0.1 l of solution (2). When the chromatograms are recorded in the prescribed conditions,
the components elute in the order indicated in the composition of the solution. Record the retention
times of these substances. The test is not valid unless the number of theoretical plates calculated from
the limonene peak is at least 30,000 and the resolution factor between the peaks corresponding to
limonene and cineole is at least 1.5.
Inject 0.1 l of solution (1). Using the retention times determined from the chromatogram
obtained with solution (2) locate the components of the reference solution on the chromatogram
obtained with solution (1) (disregard the peak due to hexane). Determine the percentage content of
the components by normalisation. The percentages are within the following ranges:
Limonene 1.5 to 7.0%,
Cineole Less than 1.5%,
Menthone 17 to 35%,
Menthofuran Less than 1.0%,
Isomenthone 5.0 to 13%,
Menthyl acetate 1.0 to 7.0%,
Pulegone Less than 1.5% (Brazilian-type oil); less than 3.5% (Chinese-type oil),
Menthol 30 to 45%,
Carvone Less than 1.0%.
The ratio of cineole content to limonene content is less than 1.
Storage Dementholised Mint Oil should be kept in a well-filled, well-closed container, protected
from light and stored at a temperature not exceeding 25.
Labelling The label states whether the oil is Brazilian-type oil or Chinese-type oil.
27-36
Mitobronitol
CH 2Br
HO
HO
OH
OH
CH 2Br
C6H12Br2O4
308.0
488-41-5
Definition Mitobronitol is 1,6-dibromo-1,6-dideoxy-D-mannitol. It contains not less than 98.5%

and not more than 101.0% of C6H12Br2O4 calculated with reference to the dried substance.
Characteristics A white or almost white, crystalline solid.
Slightly soluble in water, in acetone and in ethanol (96%); practically insoluble in chloroform.
Identification
mitobronitol (RS 236).
B. Dissolve 0.1 g in 10 ml of 1M sodium hydroxide, boil, cool, acidify with 2M nitric acid and add 1 ml
of silver nitrate solution. A pale yellow, curdy precipitate is produced.
C. Dissolve 20 mg in 2 ml of a mixture of 1 volume of periodic acid solution and 24 volumes of water.
Add 1 ml of 0.25M barium chloride and shake well. A white, flocculent precipitate is produced.
Acidity Shake 2 g with 50 ml of carbon dioxide-free water for 15 minutes and filter. 40 ml of the
filtrate requires not more than 0.3 ml of 0.02M sodium hydroxide VS for neutralisation using
phenolphthalein solution R1 as indicator.
Clarity and colour of solution A 4.0% w/v solution in dimethylformamide is clear, Appendix IV A,
and colourless, Appendix IV B, Method I.
Heavy metals 1.0 g complies with limit test C for heavy metals, Appendix VII (20 ppm). Use 2 ml of
lead standard solution (10 ppm Pb) to prepare the standard.
Ionic halide Shake 0.20 g with 30 ml of water for 5 minutes and filter. 15 ml of the filtrate complies
with the limit test for chlorides, Appendix VII (500 ppm, calculated as Cl).
Assay Dissolve 0.2 g in 20 ml of 1M sodium hydroxide by heating gently, cool, add 25 ml of 0.1M
silver nitrate VS and acidify with 5 ml of 5M nitric acid. Add 2 ml of ammonium iron(III) sulphate
solution R2 and titrate the excess of silver nitrate with 0.1M ammonium thiocyanate VS. Each ml of
0.1M silver nitrate VS is equivalent to 15.40 mg of C6H12Br2O4.
Storage Mitobronitol should be kept in a well-closed container and protected from light.
Preparation
Mitobronitol Tablets
27-37
Mitoxantrone Hydrochloride / Mitozantrone Hydrochloride

H
N
HO
NH
OH
,2HCl
HO
NH
OH
N
H
C22H30Cl2N4O6
517.4
70476-82-3
Mitoxantrone Hydrochloride complies with the requirements of the 3rd edition of the European
Preparation
Mitoxantrone Intravenous Infusion/Mitozantrone Intravenous Infusion
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Mitoxantrone hydrochloride contains not less than 97.0 per cent and not more than the equivalent of
102.0 per cent of 1,4-dihydroxy-5,8-bis[2-(2-hydroxyethylamino)ethylamino]anthracene-9,10-dione
dihydrochloride, calculated with reference to the anhydrous, ethanol-free substance.
CHARACTERS
A dark blue, electrostatic powder, hygroscopic, sparingly soluble in water, slightly soluble in
methanol, practically insoluble in acetone.
CAUTION: Mitoxantrone hydrochloride and impurity A are electrostatic. The use of an antistatic gun or
other suitable method to discharge the solids before weighing or transfer is recommended.
IDENTIFICATION
A. Dissolve 2 mg to 3 mg in 1 ml of methanol R by warming in a water bath at 40C to 50C. Evaporate the solution to dryness under a stream of dry nitrogen, warming gently if necessary. Examine the
residue by infrared absorption spectrophotometry (2.2.24), comparing with the Ph. Eur. reference
spectrum of mitoxantrone hydrochloride.
B. It gives reaction (b) of chlorides (2.3.1).
TESTS
Ethanol Not more than 1.6 per cent m/m of ethanol, determined by gas chromatography (2.2.28)
using propanol R as the internal standard.
Internal standard solution. Dilute 2.0 ml of propanol R to 100 ml with water R. Dilute 5.0 ml of this
solution to 100 ml with water R.
Test solution. Mix 0.100 g of the substance to be examined with 2.0 ml of the internal standard
solution and dilute to 5.0 ml with water R. Place the flask in a sonic bath for 2 min then shake the
flask for 2 min. If necessary repeat the sonication and shaking until dissolution is complete.
Reference solution. Dilute 2.0 ml of ethanol R to 100.0 ml with water R. Dilute 5.0 ml of this solution
to 100.0 ml with water R. Dilute 10.0 ml of this solution and 10.0 ml of the internal standard
solution to 25.0 ml with water R.
a column 2 m long and 3 mm in internal diameter packed with ethylvinylbenzenedivinylbenzene
copolymer R,
helium for chromatography R as the carrier gas at a flow rate of 19 ml per minute,
27-38
maintaining the column at a temperature of 120C, the temperature of the injector at 175C and
the temperature of the detector at 210C.
Inject 1 l of the test solution and 1 l of the reference solution. The retention times are about
1 min for ethanol and about 2 min for propanol. The test is not valid unless the resolution between
the peaks due to ethanol and propanol in the chromatogram obtained with the reference solution is at
least six. Calculate the content of ethanol taking its density (2.2.5) to be 0.790 g/ml at 20C.
Related substances Examine by liquid chromatography (2.2.29) as prescribed under Assay. In the
chromatogram obtained with the test solution, the area of any peak apart from the principal peak is
not greater than the area of the peak in the chromatogram obtained with reference solution (b) (1 per
cent) and the sum of the areas of all the peaks apart from the principal peak is not greater than twice
the area of the peak in the chromatogram obtained with reference solution (b) (2 per cent). Disregard
any peak with an area less than that of the principal peak in the chromatogram obtained with reference solution (d).
ASSAY
Test solution. Dissolve 20.0 mg of the substance to be examined in about 40 ml of the mobile phase, if
necessary with the aid of ultrasonication, and dilute to 50.0 ml with the mobile phase.
Reference solution (a). Dissolve 20.0 mg of mitoxantrone hydrochloride CRS in about 40 ml of the
mobile phase, if necessary with the aid of ultrasonication, and dilute to 50.0 ml with the mobile
phase.
Reference solution (b). Dilute 1 ml of the test solution to 100 ml with the mobile phase.
Reference solution (c). Dissolve 2.0 mg of mitoxantrone impurity A CRS in 1.0 ml of reference solution
(a).
Reference solution (d). Dilute 1 ml of reference solution (b) to 10 ml with the mobile phase.
a column 0.30 m long and 3.0 mm in internal diameter packed with phenyl silica gel for chromatography R (10 m),
as mobile phase at a flow rate of 3 ml per minute a mixture of 750 volumes of water R, 250
volumes of acetonitrile R and 25 volumes of a solution prepared as follows: dissolve 22.0 g of
sodium heptanesulphonate R in about 150 ml of water R and filter through a 0.45 m filter; wash
the filter with water R and combine the filtrate and washings; add 32.0 ml of glacial acetic acid R
and dilute to 250 ml with water R,
a loop injector.
Inject separately 50 l of each solution. The test is not valid unless the resolution between the two
principal peaks in the chromatogram obtained with reference solution (c) is at least 3.0. Record the
chromatogram obtained with the test solution for three times the retention time of the principal peak.
Calculate the content of C22H30Cl2N4O6 from the peak areas in the chromatograms obtained with
the test solution and reference solution (a) and the declared content of C22H30Cl2N4O6 in
mitoxantrone hydrochloride CRS.
STORAGE
IMPURITIES
HO
H
N
NH O
OH
R1
R3
NH O
R2
A. R1 = R3 = H, R2 = OH: 1-amino-5,8-dihydroxy-4-[[2-[(2-hydroxyethyl)amino]ethyl]amino]anthracene-9,10-dione,
B. R1 = R2 = H, R3 = CH2-CH2-NH-CH2-CH2OH:
5-hydroxy-1,4-bis[[2-[(2-hydroxyethyl)amino]ethyl]amino]anthracene-9,10-dione,
C. R1 = Cl, R2 = OH, R3 = CH2-CH2NH-CH2CH2OH: 2-chloro-1,4-dihydroxy-5,8-bis[[2-[(2hydroxyethyl)amino]ethyl]-amino]anthracene-9,10-dione,
27-39
HO
HO
H
N
NH O
OH
NH O
OH
D. 8,11-dihydroxy-4-(2-hydroxyethyl)-6-[[2-[(2-hydroxyethyl)-amino]ethyl]amino]1,2,3,4tetrahydronaphtho[2,3-f]quinoxaline-7,12-dione.
_______________________________________________________________________________________________________________________ Ph Eur
27-40
Mometasone Furoate
Cl
HO
Me
O
Me
H
Cl
Me
H
O
C27H30Cl2O6
521.4
83919-23-7
Mometasone Furoate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1449].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Mometasone furoate contains not less than 97.0 per cent and not more than the equivalent of
103.0 per cent of 9,21-dichloro-11,17-dihydroxy-16-methylpregna-1,4-diene-3,20-dione 17furane-2-carboxylate, calculated with reference to the dried substance.
CHARACTERS
A white or almost white powder, practically insoluble in water, freely soluble in acetone and in
methylene chloride, slightly soluble in alcohol.
IDENTIFICATION
obtained with mometasone furoate CRS. Examine the substances prepared as discs.
Reference solution (a). Dissolve 20 mg of mometasone furoate CRS in methylene chloride R and dilute to
Reference solution (b). Dissolve 10 mg of beclometasone dipropionate CRS in reference solution (a) and
examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test
solution is similar in position and size to the principal spot in the chromatogram obtained with
reference solution (a). Spray with alcoholic solution of sulphuric acid R. Heat at 120C for 10 min or
until the spots appear. Allow to cool. Examine the chromatograms in daylight and in ultraviolet light
at 365 nm. The principal spot in the chromatogram obtained with the test solution is similar in
position, colour in daylight, fluorescence in ultraviolet light at 365 nm and size to the principal spot
in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows two spots which, when examined in ultraviolet light
at 365 nm, may not be completely separated.
C. Add about 2 mg to 2 ml of sulphuric acid R and shake to dissolve. Within 15 min a light yellow
colour develops. When examined in ultraviolet light at 365 nm, no fluorescence is seen. Add the
solution to 10 ml of water R and mix. The colour fades and there is no fluorescence.
D. Mix 80 mg with 0.30 g of anhydrous sodium carbonate R and ignite in a crucible until an almost
white residue is obtained. Allow to cool and dissolve the residue in 5 ml of dilute nitric acid R. Filter.
To 1 ml of the filtrate add 1 ml of water R. The solution gives reaction (a) of chlorides (2.3.1).
TESTS
Specific optical rotation (2.2.7). Dissolve 50.0 mg in alcohol R and dilute to 10.0 ml with the same
27-41
substance.
Related substances Examine by liquid chromatography (2.2.29). Prepare the solutions immediately
before use.
Solvent solution. Prepare a mixture of equal volumes of acetonitrile R and water R to which is added 0.1
volume of acetic acid R.
Test solution. Dissolve 20.0 mg of the substance to be examined in the solvent solution and dilute to
Reference solution (a). Dissolve 2 mg of mometasone furoate CRS and 6 mg of beclometasone dipropionate
CRS in the solvent solution and dilute to 10.0 ml with the same solvent. Dilute 0.25 ml of the
solution to 10.0 ml with the solvent solution.
Reference solution (b). Dilute 1.0 ml of the test solution to 20.0 ml with the solvent solution. Dilute
1.0 ml of the solution to 10.0 ml with the solvent solution.
as mobile phase at a flow rate of 1 ml/min a mixture of equal volumes of acetonitrile R and
water R,
conditions, the retention times are: mometasone furoate about 17 min and beclometasone
dipropionate about 22 min. The test is not valid unless the resolution between the peaks corresponding to mometasone furoate and beclometasone dipropionate is at least 6. If necessary adjust the
concentration of acetonitrile in the mobile phase.
chromatography for twice the retention time of the principal peak in the chromatogram obtained with
the test solution. In the chromatogram obtained with the test solution: the area of any peak apart
from the principal peak, is not greater than 0.6 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.3 per cent); the sum of the areas of all peaks apart from
the principal peak, is not greater than 1.2 times the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.6 per cent). Disregard any peak due to the solvent and any
100C to 105C.
ASSAY
Dissolve 50.0 mg in alcohol R and dilute to 100.0 ml with the same solvent. Dilute 2.0 ml of this
Calculate the content of C27H30Cl2O6 taking the specific absorbance to be 481.
IMPURITIES
Cl
O
Me
Me
Me
H
A. 21-chloro-16-methyl-3,20-doxopregna-1,4,9(11)-trien-17-yl furan-2-carboxylate,
O2S
Me
HO
H
Me
Cl
O
O
O
Me
B. 9-chloro-11,17-dihydroxy-16-methyl-17-(5H-1,2-oxathiol-4-yl)androsta-1,4-dien-3-one
S,S-dioxide 17-furan-2-carboxylate,
27-42
Cl
O
Me
O
Me
H
H
Me
H
C. 21-chloro-16-methyl-3,11,20-trioxopregna-1,4-dien-17-yl furan-2-carboxylate,
Cl
O
Me
H
Me
O
H
Me
H
D. 21-chloro-9,11-epoxy-16-methyl-3,20-dioxo-9-pregna-1,4-dien-17-yl furan-2carboxylate,
R4
O
Me
R2O
Me
Cl
Fur =
OR3
Me
O
O
O
R1
E. R1 = H2, R2 = R3 = Fur, R4 = Cl: 9,21-dichloro-11,17-dihydroxy-16-methylpregna-1,4-diene3,20-dione 11,17-bis(furan-2-carboxylate),

F. R1 = O, R2 = H, R3 = Fur, R4 = Cl: 9,21-dichloro-11,17-dihydroxy-16-methylpregna-1,4-diene3,6,20-trione 17-furan-2-carboxylate,
G. R1 = H2, R2 = R3 = H, R4 = Cl: 9,21-dichloro-11,17-dihydroxy-16-methylpregna-1,4-diene3,20-dione (mometasone),
H. R1 = H2, R2 = H, R3 = Fur, R4 = OH: 9-chloro-11,17,21-trihydroxy-16-methylpregna-1,4-diene3,20-dione 17-furan-2-carboxylate,
Cl Me
HO
Me
Cl
H
O
O
O
Me
OAc
I. 6-acetoxy-9,21-dichloro-11,17-dihydroxy-16-methylpregn-1-ene-3,20-dione 17-furan2-carboxylate.
__________________________________________________________________________________________________________ Ph Eur
27-43
Morphine Hydrochloride
corrected 1/01
HO
O
H
HO
H
NMe ,HCl
C17H19NO3,HCl,3H2O
375.8
52-26-6 (anhydrous)
Morphine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparations
Chloroform and Morphine Tincture
Morphine Suppositories
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Morphine hydrochloride contains not less than 98.0 per cent and not more than the equivalent of
101.0 per cent of 7,8-didehydro-4,5-epoxy-17-methylmorphinane-3,6-diol hydrochloride
CHARACTERS
A white or almost white, crystalline powder or colourless, silky needles or cubical masses, efflorescent
in a dry atmosphere, soluble in water and in glycerol, slightly soluble in alcohol.
IDENTIFICATION
A. Dissolve 10 mg in water R and dilute to 100.0 ml with the same solvent. Examined between
250 nm and 350 nm (2.2.25), the solution shows a single absorption maximum, at 285 nm. The
specific absorbance at the maximum is about 41.
B. Dissolve 10 mg in 0.1M sodium hydroxide and dilute to 100.0 ml with the same solvent. Examined
between 265 nm and 350 nm (2.2.25), the solution shows a single absorption maximum, at 298 nm.
The specific absorbance at the maximum is about 70.
C. To about 1 mg of powdered substance in a porcelain dish add 0.5 ml of sulphuric acid-formaldehyde
reagent R. A purple colour develops and becomes violet.
D. Dissolve about 5 mg in 5 ml of water R and add 0.15 ml of a freshly prepared 10 g/l solution of
potassium ferricyanide R and 0.05 ml of ferric chloride solution R1. A blue colour is produced
immediately.
E. Dissolve about 5 mg in 5 ml of water R and add 1 ml of dilute hydrogen peroxide solution R, 1 ml of
dilute ammonia R1 and 0.05 ml of a 40 g/l solution of copper sulphate R. A red colour develops.
F. It gives reaction (a) of chlorides (2.3.1).
G. It gives the reaction of alkaloids (2.3.1).
TESTS
Solution S Dissolve 0.50 g in water R and dilute to 25.0 ml with the same solvent.
solution Y6 or BY6 (Method II, 2.2.2).
Acidity or alkalinity To 10 ml of solution S add 0.05 ml of methyl red solution R. Not more than
0.2 ml of 0.02M sodium hydroxide or 0.02M hydrochloric acid is required to change the colour of the
indicator.
Specific optical rotation (2.2.7). 110 to 115, determined on solution S and calculated with
Related substances Examine by thin-layer chromatography (2.2.27), using a TLC silica gel G plate R.
Test solution. Dissolve 0.10 g of the substance to be examined in a mixture of equal volumes of
alcohol R and water R and dilute to 10 ml with the same mixture of solvents.
27-44
Reference solution. Dissolve 50 mg of codeine phosphate R in 5 ml of the test solution. Dilute 0.1 ml of
the solution to 10 ml with a mixture of equal volumes of alcohol R and water R.
Apply separately to the plate 10 l of each solution. Develop over a path of 15 cm using a freshly
prepared mixture of 2.5 volumes of concentrated ammonia R, 32.5 volumes of acetone R, 24.5 volumes
of ethanol R, 10.5 volumes of water R and 35 volumes of toluene R, prepared in the order given. Dry
the plate in a current of air. Spray with potassium iodobismuthate solution R and dry for 15 min in a
current of air. Spray with dilute hydrogen peroxide solution R. Any spot corresponding to codeine in the
chromatogram obtained with the test solution is not more intense than the corresponding spot in the
chromatogram obtained with the reference solution (1 per cent); any spot apart from the principal
spot and the spot corresponding to codeine, is not more intense than the spot corresponding to
morphine in the chromatogram obtained with the reference solution (1 per cent). The test is not valid
unless the chromatogram obtained with the reference solution shows two clearly separated spots.
Meconate To 10 ml of solution S add 1 ml of hydrochloric acid R and 0.1 ml of ferric chloride
solution R1. The absorbance (2.2.25) measured at 480 nm is not greater than 0.05 (0.2 per cent). Use
as the compensation liquid a blank solution prepared at the same time and in the same manner using
10 ml of water R.
at 130C.
Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on the residue from the test for
loss on drying.
ASSAY
Dissolve 0.350 g, heating if necessary, in 30 ml of anhydrous acetic acid R. Cool and add 6 ml of
mercuric acetate solution R. Titrate with 0.1M perchloric acid, using 0.1 ml of crystal violet solution R as
indicator.
1 ml of 0.1M perchloric acid is equivalent to 32.18 mg of C17H20ClNO3.
STORAGE
IMPURITIES
A. codeine,
Me
Me
N H
H
OH H
OH HO
H OH
B. 2,2-bimorphine (pseudomorphine),
Me
H
HO
H OH
C. morphine N-oxide,
HOOC
COOH
OH
D. 3-hydroxy-4-oxo-4H-pyran-2,6-dicarboxylic acid (meconic acid).

__________________________________________________________________________________________________________ Ph Eur
27-45
Morphine Sulphate
corrected 1/01
HO
O
H
,H2SO4
H
HO
H
C34H40N2O10S,5H2O
NMe
2
759
6211-15-0
Morphine Sulphate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1244].
Preparations
Morphine Sulphate Injection
Morphine and Atropine Injection
Morphine Suppositories
Morphine Tablets
Prolonged-release Morphine Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Morphine sulphate contains not less than 98.0 per cent and not more than the equivalent of
102.0 per cent of di(7,8-didehydro-4,5-epoxy-17-methylmorphinane-3,6-diol) sulphate,
calculated with reference to the anhydrous, ethanol-free substance.
CHARACTERS
A white or almost white, crystalline powder, soluble in water, very slightly soluble in alcohol,
practically insoluble in toluene.
IDENTIFICATION
A. Examine by infrared absorption spectrophotometry (2.2.24), after drying the substance to be
examined at 145C for 1 h, comparing with the Ph. Eur. reference spectrum of morphine sulphate.
B. Dissolve 0.100 g in water R and dilute to 100.0 ml with the same solvent (solution A). Dilute
10.0 ml to 100.0 ml with water R. Examined between 250 nm and 300 nm (2.2.25), the solution
shows a single absorption maximum, at 285 nm. The specific absorbance is in the range 37 to 43.
Dilute 10.0 ml of solution A to 100.0 ml with 0.1M sodium hydroxide. Examined between 250 nm and
350 nm (2.2.25), the solution shows a single absorption maximum, at 298 nm. The specific
absorbance is in the range 64 to 72.
C. To about 1 mg of powdered substance in a porcelain dish add 0.5 ml of sulphuric acid-formaldehyde
reagent R. A purple colour develops and becomes violet.
D. It gives the reaction of alkaloids (2.3.1).
E. It gives the reactions of sulphates (2.3.1).
TESTS
indicator.
reference to the anhydrous, ethanol-free substance.
27-46
alcohol R and water R and dilute to 10 ml with the same mixture of solvents.
Reference solution (a). Dissolve 25 mg of codeine phosphate R in 5 ml of the test solution. Dilute 0.2 ml
of the solution to 10 ml with a mixture of equal volumes of alcohol R and water R.
Reference solution (b). Dilute 0.1 ml of the test solution to 20 ml with a mixture of equal volumes of
alcohol R and water R.
Reference solution (c). Dilute 2.0 ml of reference solution (b) to 5.0 ml with a mixture of equal
volumes of alcohol R and water R.
Reference solution (d). Dilute 2.0 ml of reference solution (b) to 10.0 ml with a mixture of equal
volumes of alcohol R and water R.
prepared mixture of 2.5 volumes of concentrated ammonia R, 32.5 volumes of acetone R, 24.5 volumes
of ethanol R, 10.5 volumes of water R and 35 volumes of toluene R, prepared in the order given. Dry
the plate in a current of air. Spray with potassium iodobismuthate solution R and dry for 15 min in a
current of air. Spray with dilute hydrogen peroxide solution R. Any spot corresponding to impurity A in
the chromatogram obtained with the test solution is not more intense than the corresponding spot in
the chromatogram obtained with reference solution (a) (0.5 per cent). Any spot in the chromatogram
obtained with the test solution, apart from the principal spot and any spot corresponding to impurity
A is not more intense than the spot in the chromatogram obtained with reference solution (b)
(0.5 per cent) and not more than two such spots are more intense than the spot in the chromatogram
obtained with reference solution (c) (0.2 per cent). The test in not valid unless the chromatogram
obtained with reference solution (a) shows two such clearly separated spots and the spot in the
chromatogram obtained with reference solution (d) is clearly visible.
Ethanol (2.4.24). Not more than 0.5 per cent.
Iron (2.4.9). Dissolve the residue from the test for sulphated ash in water R and dilute to 10.0 ml
with the same solvent. The solution complies with the limit test for iron (5 ppm).
ASSAY
1 ml of 0.1M perchloric acid is equivalent to 66.88 mg of C34H40N2O10S.
STORAGE
IMPURITIES
A. codeine,
Me
Me
N H
H
OH H
OH HO
H OH
B. 2,2-bimorphine (pseudomorphine),
Me
H
HO
H OH
C. morphine N-oxide.
__________________________________________________________________________________________________________ Ph Eur
27-47
Moxisylyte Hydrochloride / Thymoxamine Hydrochloride

Me
AcO
NMe2 ,HCl
O
Me
Me
C16H25NO3,HCl
315.8
964-52-3
Definition Moxisylyte Hydrochloride is 4-(2-dimethylaminoethoxy)-5-isopropyl-2-methylphenyl

acetate hydrochloride. It contains not less than 99.0% and not more than 101.0% of
C16H25NO3,HCl, calculated with reference to the dried substance.
Freely soluble in water and in chloroform; soluble in ethanol (96%); practically insoluble in ether and
in petroleum spirit.
Identification
moxisylyte hydrochloride (RS 238).
B. Yields reaction A characteristic of chlorides, Appendix VI.
Related substances Carry out the method for liquid chromatography, Appendix III D, using
solutions in the mobile phase containing (1) 0.010% w/v of 2-(6-hydroxythymoxy)ethyldimethylamine
hydrochloride BPCRS, 0.0050% w/v of 2-thymoxyethyldimethylamine hydrochloride BPCRS and 0.020%
w/v of 2-(6-chlorothymoxy)ethyldimethylamine hydrochloride BPCRS and (2) 2.0% w/v of the substance
being examined.
The chromatographic procedure may be carried out using (a) a stainless steel column
(30 cm 3.9 mm) packed with stationary phase C (10 m) (Bondapak C18 is suitable), (b) 0.005M
sodium hexanesulphonate in a mixture of 315 volumes of methanol, 185 volumes of water and 2
volumes of glacial acetic acid as the mobile phase with a flow rate of 1.0 ml per minute and (c) a
detection wavelength of 276 nm.
The peaks in the chromatogram obtained with solution (1) are due to (a) 2-(6-hydroxythymoxy)ethyldimethylamine hydrochloride, (b) 2-thymoxyethyldimethylamine hydrochloride and (c) 2-(6chlorothymoxy)ethyldimethylamine hydrochloride in order of their elution. The areas of any peaks
corresponding to (a), (b) and (c) in the chromatogram obtained with solution (2) are not greater than
the areas of the corresponding peaks in the chromatogram obtained with solution (1) (0.5% of 2-(6hydroxythymoxy)ethyldimethylamine hydrochloride, 0.25% of 2-thymoxyethyldimethylamine hydrochloride and 1% of 2-(6-chlorothymoxy)ethyldimethylamine hydrochloride).
1 g.
C16H25NO3,HCl.
Storage Moxisylyte Hydrochloride should be protected from light.
Preparation
Moxisylyte Tablets/Thymoxamine Tablets
27-48
Mupirocin
OH
O[CH 2 ] 8 COOH
HO
Me
H3C
O
OH
C26H44O9
Me
O
500.6
12650-69-0
Mupirocin complies with the requirements of the 3rd edition of the European Pharmacopoeia [1450]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Mupirocin contains not less than 93.0 per cent and not more than the equivalent of 100.5 per cent of
9-[[(2E)-4-[(2S,3R,4R,5S)-5-[(2S,3S,4S,5S)-2,3-epoxy-5-hydroxy-4-methylhexyl]-3,4-dihydroxy3,4,5,6-tetrahydro-2H-pyran-2-yl]-3-methylbut-2-enoyl]oxy]nonanoic acid, calculated with reference
PRODUCTION
If produced by a process involving fermentation steps, mupirocin complies with the requirements of
the monograph on Products of fermentation (1468).
CHARACTERS
A white or almost white powder, slightly soluble in water, freely soluble in acetone, in ethanol and in
methylene chloride.
IDENTIFICATION
Examine by infrared absorption spectrophotometry (2.2.24), comparing with the Ph. Eur. reference
spectrum of mupirocin. The ratio of the absorbance at 840 cm1 to that at 805 cm1 and the ratio of
absorbance of the peak split at 1720 cm1 are the same as the corresponding ratios in the reference
spectrum.
TESTS
pH (2.2.3). The pH of a freshly prepared saturated solution (about 10 g/l) in carbon dioxide-free
water R is 3.5 to 4.0.
Specific optical rotation (2.2.7). Dissolve 0.50 g in methanol R and dilute to 10.0 ml with the same
solvent. The specific optical rotation is 17 to 21, calculated with reference to the anhydrous
substance.
Test solution. Dissolve 50.0 mg of the substance to be examined in a mixture of equal volumes of a
13.6 g/l solution of sodium acetate R, adjusted to pH 4.0 with acetic acid R, and methanol R. Dilute to
10.0 ml with the same mixture of solvents.
Reference solution (a). Dilute 1.0 ml of the test solution to 50.0 ml with a mixture of equal volumes of
a 13.6 g/l solution of sodium acetate R, adjusted to pH 4.0 with acetic acid R and methanol R.
Reference solution (b). Adjust 10 ml of reference solution (a) to pH 2.0 using hydrochloric acid R and
allow to stand for 20 h.
Reference solution (c). Dissolve 25 mg of mupirocin lithium CRS in a mixture of equal volumes of a
as mobile phase at a flow rate of 1 ml/min a mixture of 20 volumes of water R, 30 volumes of
tetrahydrofuran R and 50 volumes of a 10.5 g/l solution of ammonium acetate R previously
adjusted to pH 5.7 with acetic acid R,
27-49
Inject 20 l of reference solution (b). The test is not valid unless in the chromatogram obtained,
the resolution between the second of the two peaks corresponding to hydrolysis products and the
peak corresponding to mupirocin is at least 7.0.
Inject 20 l of reference solution (c). When the chromatogram is recorded in the prescribed
conditions, the retention time relative to mupirocin is about 0.75 for impurity C. Inject 20 l of the
test solution and 20 l of reference solution (a). Continue the chromatography of the test solution for
3.5 times the retention time of mupirocin. In the chromatogram obtained with the test solution: the
area of any peak corresponding to impurity C is not greater than twice the area of the principal peak
in the chromatogram obtained with reference solution (a) (4 per cent); the area of any peak, apart
from the principal peak and any peak corresponding to impurity C, is not greater than half the area of
the principal peak in the chromatogram obtained with reference solution (a) (1 per cent); the sum of
the areas of all the peaks, apart from the principal peak, is not greater than three times the area of the
principal peak in the chromatogram obtained with reference solution (a) (6 per cent). Disregard any
ASSAY
Test solution. Dissolve 25.0 mg of the substance to be examined in 5 ml of methanol R and dilute to
200.0 ml with a 7.5 g/l solution of ammonium acetate R, adjusted to pH 5.7 with acetic acid R.
Reference solution (a). Dissolve 25.0 mg of mupirocin lithium CRS in 5 ml of methanol R and dilute to
Reference solution (b). Adjust 10 ml of the test solution to pH 2.0 using hydrochloric acid R and allow
to stand for 20 h.
tetrahydrofuran R and 49 volumes of a 10.5 g/l solution of ammonium acetate R adjusted to pH
5.7 with acetic acid R,
peak corresponding to mupirocin is at least 7.0. Inject reference solution (a) six times. The test is not
valid unless the relative standard deviation of the peak area of mupirocin is at most 1.0 per cent.
Inject the test solution and reference solution (a).
STORAGE
IMPURITIES
OH
Me
HO
H3C
O
OH
Me
COOH
Me
A. 9-[[(2E)-4-[(2S,3R,4R,5S)-5-[(2S,3S,4S,5S)-2,3-epoxy-5-hydroxy-4-methylhexyl]-3,4dihydroxy-5-methyl-3,4,5,6-tetrahydro-2H-pyran-2-yl]-3-methylbut-2-enoyl]oxy]nonanoic
acid (pseudomonic acid B),
OH
Me
H3C
HO
O
O
Me
COOH
OH
B. 9-[[(2E)-4-[(2S,3R,4R,5S)-3,4-dihydroxy-5-[(E)-(4S,5S)-5-hydroxy-4-methylhex-2-enyl]3,4,5,6-tetrahydro-2H-pyran-2-yl]-3-methylbut-2-enoyl]oxy]nonanoic acid (pseudomonic

acid C),
27-50
OH
HO
Me
3C
Me
COOH
OH
C. 9-[[(2E)-4-[(2S,3R,4R,5S)-5-[(2S,3S,4S,5S)-2,3-epoxy-5-hydroxy-4-methylhexyl]-3,4dihydroxy-3,4,5,6-tetrahydro-2H-pyran-2-yl]-3-methylbut-2-enoyl]oxy]nona-4-enoic acid
(pseudomonic acid D),
OH
Me
3C
O
OH OH
Me
COOH
D. 9-[[(2E)-4-[2R,3aS,6S,7R)-2-[(2S,3S)-1,3-dihydroxy-2-methylbutyl]-7-hydroxy2,3,3a,4,6,7,7a-heptahydro-4H-furo[3,2-c]pyran-6-yl]-3-methylbut-2-enoyl]oxy]nonanoic
acid,
H3C
OH
H
Me
OH
O
O
HO
Me
COOH
E. 9-[[(2E)-4-[(2R,3RS,4aS,7S,8S,8aR)-3,8-dihydroxy-2-[(1R,2S)-2-hydroxy-1methylpropyl]-3,4,4a,7,8,8a-hexahydro-2H,5H-pyrano[3,2-c]pyran-7-yl]-3-methylbut-2enoyl]oxy]nonanoic acid,
OH
Me
HO
3C
O
O
OH
Me
COOH
F. 7-[[(2E)-4-[(2S,3R,4R,5S)-5-[(2S,3S,4S,5S)-2,3-epoxy-5-hydroxy-4-methylhexyl]-3,4dihydroxy-3,4,5,6-tetrahydro-2H-pyran-2-yl]-3-methylbut-2-enoyl]oxy]heptanoic acid.
__________________________________________________________________________________________________________ Ph Eur
27-51
Mupirocin Calcium
OH
O[CH 2 ] 8 COO
HO
Me
H3C
O
OH
C52H86O18Ca,2H2O
Me
Ca 2+
O
2
1075
11504-43-6
Mupirocin Calcium complies with the requirements of the 3rd edition of the European Pharmacopoeia [1451].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Mupirocin calcium contains not less than 93.0 per cent and not more than the equivalent of
100.5 per cent of calcium bis[9-[[(2E)-4-[(2S,3R,4R,5S)-5-[(2S,3S,4S,5S)-2,3-epoxy-5-hydroxy-4methylhexyl]-3,4-dihydroxy-3,4,5,6-tetrahydro-2H-pyran-2-yl]-3-methylbut-2enoyl]oxy]nonanoate], calculated with reference to the anhydrous substance.
PRODUCTION
If produced by a process involving fermentation steps, mupirocin calcium complies with the requirements of the monograph on Products of fermentation (1468).
CHARACTERS
A white or almost white powder, very slightly soluble in water, sparingly soluble in ethanol and in
methylene chloride.
IDENTIFICATION
spectrum of mupirocin calcium.
B. It gives reaction (a) of calcium (2.3.1).
TESTS
Specific optical rotation (2.2.7). Dissolve 0.50 g in methanol R and dilute to 10.0 ml with the same
solvent. The specific optical rotation is 16 to 20, calculated with reference to the anhydrous
substance.
Reference solution (a). Dilute 1.0 ml of the test solution to 50.0 ml with a mixture of equal volumes of
a 13.6 g/l solution of sodium acetate R, adjusted to pH 4.0 with acetic acid R and methanol R.
Reference solution (b). Adjust 10 ml of reference solution (a) to pH 2.0 using hydrochloric acid R and
allow to stand for 20 h.
Reference solution (c). Dissolve 25 mg of mupirocin lithium CRS in a mixture of equal volumes of a
tetrahydrofuran R and 50 volumes of a 10.5 g/l solution of ammonium acetate R previously
adjusted to pH 5.7 with acetic acid R,
27-52
peak corresponding to mupirocin is at least 7.0. Inject 20 l of reference solution (c). When the
chromatogram is recorded in the prescribed conditions, the retention time relative to mupirocin is
about 0.75 for impurity C. Inject 20 l of the test solution and 20 l of reference solution (a).
Continue the chromatography of the test solution for 3.5 times the retention time of mupirocin. In the
chromatogram obtained with the test solution: the area of any peak corresponding to impurity C is
not greater than 1.25 times the area of the principal peak in the chromatogram obtained with
reference solution (a) (2.5 per cent); the area of any peak, apart from the principal peak and any peak
corresponding to impurity C, is not greater than half the area of the principal peak in the chromatogram obtained with reference solution (a) (1 per cent); the sum of the areas of all the peaks, apart
from the principal peak, is not greater than 2.25 times the area of the principal peak in the chromatogram obtained with reference solution (a) (4.5 per cent). Disregard any peak with an area less than
0.05 times the area of the principal peak in the chromatogram obtained with reference solution (a).
Chlorides (2.4.4). Dissolve 10.0 mg in a mixture of 1 ml of dilute nitric acid R and 15 ml of
methanol R. The solution complies with the limit test for chlorides (0.5 per cent).
Water (2.5.12). 3.0 per cent to 4.5 per cent, determined on 0.500 g by the semi-micro determination
of water.
ASSAY
Reference solution (a). Dissolve 25.0 mg of mupirocin lithium CRS in 5 ml of methanol R and dilute to
Reference solution (b). Adjust 10 ml of the test solution to pH 2.0 using hydrochloric acid R and allow to
stand for 20 h.
tetrahydrofuran R and 49 volumes of a 10.5 g/l solution of ammonium acetate R adjusted to pH 5.7
with acetic acid R,
Inject 20 l of the reference solution (b). The test is not valid unless, in the chromatogram
obtained, the resolution between the second of the two peaks corresponding to hydrolysis products
and the peak corresponding to mupirocin is at least 7.0. Inject reference solution (a) six times. The
test is not valid unless the relative standard deviation of the peak area of mupirocin is at most 1.0 per
cent. Inject the test solution and the reference solution (a).
Calculate the percentage content of mupirocin calcium by multiplying the percentage content of
mupirocin lithium by 1.038.
IMPURITIES
OH
Me
O[CH2]8COOH
HO
3C
O
OH
Me
OH
A. 9-[[(2E)-4-[(2S,3R,4R,5S)-5-[(2S,3S,4S,5S)-2,3-epoxy-5-hydroxy-4-methylhexyl]-3,4dihydroxy-5-methyl-3,4,5,6-tetrahydro-2H-pyran-2-yl]-3-methylbut-2-enoyl]oxy]-nonanoic
acid (pseudomonic acid B),
OH
Me
HO
3C
O[CH2]8COOH
O
Me
OH
B. 9-[[(2E)-4-[(2S,3R,4R,5S)-3,4-dihydroxy-5-[(E)-(4S,5S)-5-hydroxy-4-methylhex-2-enyl]3,4,5,6-tetrahydro-2H-pyran-2-yl]-3-methylbut-2-enoyl]oxy]nonanoic acid (pseudomonic

acid C),
OH
Me
HO
3C
O[CH2]4
O
OH
Me
COOH
C. 9-[[(2E)-4-[(2S,3R,4R,5S)-5-(2S,3S,4S,5S)-2,3-epoxy-5-hydroxy-4-methylhexyl]-3,4dihydroxy-3,4,5,6-tetrahydro-2H-pyran-2-yl]-3-methylbut-2-enoyl]oxy]nona-4-enoic acid
(pseudomonic acid D),
27-53
OH
O[CH2]8COOH
Me H O *
H3C
Me
OH OH
D. 9-[[(2E)-4-[(2R,3aS,6S,7R)-2-[(2S,3S)-1,3-dihydroxy-2-methylbutyl]-7-hydroxy2,3,3a,4,6,7,7a-heptahydro-4H-furo[3,2-c]pyran-6-yl]-3-methylbut-2-enoyl]oxy]nonanoic
acid,
OH
H
H3C
OH
O
3C
H
O[CH2]8COOH
HO
Me
E. 9-[[(2E)-4-[(2R,3RS,4aS,7S,8S,8aR)-3,8-dihydroxy-2-[(1R,2S)-2-hydroxy-1methylpropyl]-3,4,4a, 7,8,8a-hexahydro-2H,5H-pyrano[3,2-c]pyran-7-yl]-3-methylbut-2enoyl]oxy]nonanoic acid,

OH
HO
Me
O[CH2] 6 COOH
3C
O
OH
Me
F. 7-[[(2E)-4-[(2S,3R,4R,5S)-5-[(2S,3S,4S,5S)-2,3-epoxy-5-hydroxy-4-methylhexyl]-3,4dihydroxy-3,4,5,6-tetrahydro-2H-pyran-2-yl]-3-methylbut-2-enoyl]oxy]heptanoic acid,
HO
Me
OH
O[CH2]8COOH
R2
H3C *
* * *
Me
OH R1
G. R1 = OH, R2 = Cl : 9-[[(2E)-4-[(2S,3R,4R,5S)-5-(2-chloro-3,5-dihydroxy-4methylhexyl)-3,4-dihydroxy-3,4,5,6-tetrahydro-2H-pyran-2-yl]-3-methylbut-2enoyl]oxy]nonanoic acid,

H. R1 = Cl, R2 = OH : 9-[[(2E)-4-[(2S,3R,4R,5S)-5-(3-chloro-2,5-dihydroxy-4-methylhexyl)3,4-dihydroxy-3,4,5,6-tetrahydro-2H-pyran-2-yl]-3-methylbut-2-enoyl]oxy]nonanoic acid,
Me HO
HO
* *
e
*
*
O
OH
O[CH2]8COOH
O
Me
I. 9-[[(2E)-4-(3,4-dihydroxy-5-(3-hydroxy-4,5-dimethyl-3,4,5,6-tetrahydrofuran-2-yl)methyl-3,4,5,6-tetrahydro-2H-pyran-2-yl]-3-methylbut-2-enoyl]oxy]nonanoic acid.
__________________________________________________________________________________________________________ Ph Eur
27-54
Myrrh
Myrrh complies with the requirements of the 3rd edition of the European Pharmacopoeia [1349]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Myrrh consists of a gum-resin, hardened in air, obtained by incision or produced by spontaneous
exudation from the stem and branches of Commiphora molmol Engler and/or other species of
Commiphora.
CHARACTERS
Myrrh has a bitter taste.
IDENTIFICATION
A. The light or dark orange-brown, irregular or roundish grains or pieces of different size show
components of various colours. Their surface is mostly covered with grey to yellowish-brown dust.
B. Reduce to a powder (355). The powder is brownish-yellow to reddish-brown. Examine under a
microscope, using chloral hydrate solution R. The powder shows only a few tissue fragments from the
original plants including the following: reddish-brown cork fragments; single or grouped polyhedral
to elongated stone cells with partly strongly thickened, pitted and lignified walls with a brownish
content; fragments of thin-walled parenchyma and sclerenchymatous fibres; irregular prismatic to
polyhedral crystals of calcium oxalate, about 10 m to 25 m in size.
C. Examine the chromatograms obtained in the test for Commiphora mukul. Spray the plate with
anisaldehyde solution R, and examine in daylight while heating at 100C to 105C for 10 min. The
chromatogram obtained with the reference solution shows in the lower third an orange-red zone
(thymol) and in the middle third a violet zone (anethole).
The chromatogram obtained with the test solution shows an intense violet zone (furanoeudesma-1,3diene), exceeding the other zones in size and intensity, above the zone of anethole in the chromatogram obtained with the reference solution; a violet zone similar in position to the zone of anethole in
the chromatogram obtained with the reference solution; two intense violet zones similar in position to
the zone of thymol in the chromatogram obtained with the reference solution, the upper one corresponding to curzerenone and the lower one to 2-methoxyfuranodiene. Further mostly violet zones are
present in the chromatogram obtained with the test solution.
ESTS
Commiphora mukul Examine by thin-layer chromatography (2.2.27) using a TLC silica gel plate R.
Test solution. To 0.5 g of the powdered drug (355) add 5.0 ml of alcohol R and warm the mixture on a
water-bath for 2 min to 3 min. Cool and filter.
Reference solution. Dissolve 10 mg of thymol R and 40 l of anethole R in 10 ml of alcohol R.
mixture of 2 volumes of ethyl acetate R and 98 volumes of toluene R. Allow the plate to dry in air.
Examine in ultraviolet light at 365 nm, the chromatogram obtained with the test solution shows no
blue to violet fluorescent zones in the lower third of the chromatogram.
Matter insoluble in alcohol Not more than 70 per cent. Place 1.00 g of the powdered drug (250)
in a flask. Add 30 ml of alcohol R and shake vigorously for 10 min. Filter the supernatant through a
tared sintered-glass filter (16) avoiding the transfer of sediment from the flask. Repeat the extraction
with two quantities, each of 20 ml, of alcohol R. Quantitatively transfer the sediment to the filter by
rinsing the flask with alcohol R. Dry the filter and the residue in an oven at 100C to 105C and
weigh.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
27-55
Nabumetone
corrected 1/01
O
CH3
MeO
C15H16O2
228.3
42924-53-8
Nabumetone complies with the requirements of the 3rd edition of the European Pharmacopoeia [1350]. These
Action and use Anti-inflammatory; analgesic.
Preparations
Nabumetone Oral Suspension
Nabumetone Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Nabumetone contains not less than 97.0 per cent and not more than the equivalent of 102.0 per cent
of 4-(6-methoxynaphthalen-2-yl)butan-2-one calculated with reference to the anhydrous substance.
CHARACTERS
A white or almost white, crystalline powder, practically insoluble in water, freely soluble in acetone,
slightly soluble in methanol.
IDENTIFICATION
with nabumetone CRS.
TESTS
Inject 20 l of test solution (a), 20 l of reference solution (b) and 20 l of reference solution (c). In
the chromatogram obtained with test solution (a): the area of any peak due to impurity F, is not
greater than the area of the principal peak in the chromatogram obtained with reference solution (c)
(0.3 per cent); the sum of the areas of any other peak, apart from the principal peak and a peak due
to impurity F, is not greater than the area of the principal peak in the chromatogram obtained with
reference solution (b) (0.5 per cent). Disregard any peak with an area less than 0.1 times the area of
the principal peak in the chromatogram obtained with reference solution (b).
ASSAY
Test solution (a). Dissolve 50.0 mg of the substance to be examined in acetonitrile R and dilute to
Test solution (b). Dilute 1.0 ml of test solution (a) to 25.0 ml with acetonitrile R. Dilute 1.0 ml of the
solution to 5.0 ml with acetonitrile R.
Reference solution (a). Dissolve 20.0 mg of nabumetone CRS in acetonitrile R and dilute to 10.0 ml with
the same solvent. Dilute 1.0 ml of the solution to 50.0 ml with acetonitrile R.
Reference solution (b). Dilute 0.5 ml of test solution (a) to 100.0 ml with acetonitrile R.
Reference solution (c). Dissolve 1.5 mg of nabumetone impurity F CRS in acetonitrile R and dilute to
Reference solution (d). Dissolve 4 mg of nabumetone impurity D CRS in acetonitrile R and dilute to
100 ml with the same solvent. To 5 ml of the solution, add 5 ml of test solution (b).
a stainless steel column 0.15 m long and 4.6 mm in internal diameter packed with base-
27-56
deactivated octadecylsilyl silica gel for chromatography R (4 m),
Mobile phase A. A mixture of 12 volumes of tetrahydrofuran R, 28 volumes of acetonitrile for
chromatography R and 60 volumes of a 0.1 per cent V/V solution of glacial acetic acid R in carbon
dioxide-free water R prepared from distilled water R,
Mobile phase B. A mixture of 24 volumes of tetrahydrofuran R, 56 volumes of acetonitrile for
chromatography R and 20 volumes of a 0.1 per cent V/V solution of glacial acetic acid R in carbon
dioxide-free water R prepared from distilled water R,
Time
(min)
Mobile phase A
(%V/V)
Mobile phase B
(% V/V)
Comment
0 12
100
isocratic
12 28
100 0
0 100
linear gradient
28 33
100
isocratic
33 34
0 100
100 0
linear gradient
34 35
100
isocratic

Inject 20 l of reference solution (b) and 20 l of reference solution (d). Adjust the sensitivity of the
system so that the height of the principal peak in the chromatogram obtained with reference solution
(b) is at least 70 per cent of the full scale of the recorder. When the chromatograms are recorded
under the prescribed conditions the retention time for nabumetone is about 11 min. The test is not
valid unless, in the chromatogram obtained with reference solution (d), the resolution between the
peaks corresponding to nabumetone and impurity D is at least 1.5. Inject reference solution (a) six
times. The test is not valid unless the relative standard deviation of the peak area for nabumetone is
at most 1.0 per cent. Inject alternately test solution (b) and reference solution (a).
Calculate the percentage content of nabumetone using the chromatogram obtained with reference
solution (a).
STORAGE
IMPURITIES
O
Me
eO
A. 3-(6-methoxynaphthalen-2-yl)-5-methylcyclohexanone,
O
Me
H
and enantiomer
eO
B. (5RS)-5-(6-methoxynaphthalen-2-yl)-3-methylcyclohex-2-enone,
H OH
CH3
and enantiomer
eO
C. (2RS)-4-(6-methoxynaphthalen-2-yl)butan-2-ol,
O
CH3
eO
D. (E)-4-(6-methoxynaphthalen-2-yl)but-3-en-2-one,
O
eO
OMe
E. 1,5-bis(6-methoxynaphthalen-2-yl)pentan-3-one,
27-57
OMe
eO
F. 6,6-dimethoxy-2,2-binaphthalenyl.
__________________________________________________________________________________________________________ Ph Eur
27-58
Nadroparin Calcium
3
/ 2 Ca
2+
OR
R2
O
COO
OH
HO
O
R3
OH
OR
O
NHR 1
OR
_
OSO3
Nadroparin Calcium complies with the requirements of the 3rd edition of the European Pharmacopoeia [1134].
Action and use Low-molecular-weight heparin; anticoagulant.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Nadroparin calcium is the calcium salt of low-molecular-mass heparin obtained by nitrous acid
depolymerisation of heparin from pork intestinal mucosa, followed by fractionation to eliminate
selectively most of the chains with a molecular mass lower than 2000. The majority of the
components have a 2-O-sulpho--L-idopyranosuronic acid structure at the non-reducing end and a
6-O-sulpho-2,5-anhydro-D-mannitol structure at the reducing end of their chain.
Nadroparin calcium complies with the monograph on Heparins, low-molecular-mass (828) with the modifications and additional requirements below.
The mass-average molecular mass ranges between 3600 and 5000 with a characteristic value of about
4300.
The degree of sulphatation is about 2 per disaccharide unit.
The potency is not less than 95 I.U. and not more than 130 I.U. of anti-factor Xa activity per
milligram, calculated with reference to the dried substance. The ratio of anti-factor Xa activity to
anti-factor IIa activity is between 2.5 and 4.0.
IDENTIFICATION
Carry out identification test C as described in the monograph on Heparins, low-molecular-mass (828).
The following requirements apply.
The mass-average molecular mass ranges between 3600 and 5000. The mass percentage of chains
lower than 2000 is not more than 15 per cent. The mass percentage of chains between 2000 and
8000 ranges between 75 per cent and 95 per cent. The mass percentage of chains between 2000 and
4000 ranges between 35 per cent and 55 per cent.
TESTS
Appearance of solution Dissolve 0.5 g of the substance to be examined in 10 ml of water R. The
solution is not more intensely opalescent than the reference suspension II (2.2.1) and not more
intensely coloured than the reference solution Y5 (Method II, 2.2.2).
Ethanol Not more than 1.0 per cent m/m, determined by head-space gas chromatography (Method
II, 2.2.28), using 2-propanol R as the internal standard.
Internal standard solution. Dilute 1.0 ml of 2-propanol R to 100.0 ml with water R. Dilute 1.0 ml of this
Reference solution. Dilute 1.0 ml of ethanol R to 100.0 ml with water R. Dilute 0.5 ml of this solution
to 20.0 ml with water R.
Vial filling. Place the following into four separate vials which can be crimp-sealed and which are
compatible with the injection system:
1.0 ml of water R (blank vial),
0.50 ml of the reference solution and 0.50 ml of the internal standard solution (reference vial),
10.0 mg of the substance to be examined. Add 1.0 ml of water R (test vial A),
10.0 mg of the substance to be examined. Add 0.50 ml of water R and 0.50 ml of the internal
standard solution (test vial B).
27-59
a nickel column 1.5 m long and 2 mm in internal diameter packed with ethylvinylbenzene
divinylbenzene copolymer R (150 m to 180 m),
helium for chromatography R or nitrogen for chromatography R as the carrier gas at a flow rate of
30 ml/min,
Maintain the temperature of the column at 150C and that of the injection port and that of the
detector at 250C.
Equilibrate each vial in the head-space system at 90C for 15 min. The pre-injection pressurisation
time is 1 min.
The chromatogram obtained with the reference vial shows two peaks which correspond in order of
increasing retention time to ethanol and 2-propanol (with retention times of approximately 2.5 and
4 mins). Calculate the content of ethanol (m/m) taking its density at 20C to be 0.792 g/ml.
Fig. 11341 Apparatus used for the assay of NNO groups

Apparatus used for the assay of N-NO groups
Bubble traps. Height: 24 cm, internal diameter: 2.5 cm, internal tubing 23 cm in length by
0.5 cm internal diameter. Centrally positioned Rotulex mounting. Equipped with torion
joints on the inlet and outlet.
Chemiluminescence detector.
Cold trap. Height: 16.5 cm, internal diameter: 4 cm, internal tubing 14 cm in length and
internal diameter 1.3 cm. Equipped with torion joints on the inlet and outlet.
Condenser. Height: 21 cm, internal diameter: 3 cm. Lower rodavis joint and upper torion joint.
Flask. Round-bottomed borosilicate glass flask equipped with a central rodavis joint, a torion
joint on the left neck and a 15 screw joint on the right neck.
Isothermic flask. Internal depth: 22 cm, internal diameter: 8 cm.
Septum. Silicone material, diameter: 14 mm: thickness: 3.5 mm.
Torion joint.
Tubing. Polytetrafluoroethylene fep material, internal diameter: 3.2 mm, thickness: 0.8 mm.
N-NO groups Not more than 0.25 ppm, determined by cleavage of the N-NO bond with hydrobromic acid in ethyl acetate under a reflux condenser and detection of the released NO by
chemiluminescence.
27-60
Description of the apparatus (Figure 1134-1). Use a 500 ml borosilicate glass round-bottomed flask,
above which is attached a condenser which is equipped with:
on one side, a torion joint through which a stream of argon R can be introduced via a cannula,
on the other side, a screw joint with a piston equipped with a septum through which the
reference solution and test solution will be injected.
The round-bottomed flask is connected in series to three bubble traps which are themselves
connected to two cold traps, which are in turn connected to a chemiluminescence detector. Suitable
tubing ensures the junctions are leak-free.
Preparation of the chemiluminescence detector. Switch on the chemiluminescence detector 48 h before
use and start the vacuum pump. The vacuum must be less than 0.5 mmHg. 1 h before use, open the
oxygen valve at a pressure of 0.2 MPa and a flow rate of 9.4 ml/min.
Preparation of the bubble trap. In each bubble trap, place 30 ml of a 300 g/l solution of sodium
hydroxide R in water R.
Preparation of the cold traps.
Trap at 120C: Slowly add liquid nitrogen to an isothermic flask containing 250 ml of ethanol R
whilst stirring with a wooden spatula until a paste is obtained. Place the cold trap in the
isothermic flask prepared as described.
Trap at 160C: Slowly add liquid nitrogen to an isothermic flask containing 250 ml of 2-methylbutane R whilst stirring with a wooden spatula until a paste is obtained. Place the cold trap in the
isothermic flask prepared as described.
Drying of the 500 ml borosilicate-glass round-bottomed flask and condenser. Boil 50 ml of ethyl acetate R
under reflux for 1 h under argon R without connecting the system to the chemiluminescence detector.
Test solution. Dry the substance to be examined for 12 h over diphosphorus pentoxide R at 60C under
vacuum. Dissolve 0.10 g of the treated substance to be examined in 1.0 ml of treated formamide R.
Shake the solution obtained for 30 min.
Reference solution. Dilute 0.1 ml of nitrosodipropylamine solution R in 6.0 ml of ethanol R. Dilute 0.1 ml
of the solution obtained in 1.0 ml of treated formamide R. (This solution is equivalent to 0.05 ppm of
N-NO groups).
Place 50 ml of treated ethyl acetate R in the dry 500 ml borosilicate glass round-bottomed flask
equipped with a septum. Connect the round-bottomed flask to the condenser which has been
previously cooled to 15C for 2 h.
Connect the argon R cannula and adjust the flow rate to 0.1 litre per minute.
Check that the system is leak-free. Only the connector to the chemiluminescence detector remains
open in order to avoid excess pressure.
Heat the treated ethyl acetate R to boiling.
Evacuate the system by slowly turning the valve of the chemiluminescence detector. At the same
time tighten the inlet on the chemiluminescence detector.
When the system is equilibrated, the vacuum reaches 4 mm Hg.
The signal of the zero adjuster on the chemiluminescence detector is set to 10 per cent of the full
scale of the recorder.
Through the septum of the 500 ml borosilicate glass round-bottomed flask, sequentially inject
0.5 ml of water R, 2.0 ml of dilute hydrobromic acid R and then another 2.0 ml of dilute hydrobromic
acid R, making sure that the recorder pen has returned to the baseline between each injection.
Inject 50.0 l of the reference solution, then 50.0 l of the test solution after the recorder pen has
returned to the baseline.
Calculate the content of N-NO groups of the substance to be examined.
Free sulphates Not more than 0.5 per cent, determined by liquid chromatography (2.2.29) using an
instrument equipped with a conductivity detector.
Test solution. Dissolve 30.0 mg of the substance to be examined in water R and dilute to 10.0 ml with
the same solvent.
Reference solution. Dissolve 1.4787 g of anhydrous sodium sulphate R in water R and dilute to 1000.0 ml
with the same solvent. Dilute 1.0 ml of this solution to 200.0 ml with distilled water R (5 ppm of
sulphate ions).
an anion separation column 50 mm long and 4.6 mm in internal diameter,
a chemical neutralisation system: neutralisation micro-membrane in line with the mobile phase
for anion detection,
elute with a 1.91 g solution of disodium tetraborate R in 1000 ml of water R as mobile phase for
15 min. Change to 100 per cent of 0.1M sodium hydroxide for a period of 0.5 min. Elute with this
solution for 10 min. Return to the initial conditions for a period of 0.5 min. The flow rate is
1.0 ml/min,
a detector with a sensitivity of 30 S.
Continuously pump the chemical neutralisation system in counter-flow with a 2.45 g/l solution of
sulphuric acid R, at a flow rate of 4 ml/min.
27-61
Inject 50 l of each solution. The chromatogram obtained with the reference solution shows a
principal peak which corresponds to the sulphate ion (retention time of about 7.5 min).
Change the composition of the mobile phase, if necessary, to obtain the prescribed retention time.
Calculate the sulphate content of the substance to be examined.
__________________________________________________________________________________________________________ Ph Eur
27-62
Naftidrofuryl Oxalate
O
*
H
O
NEt2
COOH
COOH
C24H33NO3,C2H2O4
473.6
3200-06-4
Definition Naftidrofuryl Oxalate is 2-diethylaminoethyl all rac-3-(1-naphthyl)-2tetrahydrofurfurylpropionate oxalate. It contains not less than 99.0% and not more than 101.0% of
C24H33NO3,C2H2O4, calculated with reference to the dried substance.
Characteristics A fine white powder; odour, characteristic.
Freely soluble in water and in ethanol (96%); sparingly soluble in acetone; practically insoluble in
ether.
Identification
A. Dissolve 0.1 g in 5 ml of water, add 5 ml of 2M sodium hydroxide and 5 ml of dichloromethane.
Shake well, allow to separate and filter the dichloromethane layer through phase-separating paper
(Whatman 1PS is suitable) containing anhydrous sodium sulphate. Wash the aqueous layer with a
further 5 ml of dichloromethane, allow to separate and filter the dichloromethane through the same
filter. Combine the dichloromethane filtrates, evaporate to dryness and dry the oily residue over
phosphorus pentoxide at a pressure not exceeding 2 kPa for 18 hours. The infrared absorption spectrum of
the dried residue, Appendix II A, is concordant with the reference spectrum of naftidrofuryl (RS 240).
B. Dissolve about 0.5 g in 10 ml of water and add calcium chloride solution; a white precipitate is
produced. The residue dissolves in mineral acids but is practically insoluble in 2M acetic acid and in
6M ammonia.
Light absorption Absorbance of a 15.0% w/v solution at 430 nm, not more than 0.1, Appendix II B.
Related substances Carry out the method for liquid chromatography, Appendix III D, using 20 l of
each of the following solutions in the mobile phase. Solution (1) contains 0.1% w/v of the substance
being examined. Solution (2) contains 0.0001% w/v of 3-(1-naphthyl)-2-tetrahydrofurfurylpropionic
acid BPCRS. Solution (3) contains 0.0002% w/v of 2-diethylaminoethyl-3-(1-naphthyl)-2-(1naphthylmethyl)propionate oxalate BPCRS. Solution (4) contains 0.01% w/v of naftidrofuryl oxalate
BPCRS and 0.005% w/v of 2-diethylaminoethyl 3-(1-naphthyl)-2-(1-naphthylmethyl)propionate oxalate
BPCRS.
(25 cm 4.6 mm) packed with particles of silica the surface of which has been modified by
chemically-bonded phenyl groups (5 m) (Spherisorb Phenyl is suitable), (b) a mixture of 40
volumes of 0.05M sodium acetate, adjusted to pH 4.0 with an 85% v/v solution of orthophosphoric acid,
and 60 volumes of acetonitrile as the mobile phase with a flow rate of 1 ml per minute and (c) a
The test is not valid unless in the chromatogram obtained with solution (4) the resolution factor
between the two principal peaks is at least 4.
Inject solution (1) and record the chromatography for twice the retention time of the principal
peak. In the chromatogram obtained with solution (1) the area of any peak corresponding to 3-(1naphthyl)-2-tetrahydrofurfurylpropionic acid is not greater than the area of the peak in the chromatogram obtained with solution (2) (0.1%), the area of any peak corresponding to 2-diethylaminoethyl3-(1-naphthyl)-2-(1-naphthylmethyl)propionate oxalate is not greater than the area of the peak in the
chromatogram obtained with solution (3) (0.2%), the area of any other secondary peak is not greater
than the area of the peak in the chromatogram obtained with solution (2) (0.1%) and the sum of the
areas of any secondary peaks other than those corresponding to the named impurities is not greater
than 3 times the area of the peak in the chromatogram obtained with solution (2) (0.3%).
Heavy metals Mix 1.0 g with 0.5 g of magnesium oxide in a silica crucible. Ignite to dull red heat
until a homogeneous white or greyish white mass is produced. If after 30 minutes of ignition the
mixture remains coloured, allow to cool, mix with a fine glass rod and repeat the ignition. If necessary
repeat the operation. Finally heat at 800 for about 1 hour and dissolve the residue using two 5-ml
quantities of 5M hydrochloric acid. Add 0.1 ml of phenolphthalein solution and 13.5M ammonia dropwise
27-63
until a pink colour is produced. Cool, add glacial acetic acid until the solution is decolorised and add
a further 0.5 ml, filter and dilute the solution to 20 ml with water. 12 ml of the filtrate complies with
limit test A for heavy metals, Appendix VII. Use 1 ml of lead standard solution (10 ppm Pb) to prepare
Loss on drying When dried over phosphorus pentoxide at 80 at a pressure not exceeding 2 kPa for 4
Assay Dissolve 0.2 g in 50 ml of anhydrous acetic acid and carry out Method I for non-aqueous
titration, Appendix VIII A, determining the end point potentiometrically. Each ml of 0.1M perchloric
acid VS is equivalent to 47.36 mg of C24H33NO3,C2H2O4.
Storage Naftidrofuryl Oxalate should be kept in an airtight container.
Preparation
Naftidrofuryl Capsules
IMPURITIES
O
COOH
A. 3-(1-naphthyl)-2-tetrahydro-furfurylpropionic acid
O
COOEt
B. ethyl 3-(1-naphthyl)-2-tetrahydrofurfurylpropionate
NEt 2
O
, COOH
COOH
C. 2-diethylaminoethyl 3-(1-naphthyl)-2-(1-naphthylmethyl)propionate oxalate
27-64
Nalidixic Acid
Et
Me
COOH
O
C12H12N2O3
232.2
389-08-2
Nalidixic Acid complies with the requirements of the 3rd edition of the European Pharmacopoeia [0701]. These
Preparations
Nalidixic Acid Oral Suspension
Nalidixic Acid Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Nalidixic acid contains not less than 99.0 per cent and not more than the equivalent of 101.0 per
cent of 1-ethyl-7-methyl-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid, calculated with
CHARACTERS
An almost white or pale yellow, crystalline powder, practically insoluble in water, soluble in
methylene chloride, slightly soluble in acetone and in alcohol. It dissolves in dilute solutions of alkali
hydroxides.
It melts at about 230C.
IDENTIFICATION
A. Dissolve 12.5 mg in 0.1M sodium hydroxide and dilute to 50.0 ml with the same solvent. Dilute
2.0 ml of this solution to 100.0 ml with 0.1M sodium hydroxide. Examined between 230 nm and
350 nm (2.2.25), the solution shows two absorption maxima, at 258 nm and 334 nm. The ratio of
the absorbance measured at 258 nm to that measured at 334 nm is 2.2 to 2.4.
obtained with nalidixic acid CRS. Examine the substances prepared as discs.
chromatogram obtained with the test solution (b) is similar in position and size to the principal spot
in the chromatogram obtained with reference solution (a).
D. Dissolve 0.1 g in 2 ml of hydrochloric acid R. Add 0.5 ml of a 100 g/l solution of -naphthol R in
alcohol R. An orange-red colour develops.
TESTS
Absorbance Dissolve 1.50 g in methylene chloride R and dilute to 50.0 ml with the same solvent. The
absorbance (2.2.25), measured at 420 nm, is not greater than 0.10.
plate R.
Test solution (a). Dissolve 0.20 g of the substance to be examined in methylene chloride R and dilute to
Reference solution (a). Dissolve 20 mg of nalidixic acid CRS in methylene chloride R and dilute to 20 ml
Reference solution (b). Dilute 2 ml of test solution (b) to 10 ml with methylene chloride R.
Reference solution (c). Dilute to 1 ml of reference solution (b) to 10 ml with methylene chloride R.
Reference solution (d). Dilute 1 ml of reference solution (b) to 25 ml with methylene chloride R.
10 volumes of dilute ammonia R1, 20 volumes of methylene chloride R and 70 volumes of alcohol R.
27-65
Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with the test solution (a), apart from the principal spot, is not more intense than the
spot in the chromatogram obtained with reference solution (c) (0.1 per cent) and not more than one
such spot is more intense than the spot in the chromatogram obtained with reference solution (d)
(0.04 per cent).
100C to 105C.
ASSAY
Dissolve 0.150 g in 10 ml of methylene chloride R and add 30 ml of 2-propanol R and 10 ml of carbon
dioxide-free water R. Keep the titration vessel covered and pass nitrogen R through the solution
throughout the titration. Keep the temperature of the solution between 15C and 20C. Titrate with
0.1M ethanolic sodium hydroxide, determining the end-point potentiometrically (2.2.20) using a silver
silver chloride comparison electrode with a sleeve diaphragm or a capillary tip, filled with a saturated
solution of lithium chloride R in ethanol R, and a glass electrode as indicator electrode.
STORAGE
_______________________________________________________________________________________________________________________ Ph Eur
27-66
Naloxone Hydrochloride
O
,HCl
O
H
N
CH2
OH
O
C19H21NO4,HCl,2H2O
399.9
51481-60-8
Naloxone Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Naloxone Hydrochloride Dihydrate [0729]. These requirements are reproduced after the heading Definition
below.
Action and use Opioid antagonist.
Preparations
Naloxone Injection
Neonatal Naloxone Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Naloxone hydrochloride dihydrate contains not less than 98.0 per cent and not more than the
equivalent of 102.0 per cent of 4,5-epoxy-3,14-dihydroxy-17-(prop-2-enyl)morphinan-6-one
hydrochloride, calculated with reference to the anhydrous substance.
CHARACTERS
A white or almost white, crystalline powder, hygroscopic, freely soluble in water, soluble in alcohol,
practically insoluble in toluene.
IDENTIFICATION
A. Examine by infrared absorption spectrophotometry (2.2.24) comparing with the spectrum
obtained with naloxone hydrochloride dihydrate CRS.
B. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel G plate R.
Test solution. Dissolve 8 mg of the substance to be examined in 0.5 ml of water R and dilute to 1 ml
with methanol R.
Reference solution. Dissolve 8 mg of naloxone hydrochloride dihydrate CRS in 0.5 ml of water R and
Apply to the plate 5 l of each solution. Develop protected from light over a path of 10 cm using a
mixture of 5 volumes of methanol R and 95 volumes of the upper layer from a mixture of 60 ml of
dilute ammonia R2 and 100 ml of butanol R. Dry the plate in a current of air. Spray with a freshly
prepared 5 g/l solution of potassium ferricyanide R in ferric chloride solution R1. Examine in daylight.
The principal spot in the chromatogram obtained with the test solution is similar in position, colour
and size to the principal spot in the chromatogram obtained with the reference solution.
TESTS
Acidity or alkalinity To 10.0 ml of solution S add 0.05 ml of methyl red solution R. Not more than
indicator.
Test solution. Dissolve 0.125 g of the substance to be examined in 0.1M hydrochloric acid and dilute to
25.0 ml with the same acid.
27-67
Reference solution (a). Dissolve 10.0 mg of naloxone hydrochloride dihydrate CRS and 10.0 mg of
naloxone impurity A CRS in 0.1M hydrochloric acid and dilute to 10.0 ml with the same acid. Dilute
1.0 ml to 100.0 ml with 0.1M hydrochloric acid.
Reference solution (b). Dilute 1.0 ml of the test solution to 20.0 ml with 0.1M hydrochloric acid. Dilute
1.0 ml to 10.0 ml with 0.1M hydrochloric acid.
a stainless steel column 0.125 m long and 4.0 mm in internal diameter packed with end-capped
octylsilyl silica gel for chromatography R (5 m),
as mobile phase at a flow rate of 1.5 ml/min a linear gradient programme using the following
conditions:
Mobile phase A. Mix 20 ml of acetonitrile R, 40 ml of tetrahydrofuran R and 940 ml of a solution
prepared as follows: dissolve 1.17 g of sodium octanesulphonate R in 1000 ml of water R, adjust the
pH to 2.0 with a 50 per cent V/V solution of phosphoric acid R and filter (octanesulphonic acid
solution).
Mobile phase B. Mix 170 ml of acetonitrile R, 40 ml of tetrahydrofuran R and 790 ml of the
octanesulphonic acid solution.
Time
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
(min)
0 40
40 50
1000
0
0100
100
linear gradient
isocratic
as detector a spectrophotometer set at 230 nm, maintaining the temperature of the column at
40C.
Inject separately 20 l of each solution.
The test is not valid unless: the peak corresponding to naloxone in the chromatogram obtained with
reference solution (a) has a signal-to-noise ratio of at least ten; in the chromatogram obtained with
reference solution (a), the resolution between the peaks corresponding to impurity A and naloxone is
not less than four. If necessary, adjust the chromatographic conditions.
When the chromatograms are recorded in the prescribed conditions, the retention time of naloxone
is about 11 min and the relative retention time of impurity A is about 0.8 with respect to naloxone.
solution (b) (0.5 per cent); the sum of the areas of all the peaks, apart from the principal peak, is not
greater than twice the area of the principal peak in the chromatogram obtained with reference solution
(b) (1 per cent). Disregard any peak with an area less than 0.1 times that of the principal peak in the
ASSAY
Dissolve 0.300 g in 50 ml of alcohol R and add 5.0 ml of 0.01M hydrochloric acid. Carry out a potentiometric titration (2.2.20), using 0.1M ethanolic sodium hydroxide. Read the volume added between the
1 ml of 0.1M ethanolic sodium hydroxide is equivalent to 36.38 mg of C19H22ClNO4.
STORAGE
IMPURITIES
R1
R2
O
H
N
OH R3
A. R1 = R2 = R3 = H: 4,5-epoxy-3,14-dihydroxymorphinan-6-one (noroxymorphone),
B. R1 = R3 = CH2-CH = CH2, R2 = H: 4,5-epoxy-14-hydroxy-17-(prop-2-enyl)-3-(prop-2enyloxy)morphinan-6-one (3-O-allylnaloxone),
C. R1 = H, R2 = OH, R3 = CH2CH = CH2: 4,5-epoxy-3,10,14-trihydroxy-17-(prop-2enyl)morphinan-6-one (10-hydroxynaloxone),
27-68
O
O
N
OH
CH2
D. 7,8-didehydro-4,5-epoxy-3,14-dihydroxy-17-(prop-2-enyl)morphinan-6-one
(7,8-didehydronaloxone),
H2C
N
HO
CH2
N
OH
OH HO
E. 4,5:4,5-diepoxy-3,3,14,14-tetrahydroxy-17,17-bis(prop-2-enyl)-2,2-bimorphinanyl6,6-dione (2,2-bisnaloxone).
__________________________________________________________________________________________________________ Ph Eur
27-69
Nandrolone Decanoate
Me H
OCO[CH2]8CH3
H
H
H
O
C28H44O3
428.7
360-70-3
Definition Nandrolone Decanoate is 3-oxo-estr-4-en-17-yl decanoate. It contains not less than

97.0% and not more than 103.0% of C28H44O3, calculated with reference to the dried substance.
Characteristics A white to creamy white, crystalline powder; odour, faint and characteristic.
Practically insoluble in water; freely soluble in chloroform, in ethanol (96%), in ether, in fixed oils and
in esters.
Identification
nandrolone decanoate (RS 242).
B. Carry out the method for thin-layer chromatography, Appendix III A, using a silica gel F254
precoated plate the surface of which has been modified by chemically bonded octadecylsilyl groups
(Whatman KC 18F plates are suitable) and a mixture of 60 volumes of propan-2-ol, 40 volumes of
acetonitrile and 20 volumes of water as the mobile phase. Apply separately to the plate 5 l of each of
three solutions in chloroform containing (1) 0.5% w/v of the substance being examined, (2) 0.5% w/v
of nandrolone decanoate BPCRS and (3) equal volumes of solutions (1) and (2). After removal of the
plate, allow it to dry in air until the solvent has evaporated and heat at 100 for 10 minutes. Allow to
cool and examine under ultraviolet light (254 nm). The principal spot in the chromatogram obtained
with solution (1) corresponds to that in the chromatogram obtained with solution (2). The principal
spot in the chromatogram obtained with solution (3) appears as a single, compact spot.
C. Melting point, about 35, Appendix V A.
Specific optical rotation In a 2% w/v solution in 1,4-dioxan, +32 to +36, calculated with reference to the dried substance, Appendix V A.
gel GF254 as the coating substance and a mixture of 70 volumes of heptane and 30 volumes of acetone
as the mobile phase. Apply separately to the plate 5 l of each of three solutions in chloroform
containing (1) 1.0% w/v of the substance being examined, (2) 0.0050% w/v of the substance being
examined and (3) 0.010% w/v of nandrolone BPCRS. After removal of the plate, allow it to dry in air
and examine under ultraviolet light (254 nm). In the chromatogram obtained with solution (1) any
spot corresponding to nandrolone is not more intense than the spot in the chromatogram obtained
with solution (3) and any other secondary spot is not more intense than the spot in the chromatogram
maximum at 240 nm.
Storage Nandrolone Decanoate should be stored under nitrogen at a temperature of 2 to 8 and
protected from light.
Action and use Anabolic steroid.
Preparation
Nandrolone Decanoate Injection
27-70
Nandrolone Phenylpropionate
Me H
OCOCH2CH2Ph
H
H
H
O
C27H34O3
406.6
62-90-8
Definition Nandrolone Phenylpropionate is 3-oxo-estr-4-en-17-yl 3-phenylpropionate. It

contains not less than 97.0% and not more than 103.0% of C27H34O3, calculated with reference to
Characteristics A white to creamy white, crystalline powder; odour, characteristic.
Practically insoluble in water; soluble in ethanol (96%).
Identification
nandrolone phenylpropionate (RS 243).
B. Carry out the method for thin-layer chromatography, Appendix III A, using a silica gel F254
precoated plate the surface of which has been modified by chemically-bonded octadecylsilyl groups
(Whatman KC 18F plates are suitable) and a mixture of 60 volumes of propan-2-ol, 40 volumes of
three solutions in chloroform containing (1) 0.5% w/v of the substance being examined, (2) 0.5% w/v
of nandrolone phenylpropionate BPCRS and (3) equal volumes of solutions (1) and (2). After removal
of the plate, allow it to dry in air until the solvent has evaporated and heat at 100 for 10 minutes.
Allow to cool and examine under ultraviolet light (254 nm). The principal spot in the chromatogram
obtained with solution (1) corresponds to that in the chromatogram obtained with solution (2). The
principal spot in the chromatogram obtained with solution (3) appears as a single, compact spot.
Specific optical rotation In a 1% w/v solution in 1,4-dioxan, +48 to +51, calculated with reference to the dried substance, Appendix V F.
gel GF254 as the coating substance and a mixture of 70 volumes of heptane and 30 volumes of acetone
as the mobile phase. Apply separately to the plate 5 l of each of three solutions in chloroform
containing (1) 1.0% w/v of the substance being examined, (2) 0.0050% w/v of the substance being
examined and (3) 0.010% w/v of nandrolone BPCRS. After removal of the plate, allow it to dry in air
and examine under ultraviolet light (254 nm). In the chromatogram obtained with solution (1) any
spot corresponding to nandrolone is not more intense than the spot in the chromatogram obtained
with solution (3) and any other secondary spot is not more intense than the spot in the chromatogram
maximum at 240 nm.
Storage Nandrolone Phenylpropionate should be protected from light.
Preparation
Nandrolone Phenylpropionate Injection
27-71
Naphazoline Hydrochloride
N
N
H
C14H14N2,HCl
,HCl
246.7
550-99-2
Naphazoline Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Naphazoline hydrochloride contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of 2-(naphthalen-1-ylmethyl)-4,5-dihydro-1H-imidazole hydrochloride, calculated
CHARACTERS
A white or almost white, crystalline powder, freely soluble in water, soluble in alcohol.
IDENTIFICATION
350 nm (2.2.25), the solution shows four absorption maxima, at 270 nm, 280 nm, 287 nm and
291 nm. The specific absorbances at these maxima are 230 to 245, 265 to 290, 190 to 200 and 180
to 195, respectively.
obtained with naphazoline hydrochloride CRS. Examine the substances prepared as discs.
C. Dissolve about 0.5 mg in 1 ml of methanol R and add 0.5 ml of a freshly prepared 50 g/l solution
of sodium nitroprusside R and 0.5 ml of a 20 g/l solution of sodium hydroxide R. Allow to stand for
10 min and add 1 ml of an 80 g/l solution of sodium hydrogen carbonate R. A violet colour develops.
TESTS
Acidity or alkalinity To 20 ml of solution S add 0.2 ml of 0.01M sodium hydroxide and 0.1 ml of
methyl red solution R. The solution is yellow. Not more than 0.6 ml of 0.01M hydrochloric acid is
required to change the colour of the solution to red.
Naphthylacetylethylenediamine Examine by thin-layer chromatography (2.2.27), using a TLC
silica gel G plate R.
Reference solution. Dissolve 40 mg of naphazoline hydrochloride CRS in 1 ml of methanol R (solution A).
Dissolve separately 2 mg of naphthylacetylethylenediamine CRS in 10 ml of methanol R (solution B).
Mix 0.5 ml of solution A and 0.5 ml of solution B.
1.5 volumes of concentrated ammonia R and 100 volumes of methanol R. Dry the plate at 100C to
105C for 5 min, spray with a 5 g/l solution of ninhydrin R in methanol R and heat at 100C to 105C
for 10 min. Any spot corresponding to naphthylacetylethylenediamine in the chromatogram obtained
with the test solution is not more intense than the corresponding spot in the chromatogram obtained
with the reference solution (0.5 per cent). The test is not valid unless the chromatogram obtained
with the reference solution shows two clearly separated principal spots.
27-72
100C to 105C.
ASSAY
potentiometric titration (2.2.20), using 0.1M sodium hydroxide. Read the volume of 0.1M sodium
hydroxide added between the two points of inflexion.
1 ml of 0.1M sodium hydroxide is equivalent to 24.67 mg of C14H15ClN2.
STORAGE
_______________________________________________________________________________________________________________________ Ph Eur
27-73
Naphazoline Nitrate
C14H14N2,HNO3
273.3
5144-52-5
Naphazoline Nitrate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0147].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Naphazoline nitrate contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of 2-(naphthalen-1-ylmethyl)-4,5-dihydro-1H-imidazole nitrate, calculated with
CHARACTERS
A white or almost white, crystalline powder, sparingly soluble in water, soluble in alcohol.
IDENTIFICATION
B. Dissolve 50 mg in 0.01M hydrochloric acid and dilute to 250.0 ml with the same acid. Dilute
350 nm (2.2.25), the solution shows four absorption maxima, at 270 nm, 280 nm, 287 nm and
291 nm. The specific absorbances at these maxima are about 215, 250, 175 and 170 respectively.
obtained with naphazoline nitrate CRS.
D. Dissolve about 0.5 mg in 1 ml of methanol R and add 0.5 ml of a freshly prepared 50 g/l solution
10 min and add 1 ml of an 80 g/l solution of sodium hydrogen carbonate R. A violet colour develops.
E. Dissolve about 10 mg in 5 ml of water R. Add 0.2 g of magnesium oxide R. Shake mechanically for
30 min, add 10 ml of chloroform R and shake vigorously. Allow to stand and separate, filter and
evaporate the aqueous layer to dryness. The residue gives the reaction of nitrates (2.3.1).
TESTS
Solution S Dissolve 0.5 g in carbon dioxide-free water R, warming gently, and dilute to 50 ml with the
same solvent.
Naphthylacetylethylenediamine Examine by thin-layer chromatography (2.2.27), using a TLC
silica gel G plate R.
Reference solution. Dissolve 40 mg of naphazoline nitrate CRS in 1 ml of methanol R (solution A).
Dissolve separately 2 mg of naphthylacetylethylenediamine CRS in 10 ml of methanol R (solution B).
Mix 0.5 ml of solution A and 0.5 ml of solution B.
1.5 volumes of concentrated ammonia R and 100 volumes of methanol R. Dry the plate at 100C to
105C for 5 min, spray with a 5 g/l solution of ninhydrin R in methanol R and heat at 100C to 105C
for 10 min. Any spot corresponding to naphthylacetylethylenediamine in the chromatogram obtained
with the test solution is not more intense than the corresponding spot in the chromatogram obtained
with the reference solution (0.5 per cent). The test is not valid unless the chromatogram obtained
with the reference solution shows two clearly separated spots.
Chlorides (2.4.4). 15 ml of solution S complies with the limit test for chlorides (330 ppm).
100C to 105C.
ASSAY
27-74
STORAGE
_______________________________________________________________________________________________________________________ Ph Eur
27-75
Naproxen
H
Me
COOH
MeO
C14H14O3
230.3
22204-63-1
Naproxen complies with the requirements of the 3rd edition of the European Pharmacopoeia [0731]. These
Preparations
Naproxen Oral Suspension
Naproxen Suppositories
Naproxen Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Naproxen contains not less than 98.5 per cent and not more than the equivalent of 100.5 per cent of
(S)-2-(6-methoxy-naphth-2-yl)propionic acid, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, practically insoluble in water, soluble in alcohol and in
methanol, sparingly soluble in ether.
IDENTIFICATION
Second identification: A, B, C, E.
C. Dissolve 40.0 mg in methanol R and dilute to 100.0 ml with the same solvent. Dilute 10.0 ml of
the solution to 100.0 ml with methanol R. Examined between 230 nm and 350 nm (2.2.25), the
solution shows four absorption maxima, at 262 nm, 271 nm, 316 nm and 331 nm. The specific
absorbances at the maxima are 216 to 238, 219 to 241, 61 to 69 and 79 to 87, respectively.
D. Examine by infrared absorption spectrophotometry (2.2.24) comparing with the spectrum
obtained with naproxen CRS. Examine the substances prepared as discs, using potassium bromide R.
E. Dissolve about 2 mg in 2 ml of sulphuric acid R. The solution is yellow. Add 50 mg of chloral
hydrate R and shake to dissolve. The solution becomes orange and then orange-red.
TESTS
2.2.2).
Specific optical rotation (2.2.7). Dissolve 0.500 g in chloroform R and dilute to 25.0 ml with the
same solvent. The specific optical rotation is +63 to +68.5, calculated with reference to the dried
substance.
coating substance.
the same solvent.
Reference solution. Dilute 0.5 ml of the test solution to 100 ml with methanol R.
3 volumes of glacial acetic acid R, 9 volumes of tetrahydrofuran R and 90 volumes of toluene R. Allow
the plate to dry in air and examine in ultraviolet light at 254 nm. Any spot in the chromatogram
27-76
100C to 105C for 3 h.
ASSAY
Dissolve 0.200 g in a mixture of 25 ml of water R and 75 ml of methanol R. Titrate with 0.1M sodium
hydroxide, using 1 ml of phenolphthalein solution R as indicator.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
27-77
Neohesperidin-Dihydrochalcone
1/01
OH
O
OH
OH
O
OH
OMe
OH
HO
O
OH
O
Me
OH
C28H36O15
OH
613
20702-77-6
Neohesperidin-Dihydrochalcone complies with the requirements of the 3rd edition of the European
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
1-[4-[[2-O-(6-Deoxy--L-mannopyranosyl)--D-glucopyranosyl]oxy]-2,6-dihydroxyphenyl]-3-(3hydroxy-4-methoxyphenyl)propan-1-one.
Content: 96.0 per cent to 101.0 per cent (anhydrous substance).
CHARACTERS
Appearance: white or yellowish-white powder.
Solubility: practically insoluble in water, freely soluble in dimethyl sulphoxide, soluble in methanol,
practically insoluble in methylene chloride.
IDENTIFICATION
A. Infrared absorption spectrophotometry (2.2.24).
Comparison: neohesperidin-dihydrochalcone CRS.
B. Examine the chromatograms obtained in the assay.
Results: the principal peak in the chromatogram obtained with test solution (b) is similar in retention
time and size to the principal peak in the chromatogram obtained with reference solution (a).
TESTS
Appearance of solution The solution is clear (2.2.1) and not more intensely coloured than
reference solution Y4 (2.2.2, Method II).
Dissolve 0.25 g in methanol R and dilute to 25 ml with the same solvent.
Related substances Liquid chromatography (2.2.29).
Test solution (a). Dissolve 0.10 g of the substance to be examined in dimethyl sulphoxide R and dilute
Test solution (b). Dilute 10.0 ml of test solution (a) to 20.0 ml with dimethyl sulphoxide R.
Reference solution (a). Dissolve 50.0 mg of neohesperidin-dihydrochalcone CRS in dimethyl sulphoxide R
Reference solution (b). Dissolve 4.0 mg of neohesperidin-dihydrochalcone impurity B CRS in dimethyl
sulphoxide R and dilute to 100.0 ml with the same solvent.
Reference solution (c). Dilute 1.0 ml of test solution (a) to 100.0 ml with dimethyl sulphoxide R.
Reference solution (d). In order to prepare in situ impurity F and impurity G, suspend 0.10 g of the
substance to be examined in 10.0 ml of a 100 g/l solution of sulphuric acid R. Heat the sample for
5 min on a water-bath. Dilute immediately 1.0 ml of the resulting solution to 50.0 ml with dimethyl
sulphoxide R.
Column:
size: l = 0.15 m, = 3.9 mm,
27-78
stationary phase: spherical octadecylsilyl silica gel for chromatography R (4 m) with a carbon
loading of 7 per cent,
temperature: 30C.
Mobile phase: mix 20 volumes of acetonitrile R and 80 volumes of a solution prepared by adding
5.0 ml of glacial acetic acid R to 1000.0 ml of water R.
Flow rate: 1.0 ml/min.
Detection: spectrophotometer at 282 nm.
Injection: 10 l; inject test solution (a) and reference solutions (a), (b), (c) and (d).
Run time: 5 times the retention time of neohesperidin-dihydrochalcone which is about 10 min.
Relative retention with reference to neohesperidin-dihydrochalcone: impurity B = about 0.4; impurity
D = about 0.7; impurity F = about 1.2; impurity G = about 3.7.
System suitability:
resolution: minimum of 2.5 between the first peak (neohesperidin-dihydrochalcone) and the
second peak (impurity F) in the chromatogram obtained with reference solution (d),
chromatogram obtained with reference solution (a) is similar to the chromatogram provided with
neohesperidin-dihydrochalcone CRS.
Limits:
impurity B: not more than the area of the principal peak in the chromatogram obtained with
reference solution (b) (2 per cent),
impurity D: not more than twice the area of the principal peak in the chromatogram obtained
with reference solution (c) (2 per cent),
any other impurity: not more than 0.5 times the area of the principal peak in the chromatogram
obtained with reference solution (c) (0.5 per cent),
total of all impurities apart from impurity B: not more than 2.5 times the area of the principal peak
in the chromatogram obtained with reference solution (c) (2.5 per cent),
disregard limit: 0.05 times the area of the principal peak in the chromatogram obtained with
reference solution (c) (0.05 per cent).
Heavy metals (2.4.8): maximum 10 ppm.
2.0 g complies with limit test D. Prepare the standard using 2 ml of lead standard solution (10 ppm
Pb) R.
Water (2.5.12): maximum 12.0 per cent, determined on 0.200 g.
Sulphated ash (2.4.14): maximum 0.2 per cent, determined on 1.0 g.
ASSAY
Liquid chromatography (2.2.29) as described in the test for related substances.
Injection: 10 l; inject test solution (b) and reference solutions (a) and (d).
System suitability:
resolution: minimum of 2.5 between the first peak (neohesperidin-dihydrochalcone) and the
second peak (impurity F) in the chromatogram obtained with reference solution (d),
repeatability: reference solution (a).
Calculate the percentage content of C28H36O15 using the chromatogram obtained with reference
solution (a) and the stated content of C28H36O15 in neohesperidin-dihydrochalcone CRS, correcting for
the water content of the substance to be examined.
STORAGE
Protected from light.
IMPURITIES
OH
Rh =
Me
OH OH
OH
OH
O O
O
CH3
OH
OH
O
ORh
A. 1-[4-[[2-O-(6-deoxy--L-mannopyranosyl--D-glucopyranosyl]oxy]-2,6dihydroxyphenyl]ethanone (phloroacetophenone neohesperidoside),
27-79
OH
OH
OH
O O
OH
OMe
HO
ORh
B. 7-[[2-O-(6-deoxy--L-mannopyranosyl)--D-glucopyranosyl]oxy]-5-hydroxy-2-(3hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one (neodiosmin),
OH
OH
O O
OH
OH
and enantiomer
OMe
HO
ORh
C. (2RS)-7-[[2-O-(6-deoxy--L-mannopyranosyl)--D-glucopyranosyl]oxy]-5-hydroxy-2-(3hydroxy-4-methoxyphenyl)-2,3-dihydro-4H-1-benzopyran-4-one (neohesperidin),
OH
O
OH
OH
O O
OH
OH
O
ORh
D. 1-[4-[[2-O-(6-deoxy--L-mannopyranosyl)--D-glucopyranosyl]oxy]-2,6dihydroxyphenyl]-3-(4-hydroxyphenyl)propan-1-one (naringin-dihydrochalcone),
OH
O
OH
OX
O O
OH
OMe
OH
O
OH
E. X = Rh: 1-[4-[[6-O-(6-deoxy--L-mannopyranosyl)--D-glucopyranosyl]oxy]-2,6dihydroxyphenyl]-3-(3-hydroxy-4-methoxyphenyl)propan-1-one (hesperidin-dihydrochalcone),

F. X = H: 1-[4-(-D-glucopyranosyloxy)-2,6-dihydroxyphenyl]-3-(3-hydroxy-4methoxyphenyl)propan-1-one (hesperetin-dihydrochalcone 7-glucoside),
OH
O
OH
HO
OH
OMe
G. 3-(3-hydroxy-4-methoxyphenyl)-1-(2,4,6-trihydroxyphenyl)propan-1-one (hesperetindihydrochalcone).
__________________________________________________________________________________________________________ Ph Eur
27-80
Neomycin Sulphate
corrected 1/01
CH2NH 2
O
OH
HO
H 2N
OH H2N
O
,xH2SO 4
H2N
O
HOCH2 O
O
CH2NH2
OH
HO
O
NH2
OH
neomycin B
C23H46N6O13,xH2SO4
615 (base)
1405-10-3
Neomycin Sulphate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0197].
Preparations
Hydrocortisone and Neomycin Cream
Hydrocortisone Acetate and Neomycin Ear Drops
Hydrocortisone Acetate and Neomycin Eye Drops
Neomycin Eye Drops
Hydrocortisone Acetate and Neomycin Eye Ointment
Neomycin Eye Ointment
Neomycin Oral Solution
Neomycin Tablets
When neomycin is prescribed, Neomycin Sulphate shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Neomycin sulphate is a mixture of the sulphates of substances produced by the growth of certain
selected strains of Streptomyces fradiae, the main component being the sulphate of 4-O-(2,6-diamino2,6-dideoxy--D-glucopyranosyl)-5-O-[3-O-(2,6-diamino-2,6-dideoxy--L-idopyranosyl)--Dribofuranosyl]-2-deoxy-D-streptamine (neomycin B). The potency is not less than 680 I.U. per
milligram, calculated with reference to the dried substance.
CHARACTERS
A white or yellowish-white powder, hygroscopic, very soluble in water, very slightly soluble in
alcohol, practically insoluble in acetone.
IDENTIFICATION
A. It complies with the test for neomycin C (see Tests).
B. It gives reaction (a) of sulphates (2.3.1).
TESTS
Specific optical rotation (2.2.7). Dissolve 1.00 g in water R and dilute to 10.0 ml with the same
substance.
27-81
Neamine Examine by thin-layer chromatography (2.2.27), using silica gel H R as the coating
substance.
the same solvent.
Reference solution (a). Dissolve 0.5 mg of neamine CRS in 1.0 ml of water R.
Reference solution (b). Mix 0.5 ml of the test solution with 0.5 ml of reference solution (a).
Apply separately to the plate as 5 mm bands 5 l of each solution. Dry the bands. Develop over a
path of at least 8 cm using a mixture of 10 volumes of methylene chloride R, 20 volumes of concentrated
ammonia R and 30 volumes of methanol R. Dry the plate at 100C to 105C for 10 min. Spray with
ninhydrin and stannous chloride reagent R and heat the plate at 110C for 15 min. Spray the plate again
with the same reagent and heat at 110C for 15 min. Any spot corresponding to neamine in the
chromatogram obtained with the test solution is not more intense than the spot in the chromatogram
obtained with reference solution (a) (2 per cent). The test is not valid unless the chromatogram
obtained with reference solution (b) shows two clearly separated principal spots.
Neomycin C Examine by thin-layer chromatography (2.2.27), using a suitable silica gel as the
coating substance.
Test solution. Dissolve 40 mg of the substance to be examined in water R and dilute to 5.0 ml with the
same solvent.
Reference solution (a). Dissolve 30 mg of framycetin sulphate CRS in water R and dilute to 25.0 ml with
the same solvent.
Reference solution (b). Dilute 5.0 ml of reference solution (a) to 25.0 ml with water R.
Reference solution (c). Dissolve 40 mg of neomycin sulphate CRS in water R and dilute to 5.0 ml with
the same solvent.
Apply separately to the plate as 5 mm bands 5 l of each solution. Develop over a path of at least
12 cm using a mixture of 20 volumes of methanol R and 80 volumes of a 200 g/l solution of sodium
chloride R. Dry the plate at 100C to 105C for 10 min. Spray with ninhydrin solution R1 and heat the
plate at 100C to 105C for 10 min. In the chromatogram obtained with the test solution: the
principal spot is similar in position, colour and size to the principal spot in the chromatogram
obtained with reference solution (c); the spot (neomycin C) with an Rf value slightly less than that of
the principal spot is not more intense than the spot obtained with reference solution (a) (15 per cent)
but is more intense than the spot in the chromatogram obtained with reference solution (b) (3 per
cent). The test is not valid unless in the chromatogram obtained with reference solution (c) a spot
appears with an Rf value slightly less than that of the principal spot.
Sulphate 27.0 per cent to 31.0 per cent of sulphate (SO4), calculated with reference to the dried
substance. Dissolve 0.250 g in 100 ml of water R and adjust the solution to pH 11 using concentrated
ammonia R. Add 10.0 ml of 0.1M barium chloride and about 0.5 mg of phthalein purple R. Titrate with
0.1M sodium edetate adding 50 ml of alcohol R when the colour of the solution begins to change,
continuing the titration until the violet-blue colour disappears.
1 ml of 0.1M barium chloride is equivalent to 9.606 mg of sulphate (SO4).
Loss on drying (2.2.32). Not more than 8.0 per cent, determined on 1.00 g by drying at 60C over
diphosphorus pentoxide R at a pressure not exceeding 0.7 kPa for 3 h.
ASSAY
Carry out the microbiological assay of antibiotics (2.7.2). Use neomycin sulphate for microbiological
assay CRS as the reference substance.
STORAGE
IMPURITIES
CH2NH2
O
OH
HO
H2N O
OH
H2N
CH2NH2
OH
HO
NH2 O
A. neomycin C,
NH 2
HOCH2 O
OH
27-82
CH2NH2
O
OH
HO
H2N O
OH
H2N
H2N
OH
B. neamine.
__________________________________________________________________________________________________________ Ph Eur
28-1
Neostigmine Bromide
+
NMe3
Br
OCONMe 2
C12H19BrN2O2
303.2
114-80-7
Neostigmine Bromide complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Anticholinesterase.
Preparation
Neostigmine Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Neostigmine bromide contains not less than 98.5 per cent and not more than the equivalent of
101.0 per cent of (3-dimethylcarbamoyloxyphenyl)trimethylammonium bromide, calculated with
CHARACTERS
A white, crystalline powder or colourless crystals, hygroscopic, very soluble in water, freely soluble in
alcohol.
IDENTIFICATION
A. Dissolve 20 mg in 0.5M sulphuric acid and dilute to 100 ml with the same acid. Examined between
230 nm and 350 nm, the solution shows two absorption maxima (2.2.25), at 260 nm and 266 nm.
The specific absorbances at the maxima are about 16 and about 14, respectively.
obtained with neostigmine bromide CRS.
C. Heat about 50 mg with a mixture of 0.4 g of potassium hydroxide R and 2 ml of alcohol R on a
water-bath for 3 min, replacing the evaporated alcohol. Cool and add 2 ml of water R and 2 ml of
diazobenzenesulphonic acid solution R1. An orange-red colour develops.
D. It gives the reactions of bromides (2.3.1).
TESTS
(3-Hydroxyphenyl)trimethylammonium bromide Dissolve 50 mg in a mixture of 1 ml of sodium
carbonate solution R and 9 ml of water R. The absorbance (2.2.25) measured immediately at 294 nm is
not greater than 0.25.
100C to 105C.
ASSAY
Dissolve 0.225 g in 2 ml of formic acid R. Add 50 ml of acetic anhydride R. Titrate with 0.1M perchloric
acid determining the end-point potentiometrically (2.2.20).
1 ml of 0.1M perchloric acid is equivalent to 30.32 mg of C12H19BrN2O2.
STORAGE
IMPURITIES
A. (3-hydroxyphenyl)trimethylammonium bromide.
__________________________________________________________________________________________________________ Ph Eur
28-2
Neostigmine Metilsulfate
+
NMe3
CH3SO4
OCONMe2
C13H22N2O6S
334.4
51-60-5
Neostigmine Metilsulfate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Neostigmine Injection
When neostigmine methylsulphate is prescribed or demanded, Neostigmine Metilsulfate shall be
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Neostigmine metilsulfate contains not less than 98.5 per cent and not more than the equivalent of
101.0 per cent of (3-dimethylcarbamoylphenyl)trimethylammonium methyl sulphate, calculated with
CHARACTERS
alcohol, practically insoluble in ether.
IDENTIFICATION
B. Dissolve 50 mg in 0.5M sulphuric acid and dilute to 100.0 ml with the same acid. Examined
267 nm. The ratio of the absorbance at the maximum at 267 nm to that at the maximum at 261 nm
is 0.84 to 0.87. The identification is not valid unless in the test for resolution (2.2.25) the ratio of the
absorbances is not less than 1.9.
C. Examine by infrared absorption spectrophotometry (2.2.24) comparing with the spectrum
obtained with neostigmine metilsulfate CRS. Examine the substances prepared as discs.
D. To 50 mg add 0.4 g of potassium hydroxide R and 2 ml of alcohol R and heat on a water-bath for
3 min, replacing the evaporated alcohol. Cool and add 2 ml of water R and 2 ml of
diazobenzenesulphonic acid solution R1. An orange-red colour develops.
E. Dissolve 0.1 g in 5 ml of distilled water R and add 1 ml of barium chloride solution R1. No precipitate
is formed. Add 2 ml of hydrochloric acid R and heat in a water-bath for 10 min. A fine, white
precipitate is formed.
TESTS
Acidity or alkalinity To 4.0 ml of solution S add 6.0 ml of water R and 0.1 ml of phenolphthalein
solution R1. The solution is colourless. Add 0.3 ml of 0.01M sodium hydroxide; the solution becomes
red. Add 0.4 ml of 0.01M hydrochloric acid; the solution becomes colourless. Add 0.1 ml of methyl red
solution R; the solution becomes red or yellowish-red.
(3-Hydroxyphenyl)trimethylammonium methyl sulphate Dissolve 50 mg in a mixture of 1 ml
of sodium carbonate solution R and 9 ml of water R. The absorbance (2.2.25) measured immediately at
294 nm is not greater than 0.20.
100C to 105C.
28-3
ASSAY
Dissolve 0.300 g in 150 ml of water R and add 100 ml of dilute sodium hydroxide solution R. Distil
collecting the distillate in 40 ml of a 40 g/l solution of boric acid R until the total volume in the
collecting vessel is about 250 ml. Titrate the solution in the collecting vessel with 0.1M hydrochloric
acid, using 0.25 ml of methyl red mixed solution R as indicator. Carry out a blank test.
1 ml of 0.1M hydrochloric acid is equivalent to 33.44 mg of C13H22N2O6S.
STORAGE
Store in a airtight container, protected from light.
__________________________________________________________________________________________________________ Ph Eur
28-4
Netilmicin Sulphate
corrected 1/01
HO
H 2N
O
NHMe
Me
OH
O
O
,5 H2SO4
O
H2N
OH
NHEt
NH2
C42H82N10O14,5H2SO4
1442
56391-57-2
Netilmicin Sulphate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Netilmicin sulphate is the sulphate of 4-O-(2,6-diamino-2,3,4,6-tetradeoxy--D-glycero-hex-4enopyranosyl)-1-N-ethyl-6-O-[4-C-methyl-3-(methylamino)-3-deoxy--L-arabino-pyranosyl]-2deoxy-D-streptamine, a substance obtained by synthesis from sisomicin. The potency is not less than
650 I.U. per milligram, calculated with reference to the dried substance.
CHARACTERS
A white or yellowish-white powder, very hygroscopic, very soluble in water, practically insoluble in
acetone and in alcohol.
IDENTIFICATION
A. Examine the chromatograms obtained in the test for related substances. The principal spot in the
B. It gives reaction (a) of sulphates (2.3.1).
TESTS
Appearance of solution Solution S is clear (2.2.1). The absorbance of solution S measured at
400 nm (2.2.25) is not greater than 0.08.
substance.
Related substances Examine by thin-layer chromatography (2.2.27), using a TLC silica gel plate R.
Test solution (a). Dissolve 0.30 g of the substance to be examined in water R and dilute to 2.0 ml with
the same solvent.
Test solution (b). Dilute 1.0 ml of test solution (a) to 50 ml with water R.
Reference solution (a). Dissolve 30 mg of netilmicin sulphate CRS in water R and dilute to 10 ml with
the same solvent.
Reference solution (c). Dissolve 36 mg of sisomicin sulphate CRS in water R and dilute to 25 ml with the
same solvent.
Reference solution (d). Dissolve 31 mg of 1-N-ethylgaramine sulphate CRS in water R and dilute to
Reference solution (e). Dissolve 20 mg of netilmicin sulphate CRS, 20 mg of sisomicin sulphate CRS and
20 mg of 1-N-ethylgaramine sulphate CRS in water R and dilute to 10 ml with the same solvent.
28-5
Apply separately to the plate 2 l of each solution and develop over a path of 10 cm using a mixture
of 20 volumes of concentrated ammonia R, 40 volumes of methanol R and 40 volumes of methylene
chloride R. Dry the plate in a current of warm air and spray with ninhydrin and stannous chloride
reagent R. Heat the plate at 110C for 20 min. In the chromatogram obtained with test solution (a):
any spot corresponding to sisomicin is not more intense than the spot in the chromatogram obtained
with reference solution (c) (1 per cent); any spot corresponding to 1-N-ethylgaramine is not more
intense than the spot in the chromatogram obtained with reference solution (d) (1 per cent); any spot
with an Rf higher than that of the principal spot is not more intense than the spot in the chromatogram obtained with reference solution (a) (2 per cent); no other secondary spot is more intense than
the spot in the chromatogram obtained with reference solution (b) (1 per cent). The test is not valid
unless the chromatogram obtained with reference solution (e) shows three clearly separated spots.
0.1M sodium edetate adding 50 ml of alcohol R when the colour of the solution begins to change and
continue the titration until the violet-blue colour disappears.
Loss on drying (2.2.32). Not more than 15.0 per cent, determined on 0.500 g by drying at 110C
under high vacuum for 3 h.
ASSAY
Carry out the microbiological assay of antibiotics (2.7.2), using the diffusion method.
STORAGE
LABELLING
The label states:
IMPURITIES
HO
O
NHMe
Me
H2N
HO
O
O
H2N
OH
NH2
NH2
A. 4-O-(2,6-diamino-2,3,4,6-tetradeoxy--d-glycero-hex-4-enopyranosyl)-6-O-[4-C-methyl-3(methylamino)-3-deoxy--L-arabino-pyranosyl]-2-deoxy-D-streptamine (sisomicin),
HO
O
NHMe
Me
HO
O
HO
OH
NHEt
NH2
B. 1-N-ethyl-6-O-[4-C-methyl-3-(methylamino)-3-deoxy--L-arabino-pyranosyl]-2-deoxy-Dstreptamine (1-N-ethylgaramine).
__________________________________________________________________________________________________________ Ph Eur
28-6
Anhydrous Niclosamide
Niclosamide
OH
NO2
O
N
H
Cl
C13H8Cl2N2O4
Cl
327.1
50-65-7
Anhydrous Niclosamide complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Niclosamide Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Anhydrous niclosamide contains not less than 98.0 per cent and not more than the equivalent of
101.0 per cent of 5-chloro-N-(2-chloro-4-nitrophenyl)-2-hydroxybenzamide, calculated with reference to the dried substance.
CHARACTERS
Yellowish-white or yellowish, fine crystals, practically insoluble in water, sparingly soluble in acetone,
slightly soluble in ethanol.
IDENTIFICATION
obtained with anhydrous niclosamide CRS. Examine as discs prepared using about 0.5 mg of substance
and 0.3 g of potassium bromide R.
C. To 50 mg add 5 ml of 1M hydrochloric acid and 0.1 g of zinc powder R, heat in a water-bath for
10 min, cool and filter. To the filtrate add 1 ml of a 5 g/l solution of sodium nitrite R and allow to
stand for 3 min; add 2 ml of a 20 g/l solution of ammonium sulphamate R, shake, allow to stand for
3 min and add 2 ml of a 5 g/l solution of naphthylethylenediamine dihydrochloride R. A violet colour is
produced.
D. Heat the substance on a copper wire in a non-luminous flame. The flame becomes green.
E. It complies with the test for loss on drying (see Tests).
TESTS
Test solution. Dissolve 50 mg of the substance to be examined in methanol R, heating gently, cool and
Reference solution. Dilute 1.0 ml of the test solution to 100.0 ml with acetonitrile R. Dilute 1.0 ml of
this solution to 20.0 ml with acetonitrile R.
as mobile phase at a flow-rate of 1.0 ml per minute, a mixture of equal volumes of acetonitrile R
and a solution containing 2 g/l of potassium dihydrogen phosphate R, 1 g/l of disodium hydrogen
phosphate R and 2 g/l of tetrabutylammonium hydrogen sulphate R,
Adjust the sensitivity so that the height of the peak corresponding to niclosamide in the chromatogram obtained with the reference solution is not less than 20 per cent of the full scale of the recorder.
Inject 20 l of each solution and record the chromatogram for twice the retention time of
niclosamide. In the chromatogram obtained with the test solution, the sum of the areas of the peaks,
28-7
apart from the peak corresponding to niclosamide and the peak due to the solvent, is not greater than
four times the area of the principal peak in the chromatogram obtained with the reference solution
(0.2 per cent). Disregard any peak with an area less than 10 per cent of the area of the peak corresponding to niclosamide in the chromatogram obtained with the reference solution.
5-Chlorosalicylic acid
Test solution. To 1.0 g of the substance to be examined add 15 ml of water R, boil for 2 min, cool,
filter through a membrane filter (nominal pore size: 0.45 m), wash the filter and dilute the
combined filtrate and washings to 20.0 ml with water R.
Reference solution. Dissolve 30 mg of 5-chlorosalicylic acid R in 20 ml of methanol R and dilute to
100.0 ml with water R. Dilute 1.0 ml of the solution to 100.0 ml with water R.
To 10.0 ml of the test solution and to 10.0 ml of the reference solution add separately 0.1 ml of ferric
chloride solution R2. Any violet colour in the test solution is not more intense than that in the reference
solution (60 ppm).
2-Chloro-4-nitroaniline
Test solution. To 0.250 g of the substance to be examined add 5 ml of methanol R, heat to boiling,
cool, add 45 ml of 1M hydrochloric acid, heat again to boiling, cool, filter and dilute the filtrate to
50.0 ml with 1M hydrochloric acid.
Reference solution. Dissolve 50 mg of 2-chloro-4-nitro-aniline R in methanol R and dilute to 100.0 ml
with the same solvent. Dilute 1.0 ml of the solution to 100.0 ml with methanol R. Dilute 2.0 ml of
this solution to 20.0 ml with 1M hydrochloric acid.
To 10.0 ml of the test solution and to 10.0 ml of the reference solution add separately 0.5 ml of a
5 g/l solution of sodium nitrite R and allow to stand for 3 min. Add 1 ml of a 20 g/l solution of
ammonium sulphamate R, shake, allow to stand for 3 min and add 1 ml of a 5 g/l solution of
naphthylethylenediamine dihydrochloride R. Any pinkish-violet colour in the test solution is not more
intense than that in the reference solution (100 ppm).
Chlorides (2.4.4). To 2 g add a mixture of 1.2 ml of acetic acid R and 40 ml of water R, boil for
2 min, cool and filter. 2 ml of the filtrate diluted to 15 ml with water R complies with the limit test for
100C to 105C for 4 h.
ASSAY
Dissolve 0.3000 g in 80 ml of a mixture of equal volumes of acetone R and methanol R. Titrate with
0.1M tetrabutylammonium hydroxide, determining the end-point potentiometrically (2.2.20).
STORAGE
__________________________________________________________________________________________________________ Ph Eur
28-8
Niclosamide Monohydrate
C13H8Cl2N2O4,H2O
345.1
50-65-7 (anhydrous)
Niclosamide Monohydrate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Niclosamide Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Niclosamide monohydrate contains not less than 98.0 per cent and not more than the equivalent of
101.0 per cent of 5-chloro-N-(2-chloro-4-nitrophenyl)-2-hydroxybenzamide, calculated with reference to the dried substance.
CHARACTERS
Yellowish, fine crystals, practically insoluble in water, sparingly soluble in acetone, slightly soluble in
ethanol.
IDENTIFICATION
A. Melting point (2.2.14): 227C to 232C, determined after drying at 100C to 105C for 4 h.
B. Dry the substance to be examined at 100C to 105C for 4 h. Examine by infrared absorption
spectrophotometry (2.2.24), comparing with the spectrum obtained with anhydrous niclosamide CRS.
Examine as discs prepared using about 0.5 mg of substance and 0.3 g of potassium bromide R.
C. To 50 mg add 5 ml of 1M hydrochloric acid and 0.1 g of zinc powder R, heat in a water-bath for
10 min, cool and filter. To the filtrate add 1 ml of a 5 g/l solution of sodium nitrite R and allow to
stand for 3 min; add 2 ml of a 20 g/l solution of ammonium sulphamate R, shake, allow to stand for
3 min and add 2 ml of a 5 g/l solution of naphthylethylenediamine dihydrochloride R. A violet colour is
produced.
D. Heat the substance on a copper wire in a non-luminous flame. The flame becomes green.
E. It complies with the test for loss on drying (see Tests).
TESTS
Test solution. Dissolve 50 mg of the substance to be examined in methanol R, heating gently, cool and
Reference solution. Dilute 1.0 ml of the test solution to 100.0 ml with acetonitrile R. Dilute 1.0 ml of
this solution to 20.0 ml with acetonitrile R.
a stainless steel column 0.125 m long and 4 mm in internal diameter, packed with octadecylsilyl
as mobile phase at a flow-rate of 1.0 ml per minute, a mixture of equal volumes of acetonitrile R
and a solution containing 2 g/l of potassium dihydrogen phosphate R, 1 g/l of disodium hydrogen
phosphate R and 2 g/l of tetrabutylammonium hydrogen sulphate R,
Adjust the sensitivity so that the height of the peak corresponding to niclosamide in the chromatogram obtained with the reference solution is not less than 20 per cent of full-scale of the recorder.
Inject 20 l of each solution and record the chromatogram for twice the retention time of
niclosamide. In the chromatogram obtained with the test solution, the sum of the areas of the peaks,
apart from the peak corresponding to niclosamide and the peak due to the solvent, is not greater than
four times the area of the principal peak in the chromatogram obtained with the reference solution
(0.2 per cent) Disregard any peak with an area less than 10 per cent of the area of the peak corresponding to niclosamide in the chromatogram obtained with the reference solution.
5-Chlorosalicylic acid
Test solution. To 1.0 g of the substance to be examined add 15 ml of water R, boil for 2 min, cool,
filter through a membrane filter (nominal pore size: 0.45 m), wash the filter and dilute the
combined filtrate and washings to 20.0 ml with water R.
Reference solution. Dissolve 30 mg of 5-chlorosalicylic acid R in 20 ml of methanol R and dilute to
100.0 ml with water R. Dilute 1.0 ml of the solution to 100.0 ml with water R.
28-9
To 10.0 ml of the test solution and to 10.0 ml of the reference solution add separately 0.1 ml of ferric
chloride solution R2. Any violet colour produced in the test solution is not more intense than that in
the reference solution (60 ppm).
2-Chloro-4-nitroaniline
Test solution. To 0.250 g of the substance to be examined add 5 ml of methanol R, heat to boiling,
cool, add 45 ml of 1M hydrochloric acid, heat again to boiling, cool, filter and dilute the filtrate to
50.0 ml with 1M hydrochloric acid.
Reference solution. Dissolve 50 mg of 2-chloro-4-nitroaniline R in methanol R and dilute to 100.0 ml
with the same solvent. Dilute 1.0 ml of the solution to 100.0 ml with methanol R. Dilute 2.0 ml of
this solution to 20.0 ml with 1M hydrochloric acid.
To 10.0 ml of the test solution and to 10.0 ml of the reference solution add separately 0.5 ml of a
5 g/l solution of sodium nitrite R and allow to stand for 3 min. Add 1 ml of a 20 g/l solution of
ammonium sulphamate R, shake, allow to stand for 3 min and add 1 ml of a 5 g/l solution of naphthylethylenediamine dihydrochloride R. Any pinkish-violet colour produced in the test solution is not more
intense than that in the reference solution (100 ppm).
Chlorides (2.4.4). To 2 g add a mixture of 1.2 ml of acetic acid R and 40 ml of water R, boil for
2 min, cool and filter. 2 ml of the filtrate diluted to 15 ml with water R complies with the limit test for
Loss on drying (2.2.32). 4.5 per cent to 6.0 per cent, determined on 1.000 g by drying in an oven at
100C to 105C for 4 h.
ASSAY
Dissolve 0.3000 g in 80 ml of a mixture of equal volumes of acetone R and methanol R. Titrate with
0.1M tetrabutylammonium hydroxide, determining the end-point potentiometrically (2.2.20).
STORAGE
__________________________________________________________________________________________________________ Ph Eur
28-10
Nicotinamide
N
CONH2
C6H6N2O
122.1
98-92-0
Nicotinamide complies with the requirements of the 3rd edition of the European Pharmacopoeia [0047].
Action and use Component of vitamin B.
Preparations
Nicotinamide Tablets
Vitamins B and C Injection
When niacinamide is prescribed or demanded, Nicotinamide shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Nicotinamide contains not less than 99.0 per cent and not more than the equivalent of 101.0 per
cent of pyridine-3-carboxamide, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder or colourless crystals, freely soluble in water and in ethanol.
IDENTIFICATION
obtained with nicotinamide CRS.
C. Boil 0.1 g with 1 ml of dilute sodium hydroxide solution R. Ammonia is evolved which is
recognisable by its odour.
D. Dilute 2 ml of solution S (see Tests) to 100 ml with water R. To 2 ml of the solution, add 2 ml of
cyanogen bromide solution R and 3 ml of a 25 g/l solution of aniline R and shake. A yellow colour
develops.
TESTS
plate R.
alcohol R and water R and dilute to 5.0 ml with the same mixture of solvents.
Reference solution. Dilute 0.5 ml of the test solution to 200 ml with a mixture of equal volumes of
alcohol R and water R.
volumes of water R, 45 volumes of ethanol R and 48 volumes of chloroform R. Allow the plate to dry
and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with the test
Heavy metals (2.4.8). Dilute 12 ml of solution S to 18 ml with water R. 12 ml of the solution
18 h.
28-11
ASSAY
Dissolve 0.250 g in 20 ml of anhydrous acetic acid R, heating slightly if necessary, and add 5 ml of
acetic anhydride R. Titrate with 0.1M perchloric acid, using crystal violet solution R as indicator until the
colour changes to greenish-blue.
__________________________________________________________________________________________________________ Ph Eur
28-12
Nicotine
N
H
Me
N
C10H14N2
162.2
54-11-5
Nicotine complies with the requirements of the 3rd edition of the European Pharmacopoeia [1452]. These
Action and use Aid to smoking cessation.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Nicotine contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of
3-[(2S)-1-methylpyrrolidin-2-yl]pyridine, calculated with reference to the anhydrous substance.
CHARACTERS
A colourless or brownish viscous liquid, volatile, hygroscopic, soluble in water, miscible with ethanol.
IDENTIFICATION
B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the Ph. Eur. reference
spectrum of nicotine.
TESTS
solution is clear (2.2.1) and not more intensely coloured than reference solution Y5, BY5 or R5
(Method II, 2.2.2).
Specific optical rotation (2.2.7). Dissolve 1.00 g in ethanol R and dilute to 50.0 ml with the same
solvent. The specific optical rotation is 140 to 152.
Reference solution (a). Dissolve 4 mg of nicotine ditartrate CRS and 2 mg of myosmine R in the mobile
phase and dilute to 50.0 ml with the mobile phase.
as mobile phase at a flow rate of 1.5 ml/min a solution prepared as follows: dissolve 2.31 g of
sodium dodecyl sulphate R in a mixture of 250 ml of acetonitrile R and 750 ml of a 13.6 g/l solution
of potassium dihydrogen phosphate R, adjusted to pH 4.5 with sodium hydroxide R or phosphoric
acid R,
conditions the retention times are: nicotine about 13 min and impurity D about 11 min. The test is
not valid unless the resolution between the impurity D peak eluting closest to the nicotine peak and
the peak due to nicotine is at least 1.5; if necessary, adjust the concentration of acetonitrile in the
mobile phase.
for twice the retention time of the principal peak. In the chromatogram obtained with the test
solution: the area of any peak, apart from the principal peak, is not greater than the area of the
the areas of all the peaks, apart from the principal peak, is not greater than twice the area of the
any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained
28-13
of water.
ASSAY
Dissolve 60.0 mg in 30 ml of anhydrous acetic acid R. Titrate with 0.1M perchloric acid determining the
STORAGE
Store, under nitrogen, in an airtight container, protected from light.
IMPURITIES
N
H
HN
A. 3-[(2S)-1,2,3,6-tetrahydropyridin-2-yl]pyridine (anatabine),
N
Me
B. 3-(1-methyl-1H-pyrrol-2-yl)pyridine (-nicotyrine),
N
H
Me
N
O
C. (5S )-1-methyl-5-(pyridin-3-yl)pyrrolidin-2-one (cotinine),

N
D. 3-(4,5-dihydro-3H-pyrrol-2-yl)pyridine (myosmine),
N
H
and epimer at N*
N*
Me
E. 3-[(1RS,2S)-1-methylpyrrolidin-2-yl 1-oxide]pyridine (nicotine N-oxide).

__________________________________________________________________________________________________________ Ph Eur
28-14
Nicotinic Acid
N
COOH
C6H5NO2
123.1
59-67-6
Nicotinic Acid complies with the requirements of the 3rd edition of the European Pharmacopoeia [0459].
Preparation
Nicotinic Acid Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Nicotinic acid contains not less than 99.5 per cent and not more than the equivalent of 100.5 per
cent of pyridine-3-carboxylic acid, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder, soluble in boiling water and in boiling alcohol, sparingly soluble in water,
practically insoluble in ether. It dissolves in dilute solutions of the alkali hydroxides and carbonates.
IDENTIFICATION
obtained with nicotinic acid CRS.
C. Dissolve about 10 mg in 10 ml of water R. To 2 ml of the solution add 2 ml of cyanogen bromide
solution R and 3 ml of a 25 g/l solution of aniline R and shake. A yellow colour develops.
TESTS
coating substance.
Test solution. Dissolve 0.5 g of the substance to be examined in water R, warming slightly if necessary,
Reference solution. Dilute 0.5 ml of the test solution to 100 ml with water R.
volumes of water R, 10 volumes of anhydrous formic acid R and 85 volumes of propanol R. Dry the
plate at 100C to 105C for 10 min and examine in ultraviolet light at 254 nm. Any spot in the
chromatogram obtained with the test solution, apart from the principal spot, is not more intense than
the spot in the chromatogram obtained with the reference solution (0.5 per cent).
Chlorides (2.4.4). Dissolve 0.25 g in water R, heating on a water-bath, and dilute to 15 ml with the
same solvent. The solution complies with the limit test for chlorides (200 ppm).
100C to 105C for 1 h.
ASSAY
Dissolve 0.250 g in 50 ml of water R. Titrate with 0.1M sodium hydroxide, using 0.25 ml of
phenolphthalein solution R as indicator, until a pink colour is obtained. Carry out a blank titration.
1 ml of 0.1M sodium hydroxide is equivalent to 12.31 mg of C6H5NO2.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
28-15
Nicotinyl Alcohol Tartrate

H
OH
COOH
, HOOC
CH2OH
C6H7NO,C4H6O6
H
259.2
OH
6164-87-0
Definition Nicotinyl Alcohol Tartrate is 3-pyridylmethanol hydrogen (2R,3R)-tartrate. It contains

not less than 98.5% and not more than 101.5% of C6H7NO,C4H6O6, calculated with reference to
Characteristics A white or almost white crystalline powder; odourless or almost odourless.
Freely soluble in water; slightly soluble in ethanol (96%); practically insoluble in chloroform and in
ether.
Identification
A. The light absorption, Appendix II B, in the range 230 to 350 nm of a 0.004% w/v solution in 0.1M
hydrochloric acid exhibits a maximum only at 261 nm. The absorbance at 261 nm is about 0.84.
C. Yields reaction B characteristic of tartrates, Appendix VI.
Clarity and colour of solution A 5.0% w/v solution is clear, Appendix IV A, and not more intensely
coloured than reference solution Y5, Appendix IV B, Method II.
Heavy metals Dissolve the residue obtained in the test for Sulphated ash in 1 ml of 2M hydrochloric
acid and dilute to 20 ml with water. 12 ml of the solution complies with limit test A for heavy metals,
Appendix VII. Use lead standard solution (2 ppm) to prepare the standard (20 ppm).
Nicotinaldehyde Mix 10 ml of a 10% w/v solution of the substance being examined with 10 ml of a
1.0% w/v solution of phenylhydrazine hydrochloride in 3.6M orthophosphoric acid, dilute to 50 ml with
water and allow to stand for 30 minutes. Measure the absorbance of the solution at the maximum at
370 nm, Appendix II B, using in the reference cell a 0.20% w/v solution of phenylhydrazine hydrochloride in 0.72M orthophosphoric acid. The absorbance is not more than that of a solution prepared by
treating 10 ml of a 0.0010% w/v solution of pyridine-3-carboxaldehyde in the same manner.
gel GF254 as the coating substance and a mixture of 50 volumes of dichloromethane, 30 volumes of
1,4-dioxan, 16 volumes of methanol and 4 volumes of 13.5M ammonia as the mobile phase. Apply
separately to the plate 5 l of each of four solutions in 0.1M ammonia containing (1) 25% w/v of the
substance being examined, (2) 0.050% w/v of the substance being examined, (3) 0.050% w/v of
3-(aminomethyl)pyridine and (4) 0.050% w/v of nicotinyl alcohol tartrate BPCRS. After removal of the
plate, allow it to dry in air and examine under ultraviolet light (254 nm). Spray with a 2% w/v solution
of chloro-2,4,6-trinitrobenzene in absolute ethanol, dry in a current of air and spray with a 5% w/v
solution of sodium carbonate. Any spot corresponding to 3-(aminomethyl)pyridine in the chromatogram obtained with solution (1) is not more intense than the spot in the chromatogram obtained with
solution (3) (0.2%). Any other secondary spot in the chromatogram obtained with solution (1) is not
more intense than the spot in the chromatogram obtained with solution (2) (0.2%). Disregard any
spot due to tartaric acid on the line of application.
Sulphated ash Not more than 0.1%, Appendix IX A. Use 2.0 g.
C6H7NO,C4H6O6.
Preparation
Nicotinyl Alcohol Tablets
28-16
Nifedipine
H
N
Me
MeOOC
Me
COOMe
H
NO2
C17H18N2O6
346.3
21829-25-4
Nifedipine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0627]. These
Action and use Calcium antagonist.
Preparation
Nifedipine Capsules
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Nifedipine contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent of
dimethyl 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)pyridine-3,5-dicarboxylate, calculated with
CHARACTERS
A yellow, crystalline powder, practically insoluble in water, freely soluble in acetone, sparingly soluble
in ethanol. When exposed to daylight and to artificial light of certain wavelengths, it readily converts
to a nitrosophenylpyridine derivative. Exposure to ultraviolet light leads to the formation of a nitrophenylpyridine derivative. Prepare solutions immediately before use in the dark or under longwavelength light (>420 nm) and protect them from light.
IDENTIFICATION
obtained with nifedipine CRS. Examine the substances in the solid state.
the same solvent.
Reference solution. Dissolve 10 mg of nifedipine CRS in methanol R and dilute to 10 ml with the same
solvent.
15 cm using a mixture of 40 volumes of ethyl acetate R and 60 volumes of cyclohexane R. Allow the
plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram
obtained with the test solution is similar in position, appearance at 254 nm and size to the principal
spot in the chromatogram obtained with reference solution.
D. To 25 mg in a test tube, add 10 ml of a mixture of 1.5 volumes of hydrochloric acid R, 3.5 volumes
of water R and 5 volumes of alcohol R and dissolve with gentle heating. Add 0.5 g of zinc R in
granules and allow to stand for 5 min with occasional swirling. Filter into a second test tube, add
5 ml of a 10 g/l solution of sodium nitrite R to the filtrate and allow to stand for 2 min. Add 2 ml of a
50 g/l solution of ammonium sulphamate R, shake vigorously with care and add 2 ml of a 5 g/l solution
of naphthylethylenediamine dihydrochloride R. An intense red colour develops which persists for not less
than 5 min.
TESTS
Test solution. Dissolve 0.200 g of the substance to be examined in 20 ml of methanol R and dilute to
28-17
Reference solution (a). Dissolve 10 mg of nifedipine impurity A CRS in methanol R and dilute to 25.0 ml
Reference solution (b). Dissolve 10 mg nifedipine impurity B CRS in methanol R and dilute to 25.0 ml
Reference solution (c). Mix 1.0 ml of reference solution (a), 1.0 ml of reference solution (b) and 0.1 ml
of the test solution and dilute to 20.0 ml with the mobile phase. Dilute 2.0 ml of this solution to
a stainless steel column 0.15 m long and 4.6 mm in internal diameter, packed with octadecylsilyl
as mobile phase at a flow rate of about 1.0 ml per minute a mixture of 9 volumes of acetonitrile R, 36 volumes of methanol R and 55 volumes of water R,
as detector a spectrophotometer set at 235 nm; the use of an electronic integrator is advisable.
Inject 20 l of reference solution (c). When the chromatogram is recorded in the conditions
described above, the substances elute in the following order: impurity A, impurity B and nifedipine;
the retention time of nifedipine is about 15.5 min. The test is not valid unless in the chromatogram
obtained with reference solution (c): the resolution between the peaks corresponding to impurity A
and impurity B is greater than 1.5; the resolution between the peaks corresponding to impurity B and
nifedipine is greater than 1.5; the peak corresponding to impurity A has a height not less than 20 per
cent of the full scale deflection of the recorder. Inject separately 20 l of the test solution and of
reference solution (c) and record the chromatograms for twice the retention time of nifedipine. In the
chromatogram obtained with the test solution: none of the peaks apart from the principal peak and
the peaks corresponding to impurity A and impurity B has an area greater than that of the peak
corresponding to nifedipine in the chromatogram obtained with reference solution (c) (0.1 per cent);
the areas of the peaks corresponding to impurity A and impurity B are not greater than the corresponding peaks in the chromatogram obtained with reference solution (c) (0.1 per cent); the total
amount of related substances does not exceed 0.3 per cent. Disregard any peak whose area is less
than 10 per cent of the area of the peak corresponding to nifedipine in the chromatogram obtained
with reference solution (c).
100C to 105C for 2 h.
ASSAY
Dissolve 0.1300 g in a mixture of 25 ml of 2-methyl-2-propanol R and 25 ml of perchloric acid
solution R. Titrate with 0.1M ammonium and cerium sulphate using 0.1 ml of ferroin R as indicator, until
the pink colour disappears. Titrate slowly towards the end of the titration. Carry out a blank titration.
1 ml of 0.1M ammonium and cerium sulphate is equivalent to 17.32 mg of C17H18N2O6.
STORAGE
IMPURITIES
Me
Me
MeO
Me
NO2
A. Dimethyl 2,6-dimethyl-4-(2-nitrophenyl)pyridine-3,5-dicarboxylate (nitrophenylpyridine

analogue),
Me
Me
MeO
O
O
Me
O
NO
B. Dimethyl 2,6-dimethyl-4-(2-nitrosophenyl)pyridine-3,5-dicarboxylate (nitrosophenylpyridine

analogue).
__________________________________________________________________________________________________________ Ph Eur
28-18
Nikethamide
N
CONEt2
C10H14N2O
178.2
59-26-7
Nikethamide complies with the requirements of the 3rd edition of the European Pharmacopoeia [0233]. These
Action and use Respiratory stimulant.
Preparation
Nikethamide Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Nikethamide contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of N,N-diethylpyridine-3-carboxamide, calculated with reference to the anhydrous substance.
CHARACTERS
An oily liquid or a crystalline mass, colourless or slightly yellowish, miscible with water, with alcohol
and with ether.
IDENTIFICATION
A. Dissolve 0.15 g in 0.01M hydrochloric acid and dilute to 100.0 ml with the same acid. Dilute 1.0 ml
of this solution to 100.0 ml with 0.01M hydrochloric acid. Examined between 230 nm and 350 nm
(2.2.25) in a 2 cm cell, the solution shows a single absorption maximum, at 263 nm. The specific
absorbance at the maximum is about 285.
obtained with nikethamide CRS.
C. Heat 0.1 g with 1 ml of dilute sodium hydroxide solution R. Diethylamine is evolved progressively
and is recognisable by its characteristic odour and by its turning red litmus paper R blue.
D. Dilute 1 ml of solution S (see Tests) to 250 ml with water R. To 2 ml of this solution add 2 ml of
cyanogen bromide solution R. Add 3 ml of a 25 g/l solution of aniline R and shake. A yellow colour
develops.
TESTS
Appearance The substance to be examined, in liquid form or liquefied by slight heating, is clear
(2.2.1) and not more intensely coloured than reference solution Y5 (Method II, 2.2.2).
Refractive index (2.2.6). 1.524 to 1.526.
coating substance.
the same solvent.
Reference solution (a). Dissolve 40 mg of ethylnicotinamide CRS in methanol R and dilute to 100 ml
25 volumes of propanol R and 75 volumes of chloroform R. Allow the plate to dry in air and examine in
ultraviolet light at 254 nm. In the chromatogram obtained with the test solution, any spot corresponding to ethylnicotinamide is not more intense than the spot in the chromatogram obtained with
reference solution (a) (1.0 per cent) and any spot, apart from the principal spot and the spot corresponding to ethylnicotinamide, is not more intense than the spot in the chromatogram obtained with
reference solution (b) (0.1 per cent).
Heavy metals (2.4.8). Dilute 10 ml of solution S to 25 ml with water R. 12 ml of this solution
28-19
of water.
ASSAY
Dissolve 0.150 g in a mixture of 5 ml of acetic anhydride R and 20 ml of anhydrous acetic acid R.
__________________________________________________________________________________________________________ Ph Eur
28-20
Nimesulide
1/01
NHSO2CH3
O
NO2
C13H12N2O5S
308.3
51803-78-2
Nimesulide complies with the requirements of the 3rd edition of the European Pharmacopoeia [1548]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
N-(4-Nitro-2-phenoxyphenyl)methanesulphonamide.
Content: 98.5 per cent to 101.5 per cent (dried substance).
CHARACTERS
Appearance: yellowish crystalline powder.
Solubility: practically insoluble in water, freely soluble in acetone, slightly soluble in ethanol.
mp: about 149C.
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Preparation: discs.
Comparison: nimesulide CRS.
If the spectra obtained show differences, dissolve the substance to be examined and the reference
substance separately in acetone R, evaporate to dryness and record new spectra using the residues.
TESTS
Absorbance (2.2.25): maximum 0.50 at 450 nm.
Dissolve 1.0 g in acetone R and dilute to 10.0 ml with the same solvent.
Test solution. Dissolve 20 mg of the substance to be examined in 8 ml of acetonitrile R and dilute to
Reference solution (a). Dissolve 10 mg of nimesulide impurity C CRS and 10 mg of nimesulide impurity D
CRS in 20 ml of acetonitrile R and dilute to 50.0 ml with water R. Dilute 1.0 ml of the solution to
Column:
dimensions: l = 0.125 m, = 4.0 mm,
stationary phase: octadecylsilyl silica gel for chromatography R.
Mobile phase: a mixture of 35 volumes of acetonitrile R and 65 volumes of a 1.15 g/l solution of
ammonium dihydrogen phosphate R adjusted to pH 7.0 with ammonia R.
Injection: 20 l.
Run time: 7 times the retention time of nimesulide.
System suitability:
resolution: minimum of 2.0 between the 2 principal peaks in the chromatogram obtained with
28-21
Limits:
any impurity: not more than the area of the principal peak in the chromatogram obtained with
reference solution (b) (0.1 per cent),
total: not more than 5 times the area of the principal peak in the chromatogram obtained with
1.0 g complies with limit test D. Prepare the standard using 2 ml of lead standard solution (10 ppm
Pb) R.
Loss on drying (2.2.32): maximum 0.5 per cent, determined on 1.000 g by drying in an oven at
100-105C for 4 h.
ASSAY
Dissolve 0.240 g in 30 ml of previously neutralised acetone R and add 20 ml of water R. Titrate with
0.1M sodium hydroxide, determining the end-point potentiometrically (2.2.20).
1 ml of 0.1M sodium hydroxide is equivalent to 30.83 mg of C13H12N2O5S.
IMPURITIES
R4
R3
N
R3
R2
A. R1 = SO2-CH3, R2 = H, R3 = R4 = NO2: N-(2,4-dinitro-6-phenoxyphenyl)methanesulphonamide,

B. R1 = SO2-CH3, R2 = R3 = R4 = H: N-(2-phenoxyphenyl)methanesulphonamide,
E. R1 = R2 = SO2-CH3, R3 = R4 = H: N,N-bis(methylsulphonyl)-2-phenoxyaniline,
C. R1 = R2 = R3 = R4 = H: 2-phenoxyaniline,
D. R1 = R2 = R4 = H, R3 = NO2: 4-nitro-2-phenoxyaniline,
F. R1 = R2 = SO2-CH3, R3 = NO2, R4 = H: N,N-bis(methylsulphonyl)-4-nitro-2-phenoxyaniline,
OH
O2N
G. 4-nitro-2-phenoxyphenol.
__________________________________________________________________________________________________________ Ph Eur
28-22
Nimodipine
H
N
Me
Me
O
i PrOOC
OMe
H
O
NO2
and enantiomer
C21H26N2O7
418.5
66085-59-4
Nimodipine complies with the requirements of the 3rd edition of the European Pharmacopoeia [1245]. These
Preparations
Nimodipine Intravenous Infusion
Nimodipine Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Nimodipine contains not less than 98.5 per cent and not more than the equivalent of 101.5 per cent
of 2-methoxyethyl 1-methylethyl (4RS)-2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5dicarboxylate, calculated with reference to the dried substance.
CHARACTERS
A light yellow or yellow, crystalline powder, practically insoluble in water, freely soluble in ethyl
acetate, sparingly soluble in ethanol.
Exposure to ultraviolet light leads to the formation of a nitrophenylpyridine impurity.
Prepare solutions immediately before use either protected from light or under long-wavelength light
(>420 nm).
IDENTIFICATION
with nimodipine CRS. If the spectra obtained in the solid state show differences, record further spectra
using 20 g/l solutions in methylene chloride R and a 0.2 mm cell.
TESTS
Solution S Dissolve 1.0 g in acetone R and dilute to 20.0 ml with acetone R.
Appearance of solution Solution S is clear (2.2.1).
Optical rotation (2.2.7). The angle of optical rotation, determined on solution S, is 0.10 to
+0.10.
Test solution. Dissolve 40.0 mg of the substance to be examined in 2.5 ml of tetrahydrofuran R and
Reference solution (b). Dissolve 20.0 mg of nimodipine impurity A CRS in 2.5 ml of tetrahydrofuran R
and dilute to 25.0 ml with the mobile phase. Dilute 1.0 ml of the solution to 20.0 ml with the mobile
phase.
Reference solution (c). Dilute 0.5 ml of the test solution to 20.0 ml with the mobile phase.
Reference solution (d). Mix 1.0 ml of reference solution (b) and 1.0 ml of reference solution (c) and
28-23
as mobile phase at a flow rate of 2.0 ml/min a mixture of 20 volumes of methanol R, 20 volumes
of tetrahydofuran R and 60 volumes of water R,
Adjust the sensitivity of the system so that the height of the peak corresponding to nimodipine in
the chromatogram obtained with 20 l of reference solution (d) is at least 50 per cent of the full scale
of the recorder.
Inject 20 l of reference solution (d). When the chromatograms are recorded in the prescribed
conditions, the retention times are: impurity A, about 7 min and nimodipine, about 8 min. The test
is not valid unless the resolution between the peaks corresponding to impurity A and nimodipine is at
least 1.5.
Inject separately 20 l of the test solution and 20 l of reference solution (a). Record the chromatogram of the test solution for four times the retention time of nimodipine. In the chromatogram
obtained with the test solution: the area of the peak due to impurity A is not greater than the corresponding peak in the chromatogram obtained with reference solution (d) (0.1 per cent); none of the
peaks, apart from the principal peak and the peak due to impurity A, has an area greater than the area
of the principal peak in the chromatogram obtained with reference solution (a) (0.2 per cent); the
sum of the areas of all the peaks, apart from the principal peak, is not greater than 2.5 times the area
of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent).
Disregard any peak due to the solvent and any peak with an area less than 0.5 times the area of the
principal peak in the chromatogram obtained with reference solution (d).
100C to 105C.
ASSAY
Dissolve 0.180 g with gentle heating in a mixture of 25 ml of 2-methyl-2-propanol R and 25 ml
of perchloric acid solution R. Add 0.1 ml of ferroin R. Titrate with 0.1M cerium sulphate. Titrate slowly
towards the end of the titration. Carry out a blank titration.
1 ml of 0.1M cerium sulphate is equivalent to 20.92 mg of C21H26N2O7.
STORAGE
IMPURITIES
Me
Me
Me
Me
OMe
NO2
A. 2-methoxyethyl 1-methylethyl 2,6-dimethyl-4-(3-nitrophenyl)pyridine-3,5-dicarboxylate,
Me
H
N
Me
RO
OR
O
O
NO2
B. R = CH(CH3)2: bis(1-methylethyl) 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5dicarboxylate,

C. R = CH2-CH2-OCH3: bis(2-methoxyethyl) 2,6-dimethyl-4-(3-nitrophenyl)-1,4dihydropyridine-3,5-dicarboxylate.
__________________________________________________________________________________________________________ Ph Eur
28-24
Nitrazepam
O
H
N
O2N
C15H11N3O3
281.3
146-22-5
Nitrazepam complies with the requirements of the 3rd edition of the European Pharmacopoeia [0415]. These
Preparations
Nitrazepam Oral Suspension
Nitrazepam Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Nitrazepam contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of 7-nitro-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one, calculated with reference to the dried
substance.
CHARACTERS
A yellow, crystalline powder, practically insoluble in water, slightly soluble in alcohol and in ether.
IDENTIFICATION
B. Protect the solutions from light and measure the absorbances immediately. Dissolve 25.0 mg in a 5 g/l
solution of sulphuric acid R in methanol R and dilute to 250.0 ml with the same solvent. Dilute 5.0 ml
of the solution to 100.0 ml with a 5 g/l solution of sulphuric acid R in methanol R. Examined between
230 nm and 350 nm (2.2.25), the solution shows an absorption maximum at 280 nm. The specific
absorbance at the maximum is 890 to 950.
obtained with nitrazepam CRS.
D. Dissolve about 20 mg in a mixture of 5 ml of hydrochloric acid R and 10 ml of water R. Boil for
5 min, cool and add 2 ml of a 1 g/l solution of sodium nitrite R. Allow to stand for 1 min and add 1 ml
of a 5 g/l solution of sulphamic acid R and mix. Allow to stand for 1 min and add 1 ml of a 1 g/l
solution of naphthylethylenediamine dihydrochloride R. A red colour is produced.
E. Dissolve about 10 mg in 1 ml of methanol R, warming if necessary, and add 0.05 ml of dilute
sodium hydroxide solution R. An intense yellow colour is produced.
TESTS
Related substances Carry out the test protected from light. Examine by thin-layer chromatography
(2.2.27), using silica gel GF254 R as the coating substance.
same solvent. Prepare the solution immediately before use.
Reference solution (a). Dissolve 10 mg of aminonitrobenzophenone R in acetone R and dilute to 100 ml
with the same solvent. Dilute 10 ml of the solution to 50 ml with acetone R.
Reference solution (b). Dissolve 10 mg of nitrazepam impurity A CRS in acetone R and dilute to 100 ml
with the same solvent. Dilute 10 ml of the solution to 50 ml with acetone R.
Reference solution (c). Dilute 1 ml of the test solution to 20 ml with acetone R. Dilute 1 ml of this
solution to 50 ml with acetone R.
28-25
15 volumes of ethyl acetate R and 85 volumes of nitromethane R. Allow the plate to dry in air and
examine in ultraviolet light at 254 nm. In the chromatogram obtained with the test solution: any spot
corresponding to aminonitrobenzophenone is not more intense than the spot in the chromatogram
obtained with reference solution (a) (0.1 per cent); any spot corresponding to nitrazepam impurity A
cent); any spot apart from the principal spot and the spots corresponding to
aminonitrobenzophenone and nitrazepam impurity A is not more intense than the spot in the
chromatogram obtained with reference solution (c) (0.1 per cent).
100C to 105C for 4 h.
ASSAY
Dissolve 0.250 g in 25 ml of acetic anhydride R. Titrate with 0.1M perchloric acid, determining the endpoint potentiometrically (2.2.20).
STORAGE
IMPURITIES
A. 3-amino-6-nitro-4-phenylquinol-2-one,
B. 2-amino-5-nitrobenzophenone.
__________________________________________________________________________________________________________ Ph Eur
28-26
Nitrendipine
H
N
Me
MeOOC
Me
COOEt
H
NO2
and enantiomer
C18H20N2O6
360.4
39562-70-4
Nitrendipine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0046]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Nitrendipine contains not less than 98.5 per cent and not more than the equivalent of 101.5 per cent
of ethyl methyl (4RS)-2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate, calculated with reference to the dried substance.
CHARACTERS
A yellow, crystalline powder, practically insoluble in water, freely soluble in ethyl acetate, sparingly
soluble in ethanol and in methanol.
Exposure to ultraviolet light leads to the formation of a nitrophenyl-pyridine derivitive.
Prepare solutions immediately before use either protected from light or under long-wavelength light
(>420 nm).
IDENTIFICATION
with nitrendipine CRS. If the spectra obtained in the solid state show differences, record further
spectra using 20 g/l solutions in methylene chloride R and a 0.2 mm cell.
TESTS
Optical rotation (2.2.7). Dissolve 0.2 g in acetone R and dilute to 10.0 ml with the same solvent.
Reference solution (b). Dissolve 20.0 mg of nitrendipine impurity A CRS in 2.5 ml of tetrahydrofuran R
and dilute to 25.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 20.0 ml with the mobile
phase.
Reference solution (c). Dilute 0.5 ml of the test solution to 20.0 ml with the mobile phase.
as mobile phase at a flow rate of 1 ml per minute a mixture of 14 volumes of acetonitrile R, 22
volumes of tetrahydrofuran R and 64 volumes of water R,
28-27
Adjust the sensitivity of the system so that the height of the nitrendipine peak in the chromatogram
obtained with 20 l of reference solution (d) is at least 50 per cent of the full scale of the recorder.
Inject 20 l of reference solution (d). When the chromatogram is recorded in the prescribed
conditions, the retention times are: nitrendipine impurity A, about 6 min, nitrendipine about 8 min.
The test is not valid unless the resolution between the peaks corresponding to nitrendipine impurity
A and nitrendipine is at least 2.0.
Inject separately 20 l of the test solution and 20 l of reference solution (a). Record the chromatogram of the test solution for five times the retention time of the principal peak. In the chromatogram
obtained with the test solution: the area of the peak corresponding to nitrendipine impurity A is not
greater than the area of the corresponding peak in the chromatogram obtained with reference
solution (d) (0.1 per cent); none of the peaks, apart from the principal peak and the peaks corresponding to nitrendipine impurity A has an area greater than that of the peak corresponding to
nitrendipine in the chromatogram obtained with reference solution (a) (0.8 per cent); the sum of the
areas of all the peaks, apart from the principal peak, is not greater than 1.5 times the area of the
principal peak in the chromatogram obtained with reference solution (a) (1.2 per cent). Disregard
any peak with an area less than 0.5 times the area of the nitrendipine peak in the chromatogram
obtained with reference solution (d).
100C to 105C.
ASSAY
Dissolve 0.160 g with gentle heating if necessary in a mixture of 25 ml of 2-methyl-2-propanol R and
25 ml of perchloric acid solution R. Titrate with 0.1M cerium sulphate, using 0.1 ml of ferroin R as
indicator. Titrate slowly towards the end of the titration. Carry out a blank titration.
1 ml of 0.1M cerium sulphate is equivalent to 18.02 mg of C18H20N2O6.
STORAGE
IMPURITIES
Me
Me
MeO
Me
NO2
A. ethyl methyl 2,6-dimethyl-4-(3-nitrophenyl)pyridine-3,5-dicarboxylate,

Me
H
N
Me
RO
OR
O
O
NO2
B. R = CH3: dimethyl 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate,

C. R = CH2-CH3: diethyl 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5dicarboxylate.
__________________________________________________________________________________________________________ Ph Eur
28-28
Nitric Acid
1/01
HNO3
63.0
7697-37-2
Nitric Acid complies with the requirements of the 3rd edition of the European Pharmacopoeia [1549]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Nitric acid contains not less than 68.0 per cent m/m and not more than 70.0 per cent m/m of HNO3.
CHARACTERS
A clear, colourless to almost colourless liquid, miscible with water.
It has a relative density of about 1.41.
IDENTIFICATION
A. Dilute 1 ml to 100 ml with water R. The solution is strongly acid (2.2.4).
B. 0.2 ml of the solution obtained in identification test A gives the reaction of nitrates (2.3.1).
TESTS
Appearance of solution Dilute 2 ml to 10 ml with water R. The solution is clear (2.2.1) and not
Chlorides (2.4.4). To 5 g add 10 ml of water R and 0.3 ml of silver nitrate solution R2 and allow to
stand for 2 min protected from light. Any opalescence is not more intense than that of a standard
prepared in the same manner using 13 ml of water R, 0.5 ml of nitric acid R, 0.5 ml of chloride
standard solution (5 ppm Cl) R and 0.3 ml of silver nitrate solution R2 (0.5 ppm).
Sulphates (2.4.13). To 15 g add 0.2 g of sodium carbonate R. After carbon dioxide has evolved,
evaporate to dryness. Dissolve the residue in 15 ml of distilled water R. The solution complies with
the limit test for sulphates (10 ppm).
Iron (2.4.9). Dissolve the residue obtained in the test for sulphated ash in 1 ml of dilute hydrochloric
acid R and dilute to 20 ml with water R. Dilute 1 ml to 10 ml with water R. The solution complies
with the limit test for iron (10 ppm).
Heavy metals (2.4.8). Carefully evaporate 10.0 g to dryness on a water-bath. Moisten the residue
with a few drops of dilute hydrochloric acid R and dilute to 20 ml with water R. 12 ml of the solution
complies with limit test A for heavy metals (2 ppm). Prepare the standard using lead standard solution
(2 ppm Pb) R.
Sulphated ash Carefully evaporate 20.00 g to dryness. Moisten the residue with a few drops of
sulphuric acid R and ignite to dull red. The residue does not exceed 0.01 per cent.
ASSAY
To 0.750 g add 50 ml of water R and titrate with 1M sodium hydroxide, determining the end-point
1 ml of 1M sodium hydroxide is equivalent to 63.0 mg of HNO3.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
28-29
Nitric Oxide
1/01
NO
30.01
10102-43-9
Nitric Oxide complies with the requirements of the 3rd edition of the European Pharmacopoeia [1550]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Nitric oxide contains not less than 99.0 per cent V/V of NO.
CHARACTERS
A colourless gas which turns brown when exposed to air. At 20C and at a pressure of 101 kPa, 1
volume dissolves in about 21 volumes of water.
PRODUCTION
Carbon dioxide Not more than 3000 ppm V/V, determined by gas chromatography (2.2.28).
Gas to be examined. The substance to be examined.
Reference gas. Use a mixture containing 3000 ppm V/V of carbon dioxide R1 in nitrogen R.
a stainless steel column 3.5 m long and 2 mm in internal diameter packed with ethylvinylbenzenedivinylbenzene copolymer R,
helium for chromatography R as the carrier gas at a flow rate of 15 ml/min,
a thermal conductivity detector,
a loop injector,
Inject the gas to be examined and the reference gas. The test is not valid unless the chromatograms
obtained show a clear separation of carbon dioxide from nitric oxide.
Calculate the carbon dioxide content in the gas to be examined from the area of the carbon dioxide
peak in the chromatogram obtained with the reference gas.
Nitrogen Not more than 3000 ppm V/V, determined by gas chromatography (2.2.28).
Reference gas. Use a mixture containing 3000 ppm V/V of nitrogen R in helium for chromatography R.
a stainless steel column 3.5 m long and 2 mm in internal diameter packed with molecular sieve for
chromatography R (0.5 nm),
a loop injector,
obtained show a clear separation of nitrogen from nitric oxide.
Calculate the nitrogen content in the gas to be examined from the area of the peak due to nitrogen
in the chromatogram obtained with the reference gas.
Nitrogen dioxide Not more than 400 ppm V/V, determined using an ultraviolet absorption
spectrophotometry analyser.
Reference gas (a). Use nitrogen R1.
Reference gas (b). Use a mixture containing 400 ppm V/V of nitrogen dioxide R in nitrogen R.
The apparatus consists of the following:
an ultraviolet-visible light source (analytical wavelength about 400 nm),
a sample gas cell through which the feed gas flows,
a closed reference gas cell containing nitrogen R1 in parallel with the sample gas cell,
a rotating chopper which feeds light alternately through the reference gas cell and the sample gas
cell,
a semiconductor detector which generates a frequency modulated output whose amplitude is a
measure of the difference of absorption of the sample gas and the reference gas.
Carry out the analysis in the following way:
set the zero of the instrument using reference gas (a) through the sample gas cell at a flow rate of
28-30
1 litre/min,
adjust the span while feeding reference gas (b) through the sample gas cell at a flow rate of 1
litre/min,
feed the gas to be examined through the sample gas cell at a flow rate of 1 litre/min, read the
value from the instrument output and calculate if necessary the concentration of nitrogen
dioxide.
Nitrous oxide Not more than 3000 ppm V/V, determined by gas chromatography (2.2.28).
Reference gas. Use a mixture containing 3000 ppm V/V of nitrous oxide R in nitrogen R.
a loop injector,
obtained show a clear separation of nitrous oxide from nitric oxide.
Calculate the nitrous oxide content in the gas to be examined from the area of the peak due to
nitrous oxide in the chromatogram obtained with the reference gas.
Water Not more than 100 ppm V/V, determined using an electrolytic hygrometer (2.5.28).
Assay Determine the content of nitric oxide by difference using the mass balance equation after
determining the sum of the impurities described under Production.
IDENTIFICATION
spectrum of nitric oxide.
STORAGE
Store compressed at a pressure not exceeding 2.5 MPa (25 bars) measured at 15C, in suitable
containers complying with the legal regulations.
IMPURITIES
A. carbon dioxide,
B. nitrogen,
C. nitrogen dioxide,
D. nitrous oxide,
E. water.
__________________________________________________________________________________________________________ Ph Eur
28-31
Nitrofurantoin
O
O2N
NH
CH
N
O
C8H6N4O5
238.2
67-20-9
Nitrofurantoin complies with the requirements of the 3rd edition of the European Pharmacopoeia [0101].
Preparations
Nitrofurantoin Oral Suspension
Nitrofurantoin Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Nitrofurantoin contains not less than 98.0 per cent and not more than the equivalent of 102.0 per
cent of 1-(5-nitrofurfurylideneamino)imidazolidine-2,4-dione, calculated with reference to the dried
substance.
CHARACTERS
A yellow, crystalline powder or yellow crystals, odourless or almost odourless, very slightly soluble in
water and in alcohol, soluble in dimethylformamide.
IDENTIFICATION
A. Carry out the test protected from bright light. Use the solution prepared for the assay. Examined
367 nm. The ratio of the absorbance at the maximum at 367 nm to that at the maximum at 266 nm
is 1.36 to 1.42.
B. Dissolve about 10 mg in 10 ml of dimethylformamide R. To 1 ml of the solution add 0.1 ml of 0.5M
alcoholic potassium hydroxide. A brown colour develops.
TESTS
coating substance.
Test solution. Dissolve 0.25 g of the substance to be examined in a minimum of dimethylformamide R
and dilute to 10 ml with acetone R.
Reference solution. Dilute 1 ml of the test solution to 100 ml with acetone R.
10 volumes of methanol R and 90 volumes of nitromethane R. Allow the plate to dry in air and heat at
100C to 105C for 5 min. Examine in ultraviolet light at 254 nm. Spray with phenylhydrazine hydrochloride solution R. Heat the plate at 100C to 105C for a further 10 min. When examined in ultraviolet light and after spraying, any spot in the chromatogram obtained with the test solution, apart
from the principal spot, is not more intense than the spot in the chromatogram obtained with the
reference solution (1.0 per cent).
100C to 105C.
ASSAY
Carry out the assay protected from bright light. Dissolve 0.120 g in 50 ml of dimethylformamide R and
dilute to 1000.0 ml with water R. Dilute 5.0 ml of the solution to 100.0 ml with a solution containing
18 g/l of sodium acetate R and 0.14 per cent V/V of glacial acetic acid R. Measure the absorbance
(2.2.25) at the absorption maximum at 367 nm, using the sodium acetate solution described above as
compensation liquid.
Calculate the content of C8H6N4O5, taking the specific absorbance to be 765.
STORAGE
Store in a well-closed container, protected from light, at a temperature below 25C.
__________________________________________________________________________________________________________ Ph Eur
28-32
Nitrofurazone
O2N
O
N
N
H
C6H6N4O4
198.1
NH2
59-87-0
Nitrofurazone complies with the requirements of the 3rd edition of the European Pharmacopoeia for Nitrofural
Action and use Antibacterial; topical antiprotozoan.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Nitrofural contains not less than 97.0 per cent and not more than the equivalent of 103.0 per cent of
5-nitro-2-furaldehyde semicarbazone, calculated with reference to the dried substance.
CHARACTERS
A yellow or brownish-yellow, crystalline powder, very slightly soluble in water, slightly soluble in
IDENTIFICATION
A. Carry out the test protected from bright light. Use the solution prepared for the assay. Examined
375 nm. The ratio of the absorbance measured at the maximum at 375 nm to that measured at the
maximum at 260 nm is 1.15 to 1.30.
obtained with nitrofural CRS. Examine the substances prepared as discs.
the same solvent.
Reference solution. Dissolve 10 mg of nitrofural CRS in methanol R and dilute to 10 ml with the same
solvent.
10 volumes of methanol R and 90 volumes of nitromethane R. Allow the plate to dry in air and spray
with phenylhydrazine hydrochloride solution R. The principal spot in the chromatogram obtained with
the test solution is similar in position, colour and size to the principal spot in the chromatogram
D. Dissolve about 1 mg in 1 ml of dimethylformamide R and add 0.1 ml of alcoholic potassium hydroxide
solution R. A violet-red colour is produced.
TESTS
pH (2.2.3). To 1.0 g add 100 ml of carbon dioxide-free water R. Shake and filter. The pH of the
filtrate is 5.0 to 7.0.
Reference solution (a). Dissolve 10.0 mg of (5-nitro-2-furyl)methylene diacetate R in the mobile phase
and dilute to 20.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 100.0 ml with the
mobile phase.
Reference solution (b). Dissolve 10 mg of nitrofural CRS and 10 mg of nitrofurantoin R in the mobile
phase and dilute to 100 ml with the mobile phase. Dilute 5 ml of this solution to 100 ml with the
mobile phase.
volumes of water R,
28-33
principal peak in the chromatogram obtained is not less than 50 per cent of the full scale of the
recorder. Inject 20 l of reference solution (b). The test is not valid unless, in the chromatogram
obtained, the resolution between the peaks due to nitrofurantoin and nitrofural is at least 2.0. Inject
20 l of the test solution and 20 l of reference solution (a). Continue the chromatography for ten
times the retention time of nitrofural, which is about 3 min. In the chromatogram obtained with the
test solution: the area of any peak, apart from the principal peak, is not greater than the area of the
principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent); the sum of
the areas of all the peaks, apart from the principal peak, is not greater than twice the area of the
at 100C to 105C.
ASSAY
Carry out the assay protected from bright light. Dissolve 60.0 mg in 20 ml of dimethylformamide R and
dilute to 500.0 ml with water R. Dilute 5.0 ml to 100.0 ml with water R. Prepare a reference solution
in the same manner using 60.0 mg of nitrofural CRS. Measure the absorbances (2.2.25) of the two
solutions at the maximum at 375 nm. Calculate the content of C6H6N4O4 from the absorbances
measured and the concentrations of the solutions.
STORAGE
IMPURITIES
O2N
CH
N N
CH
NO2
A. 5-nitro-2-furaldehyde azine,
O2N
CH(OAc)2
B. (5-nitro-2-furyl)methylene diacetate.
__________________________________________________________________________________________________________ Ph Eur
28-34
Nitrogen
N2
28.01
7727-37-9
Nitrogen complies with the requirements of the 3rd edition of the European Pharmacopoeia [1247]. These
Nitrogen should be kept in approved metal cylinders, the shoulders of which are painted black and
the remainder grey. The cylinder should carry a label stating Nitrogen.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Nitrogen contains not less than 99.5 per cent V/V of N2.
CHARACTERS
A colourless, odourless gas. At 20C and at a pressure of 101 kPa, 1 volume dissolves in about 62
volumes of water and about 10 volumes of alcohol.
PRODUCTION
Carbon dioxide Not more than 300 ppm V/V, determined using an infrared analyser (2.5.24).
Gas to be examined. The substance to be examined. It must be filtered to avoid stray light phenomena.
Reference gas (b). Use a mixture containing 300 ppm V/V of carbon dioxide R1 in nitrogen R1.
Calibrate the apparatus and set the sensitivity using reference gases (a) and (b). Measure the content
of carbon dioxide in the gas to be examined.
Carbon monoxide Not more than 5 ppm V/V, determined using an infrared analyser (2.5.25).
Reference gas (b). Use a mixture containing 5 ppm V/V of carbon monoxide R in nitrogen R1.
of carbon monoxide in the gas to be examined.
Oxygen Not more than 50 ppm V/V, determined using an oxygen analyser with a detector scale
ranging from 0 ppm V/V to 100 ppm V/V and equipped with an electrochemical cell.
The gas to be examined passes through a detection cell containing an aqueous solution of an
electrolyte, generally potassium hydroxide. The presence of oxygen in the gas to be examined
produces variation in the electric signal recorded at the outlet of the cell that is proportional to the
oxygen content.
Calibrate the analyser according to the instructions of the manufacturer. Pass the gas to be examined through the analyser using a suitable pressure regulator and airtight metal tubes and operating at
the prescribed flow-rates until constant readings are obtained.
Assay Examine by gas chromatography (2.2.28).
Reference gas (a). Use ambient air.
Reference gas (b). Use nitrogen R1.
a stainless steel column 2 m long and 2 mm in internal diameter packed with an appropriate
molecular sieve for chromatography (0.5 nm),
a loop injector,
maintaining the temperature of the column at 50C and that of the detector at 130C.
Inject reference gas (a). Adjust the injected volumes and operating conditions so that the height of
the peak due to nitrogen in the chromatogram obtained with the reference gas is at least 35 per cent
of the full scale of the recorder. The assay is not valid unless the chromatograms obtained show a
clear separation of oxygen and nitrogen.
Inject the gas to be examined and reference gas (b). In the chromatogram obtained with the gas to
be examined, the area of the principal peak is at least 99.5 per cent the area of the principal peak in
the chromatogram obtained with the reference gas (b).
28-35
IDENTIFICATION
A. Examine the chromatograms obtained in the Assay. The retention time of the principal peak in the
chromatogram obtained with the substance to be examined is approximately the same as that of the
principal peak in the chromatogram obtained with reference gas (b).
B. In a 250 ml conical flask replace the air by the substance to be examined. Place a burning or
glowing splinter of wood in the flask. The splinter is extinguished.
C. In a suitable test tube, place 0.1 g of magnesium R in turnings. Close the tube with a two-hole
stopper fitted with a glass tube reaching about 1 cm above the turnings. Pass the substance to be
examined through the glass tube for 1 min without heating, then for 15 min while heating the test
tube to a red glow. After cooling, add 5 ml of dilute sodium hydroxide solution R. The evolving vapors
change the colour of moistened red litmus paper R blue.
TESTS
Carbon dioxide Not more than 300 ppm V/V, determined using a carbon dioxide detector tube
(2.1.6).
Carbon monoxide Not more than 5 ppm V/V, determined using a carbon monoxide detector tube
(2.1.6).
Water vapour Not more than 67 ppm V/V, determined using a water vapour detector tube (2.1.6).
STORAGE
Store as a compressed gas or a liquid in appropriate containers complying with the legal regulations.
IMPURITIES
A. carbon dioxide,
B. carbon monoxide,
C. oxygen,
D. water.
__________________________________________________________________________________________________________ Ph Eur
28-36
Nitrous Oxide
corrected 1/01
N2O
44.01
10024-97-2
Nitrous Oxide complies with the requirements of the 3rd edition of the European Pharmacopoeia [0416].
Action and use General anaesthetic; analgesic.
Nitrous Oxide should be kept in approved metal cylinders which are painted blue and carry a label
stating Nitrous Oxide. In addition, Nitrous Oxide or the symbol N2O should be stencilled in
paint on the shoulder of the cylinder.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Nitrous oxide contains not less than 98.0 per cent V/V of N2O in the gaseous phase, when sampled at
15C.
CHARACTERS
A colourless gas. At 20C and at a pressure of 101 kPa, 1 volume dissolves in about 1.5 volumes of
water.
PRODUCTION
Nitrous oxide is produced from ammonium nitrate by thermic decomposition.
Examine the gaseous phase.
If the test is performed on a cylinder, keep the cylinder at room temperature for at least 6 h before carrying out
the tests. Keep the cylinder in the vertical position with the outlet valve uppermost.
Carbon dioxide Not more than 300 ppm V/V, determined by gas chromatography (2.2.28).
Reference gas. Use a mixture containing 300 ppm V/V of carbon dioxide R1 in nitrous oxide R.
a loop injector,
Inject the gas to be examined and the reference gas. Adjust the injected volumes and operating
conditions so that the height of the peak due to carbon dioxide in the chromatogram obtained with
the reference gas is at least 35 per cent of the full scale of the recorder. The test is not valid unless the
chromatograms obtained show a clear separation of carbon dioxide from nitrous oxide.
Calculate the carbon dioxide content in the gas to be examined from the area of the carbon dioxide
peak in the chromatogram obtained with the reference gas.
Carbon monoxide Not more than 5 ppm V/V, determined by gas chromatography (2.2.28).
When the test is carried out on a cylinder, use the first portion of gas to be withdrawn.
Reference gas. Use a mixture containing 5 ppm V/V of carbon monoxide R in nitrous oxide R.
a stainless steel column 2 m long and 4 mm in internal diameter packed with a suitable
molecular sieve for chromatography (0.5 nm),
a flame-ionisation detector with methaniser,
maintaining the temperature of the column at 50C and that of the injection port and of the detector
at 130C.
Inject the gas to be examined and the reference gas. Adjust the injected volumes and the operating
conditions so that the height of the peak due to carbon monoxide in the chromatogram obtained with
the reference gas is at least 35 per cent of the full scale of the recorder.
Calculate the carbon monoxide content from the area of the peak due to carbon monoxide in the
chromatogram obtained with the reference gas.
Nitrogen monoxide and nitrogen dioxide Not more than a total of 2 ppm V/V in the gas and
liquid phases, determined using a chemiluminescence analyser (2.5.26).
28-37
Reference mixture (a). Use nitrous oxide R.
Reference mixture (b). Use a mixture containing 2 ppm V/V of nitrogen monoxide R in nitrogen R1.
Calibrate the apparatus and set the sensitivity using reference mixtures (a) and (b). Measure the
content of nitrogen monoxide and nitrogen dioxide, separately examining the samples collected from
the gas phase and the liquid phase of the gas to be examined.
ASSAY Examine by gas chromatography (2.2.28).
Reference gas. Use nitrous oxide R.
a stainless steel column 2 m long and 2 mm in internal diameter packed with silica gel for chromatography R (250 m to 355 m),
maintaining the temperature of the column and that of the injection port at 60C and that of the
detector at 130C.
Inject the gas to be examined and the reference gas. Adjust the injected volumes and the operating
conditions so that the height of the peak due to nitrous oxide in the chromatogram obtained with the
reference gas is at least 35 per cent of the full scale of the recorder. The area of the peak due to
nitrous oxide in the chromatogram obtained with the gas to be examined is at least 98.0 per cent of
the peak due to nitrous oxide in the chromatogram obtained with the reference gas.
IDENTIFICATION
spectrum of nitrous oxide.
B. Place a glowing splinter of wood in the substance to be examined. The splinter bursts into flame.
C. Introduce the substance to be examined into alkaline pyrogallol solution R. A brown colour does not
develop.
TESTS
Examine the gaseous phase.
If the test is performed on a cylinder, keep the cylinder of the substance to be examined at room temperature
for at least 6 h before carrying out the tests. Keep the cylinder in the vertical position with the outlet valve
uppermost.
(2.1.6).
Nitrogen monoxide and nitrogen dioxide Not more than 2 ppm V/V, determined using a
nitrogen monoxide and nitrogen dioxide detector tube (2.1.6).
Carbon monoxide If the test is carried out on a cylinder, use the first portion of the gas to be examined.
Not more than 5 ppm V/V, determined using a carbon monoxide detector tube (2.1.6).
STORAGE
Store liquefied under pressure in suitable containers complying with the legal regulations. The taps
and valves are not greased or oiled.
IMPURITIES
A. carbon dioxide,
B. carbon monoxide,
C. nitrogen monoxide,
D. nitrogen dioxide,
E. water.
__________________________________________________________________________________________________________ Ph Eur
28-38
Nizatidine
NHMe
2N
N
H
C12H21N5O2S2
S
S
331.5
NMe2
76963-41-2
Nizatidine complies with the requirements of the 3rd edition of the European Pharmacopoeia [1453]. These
Action and use Histamine H2-receptor antagonist.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Nizatidine contains not less than 97.0 per cent and not more than the equivalent of 101.0 per cent of
(EZ)-N-[2-[[[2-[(dimethylamino)methyl]thiazol-4-yl]methyl]sulphanyl]ethyl]-N-methyl-2nitroethene-1,1-diamine, calculated with reference to the dried substance.
CHARACTERS
An almost white or slightly brownish, crystalline powder, sparingly soluble in water, soluble in
methanol.
IDENTIFICATION
B. Dissolve 0.10 g of the substance to be examined in methanol R and dilute to 100.0 ml with the
same solvent. Dilute 2.0 ml of the solution to 100.0 ml with methanol R. Examined between 220 nm
and 350 nm (2.2.25), the solution shows two absorption maxima, at 242 nm and 325 nm. The ratio
of the absorbance measured at the maximum at 325 nm to that measured at the maximum at 242 nm
is 2.2 to 2.5.
obtained with nizatidine CRS. Examine the substance prepared as discs.
D. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel plate R.
the same solvent.
Reference solution (a). Dissolve 50 mg of nizatidine CRS in methanol R and dilute to 10 ml with the
same solvent.
Reference solution (b). Dissolve 50 mg of nizatidine CRS and 50 mg of ranitidine hydrochloride CRS in
Apply to the plate 5 l of each solution. Develop over a path corresponding to two thirds of the
height of the plate using a mixture of 2 volumes of water R, 4 volumes of concentrated ammonia R1, 15
volumes of 2-propanol R and 25 volumes of ethyl acetate R. Allow the plate to dry in air and expose to
iodine vapour until the spots are clearly visible. Examine in daylight. The principal spot in the
chromatogram obtained with the test solution is similar in position and size to the principal spot in
the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows two clearly separated spots.
TESTS
Appearance of solution Dissolve 0.2 g of the substance to be examined in a 10 g/l solution of
hydrochloric acid R and dilute to 20 ml with the same acid solution. The solution is clear (2.2.1) and
pH (2.2.3). Dissolve 0.2 g of the substance to be examined in carbon dioxide-free water R and dilute to
20 ml with the same solvent. The pH of the solution is 8.5 to 10.0.
Related substances Examine by liquid chromatography (2.2.29) as described under Assay,
replacing the mixture of mobile phases by the following elution programme:
28-39
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
03
3 20
20 45
45 50
50 60
76
76 50
50
50 76
76
24
24 50
50
50 24
24
isocratic
linear gradient
isocratic
linear gradient
re-equilibration
full scale of the recorder. The test is not valid unless the retention time of nizatidine is between
10 min and 20 min and the symmetry factor of the peak due to nizatidine is not greater than 2.0.
Inject 20 l of reference solution (c). The test is not valid unless the resolution between the peak due
to nizatidine (first peak) and impurity F (second peak) is at least 2.0.
Inject 20 l of test solution (a). In the chromatogram obtained, the area of any peak apart from the
principal peak is not greater than 0.3 times the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.3 per cent) and the sum of the areas of all these peaks is not
solution (a) (1.5 per cent). Disregard any peak with an area less than 0.03 times the area of the
100C to 105C.
ASSAY
Test solution (a). Dissolve 50.0 mg of the substance to be examined in a mixture of 24 volumes of
mobile phase B and 76 volumes of mobile phase A and dilute to 10.0 ml with the same mixture of
mobile phases.
Test solution (b). Dissolve 15.0 mg of the substance to be examined in a mixture of 24 volumes of
mobile phases.
Reference solution (a). Dilute 1.0 ml of test solution (a) to 100.0 ml with a mixture of 24 volumes of
mobile phase B and 76 volumes of mobile phase A.
Reference solution (b). Dissolve 15.0 mg of nizatidine CRS in a mixture of 24 volumes of mobile phase
B and 76 volumes of mobile phase A and dilute to 50.0 ml with the same mixture of mobile phases.
Reference solution (c). Dissolve 5 mg of nizatidine CRS and 0.5 mg of nizatidine impurity F CRS in a
mixture of 24 volumes of mobile phase B and 76 volumes of mobile phase A and dilute to 100.0 ml
with the same mixture of mobile phases.
a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with octadecysilyl
as mobile phase at a flow rate of 1.0 ml/min a mixture of 35 volumes of mobile phase B and 65
volumes of mobile phase A:
Mobile phase A. Dissolve 5.9 g of ammonium acetate R in 760 ml of water R, add 1 ml of
diethylamine R, and adjust the pH to 7.5 with acetic acid R,
Mobile phase B. Methanol R,
Inject 20 l of reference solution (b). The test is not valid unless the retention time of nizatidine is
between 8 min and 10 min and the symmetry of the peak due to nizatidine is not greater than 2.0.
Inject reference solution (b) six times. The test is not valid unless the relative standard deviation of
the peak area for nizatidine is at most 2.0 per cent.
Inject 20 l of test solution (b) and 20 l of reference solution (b). Calculated the percentage
content of nizatidine from the area of the peaks and the declared content of nizatidine CRS.
STORAGE
28-40
IMPURITIES
S
=
NMe2
N
NHMe
NHMe
2N
A. (EZ)-N,N-dimethyl-2-nitroethene-1,1-diamine,
NHMe
SMe
2N
B. (EZ)-N-methyl-1-(methylsulphanyl)-2-nitroethen-1-amine,
O
NHMe
N
H
2N
C. (EZ)-N-[2-[[[2-[(dimethylamino)methyl]thiazol-4-yl]methyl]sulphinyl]ethyl]-N-methyl-2nitroethene-1,1-diamine,
D. RSCH2CH2NH2: N,N-dimethyl[4-[[(2-aminoethyl)sulphanyl]methyl]thiazol-2yl]methanamine,
O
2N
N
H
E. N-[2-[[[2-[(dimethylamino)methyl]thiazol-4-yl]methyl]sulphanyl]ethyl]-2-nitroacetamide,
NHMe
N
H
O2N
F.
S
S
NHMe
S
N
H
O2N
(EZ)-N1,N1[thiazole-2,4-diylbis(methylenesulphanediylethylene)]bis(N-methyl-2nitroethene-1,1-diamine),
S
HN
N
H
2N
R
S
G. (EZ)-N,N-bis[2-[[[2-[(dimethylamino)methyl]thiazol-4-yl]methyl]sulphanyl]ethyl]-2nitroethene-1,1-diamine,
S
Me2N
H2N
H. 2-(dimethylamino)thioacetamide,
O
eHN
N
H
I. N-[2-[[[2-[(dimethylamino)methyl]thiazol-4-yl]methyl]-sulphanyl]ethyl]-N-methylurea,
J. R-OH : [2-[(dimethylamino)methyl]thiazol-4-yl]methanol,
OH
N
eHN
K. 3-(methylamino)-5,6-dihydro-2H-1,4-thiazin-2-one oxime.
__________________________________________________________________________________________________________ Ph Eur
28-41
Nomegestrol Acetate
1/01
O
CH3
Me
OAc
H
H
H
O
Me
C23H30O4
370.5
58652-20-3
Nomegestrol Acetate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Nomegestrol acetate contains not less than 97.0 per cent and not more than the equivalent of
103.0 per cent of 6-methyl-3,20-dioxo-19-norpregna-4,6-dien-17-yl acetate, calculated with
CHARACTERS
soluble in alcohol.
IDENTIFICATION
with nomegestrol acetate CRS.
TESTS
Appearance of solution Dissolve 1.0 g in methylene chloride R and dilute to 10 ml with the same
solvent. The solution is clear (2.2.1) and not more intensely coloured than reference solution Y5
(Method II, 2.2.2).
solvent. The specific optical rotation is 60.0 to 64.0, calculated with reference to the dried
substance.
Test solution. Dissolve 25.0 mg of the substance to be examined in methanol R and dilute to 50.0 ml
Reference solution (b). Dissolve 25.0 mg of nomegestrol acetate impurity A CRS in methanol R and dilute
Reference solution (c). Dissolve 25.0 mg of nomegestrol acetate CRS in 20 ml of methanol R, add
0.25 ml of reference solution (b) and dilute to 50.0 ml with the mobile phase.
as mobile phase at a flow rate of 1.3 ml/min a mixture of 24 volumes of acetonitrile R, 38
volumes of methanol R and 38 volumes of water R,
as detector, a variable wavelength spectrophotometer capable of operating at 245 nm and at
290 nm.
Inject 10 l of reference solution (c) and record the chromatogram with the detector set at 245 nm.
When the chromatogram is recorded in the prescribed conditions, the retention times are:
nomegestrol acetate about 17 min and impurity A about 18.5 min. Adjust the sensitivity of the
system at 245 nm so that the height of the peak due to impurity A in the chromatogram obtained
with reference solution (c) is at least 50 per cent of the full scale of the recorder.
Measure the height hp above the baseline of the peak due to impurity A and the height hv above the
28-42
baseline of the lowest point of the curve separating this peak from the peak due to nomegestrol
acetate. The test is not valid unless hp is greater than 5 times hp.
Inject 10 l of reference solution (a) and record the chromatogram with the detector set at 290 nm.
Adjust the sensitivity of the system at 290 nm so that the height of the principal peak in the chromatogram obtained with reference solution (a) is at least 50 per cent of the full scale of the recorder.
Inject 10 l of the test solution and record the chromatograms at 245 nm and 290 nm for 1.5 times
the retention time of the principal peak.
In the chromatogram obtained with the test solution at 290 nm: the area of any peak, apart from
obtained with reference solution (a) (0.1 per cent). Disregard any peak with an area less than 0.04
times that of the principal peak in the chromatogram obtained with reference solution (a) (0.02 per
cent). In the chromatogram obtained with the test solution at 245 nm: the area of any peak corresponding to impurity A is not greater than 0.4 times the area of the peak due to impurity A in the
chromatogram obtained with reference solution (c) (0.2 per cent); the area of any peak, apart from
the principal peak and any peak corresponding to impurity A, is not greater than 0.2 times the area of
the peak due to impurity A in the chromatogram obtained with reference solution (c) (0.1 per cent).
Disregard any peak with an area less than 0.1 times that of the peak due to impurity A in the chromatogram obtained with reference solution (c) (0.05 per cent).
In the chromatograms obtained at 290 nm and 245 nm, the sum of the related substances apart
from impurity A is not greater than 0.3 per cent.
100105C.
ASSAY
Dissolve 50.0 mg in ethanol R and dilute to 100.0 ml with the same solvent. Dilute 2.0 ml of the
solution to 100.0 ml with ethanol R. Measure the absorbance (2.2.25) at the maximum at 287 nm.
STORAGE
IMPURITIES
O
Me
CH3
OAc
H
H
O
Me
A. 6-methyl-3,20-dioxo-19-norpregn-4-en-17-yl acetate.
__________________________________________________________________________________________________________ Ph Eur
28-43
Nonoxinol 9
corrected 1/01
O
OH
n
3C
26027-38-3
Nonoxinol 9 complies with the requirements of the 3rd edition of the European Pharmacopoeia [1454]. These
Action and use Pharmaceutical aid; spermicide.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Nonoxinol 9 is a liquid mixture consisting mainly of monononylphenyl ethers of macrogols corresponding to the formula: C9H19C6H4-(OCH2-CH2)n-OH where the average value of n is about 9 but
can be between 4 and 16. Nonoxinol 9 contains not less than 95.0 per cent and not more than
105.0 per cent of the equivalent of -(4-nonylphenyl)--hydroxynona(oxyethylene) calculated with
CHARACTERS
A clear, colourless to light yellow, viscous liquid, miscible with water, with alcohol and with olive oil.
IDENTIFICATION
obtained with nonoxinol CRS. Examine the substances as thin films between sodium chloride R plates.
B. Examine the chromatograms obtained in the assay. The retention times of the peaks in the
chromatogram obtained with the test solution are approximately the same as those of the peaks in the
TESTS
Acid value (2.5.1). Not more than 0.2, determined on 10.0 g dissolved in 50 ml of the prescribed
Macrogol Not more than 1.6 per cent. Transfer an accurately weighed sample of about 10 g to a
250 ml beaker. Add 100 ml of ethyl acetate R and stir continuously using a magnetic stirrer to
dissolve. Transfer to a 500 ml separating funnel fitted with a glass stopper with the aid of 100 ml of a
292 g/l solution of sodium chloride R. Insert the stopper and shake vigorously for 1 min. Remove the
stopper carefully. Immerse a thermometer in the mixture and support the separating funnel so that it
is partially immersed in a water-bath maintained at 50C. Swirl the separating funnel gently while
letting the internal temperature rise to between 40C and 45C, then immediately remove the
separating funnel from the bath, dry the outside surface and drain the lower layer into another
500 ml separating funnel. In the same manner, extract the ethyl acetate layer R a second time with
100 ml of a freshly prepared 292 g/l solution of sodium chloride R, combining the two lower extracts.
Wash the combined lower layers with 100 ml of ethyl acetate R and drain the lower layer into a clear
500 ml separating funnel. Discard the ethyl acetate layer. Shake the lower layer with two quantities,
each of 100 ml, of methylene chloride R, drain the lower layers through folded filter paper and combine
them in a 250 ml beaker. Evaporate on a water-bath to dryness and continue heating until the odour
of methylene chloride is no longer perceptible. Allow the beaker to cool. Add 25 ml of acetone R and
dissolve the residue using a magnetic stirrer. Filter through folded filter paper into a tared 250 ml
beaker, rinsing with two 25 ml portions of acetone R. Evaporate to dryness on a water-bath. Dry in
vacuo at 60C for 1 h. Allow the beaker to cool and weigh.
Cloud point Between 52C and 56C. Transfer 1.0 g to a 250 ml beaker, add 99 g of water R and
mix to dissolve. Transfer about 30 ml of the solution into a 70 ml test tube. Heat the test tube on a
water-bath stirring continuously with a thermometer until the solution becomes cloudy. Remove the
test tube from the bath immediately so that the temperature rises no more than 2C further and
continue stirring.
The cloud point is the temperature at which the solution becomes sufficiently clear that the entire
thermometer bulb is plainly seen.
28-44
Ethylene oxide and dioxan (2.4.25). Not more than 1 ppm of ethylene oxide and not more than
50 ppm of dioxan.
of water.
ASSAY
Test solution. Dissolve 50.0 mg of the substance to be examined in a mixture of 20 volumes of ethyl
acetate R and 80 volumes of hexane R and dilute to 25.0 ml with the same mixture of solvents and
mix.
Reference solution. Dissolve 50.0 mg of nonoxinol 9 CRS in a mixture of 20 volumes of ethyl acetate R
and 80 volumes of hexane R and dilute to 25.0 ml with the same mixture of solvents.
a stainless steel column 0.25 m long and 4 mm in internal diameter packed with diol silica gel for
Mobile phase A. A mixture of 20 volumes of ethyl acetate R and 80 volumes of hexane R,
Mobile phase B. A mixture of 2.5 volumes of methanol R and 97.5 volumes of ethyl acetate R,
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
02
2 10
10 20
20 30
100
100 84
84 70
0 16
16 30
70 62
62 57
30 38
38 43
equilibration
linear gradient
linear gradient
linear gradient
57 54
43 46
54 50
50
50 100
46 50
50
50 0
30 40
40 50
50 70
70 75
75 76
linear gradient
linear gradient
linear gradient
isocratic
re-equilibration

Inject 100 l of the test solution and 100 l of the reference solution. The nonoxinol oligomers
elute as sharp distinct peaks. Use the retention times for the determination of the oligomers.
Measure the responses for each peak.
The sum of the areas of any peaks corresponding to nonoxinols with chain lengths n < 4 or n >16 is
not greater than 1.0 per cent of the sum of the areas of the peaks corresponding to nonoxinols with
chain lengths n = 4 to n = 16.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
28-45
Noradrenaline Acid Tartrate / Norepinephrine Acid Tartrate

together when the substance is stated as the active ingredient in a formulated preparation (see Supplementary
H
OH
HO
NH2
OH
COOH
, HOOC
HO
C8H11NO3,C4H6O6,H2O
337.3
OH
69815-49-2
Noradrenaline Acid Tartrate complies with the requirements of the 3rd edition of the European
Pharmacopoeia for Noradrenaline Tartrate [0285]. These requirements are reproduced after the heading
Definition below.
Action and use Alpha-adrenoceptor agonist.
Preparation
Noradrenaline Injection/Norepinephrine Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Noradrenaline tartrate contains not less than 98.5 per cent and not more than the equivalent of
101.0 per cent of (R)-2-amino-1-(3,4-dihydroxyphenyl)ethanol hydrogen tartrate, calculated with
CHARACTERS
A white or almost white, crystalline powder, odourless, freely soluble in water, slightly soluble in
IDENTIFICATION
First identification: A, C, F.
Second identification: A, B, D, E, F.
A. Dissolve 2 g in 20 ml of a 5 g/l solution of sodium metabisulphite R and make alkaline by addition of
ammonia R. Keep in iced water for 1 h and filter. Reserve the filtrate for identification test F. Wash
the precipitate with three quantities, each of 2 ml, of water R, with 5 ml of alcohol R and finally with
5 ml of ether R and dry in vacuo for 3 h. The specific optical rotation (2.2.7) of the precipitate
(noradrenaline base) is 44 to 48, determined using a 20.0 g/l solution in 0.5M hydrochloric acid.
300 nm (2.2.25), the solution shows an absorption maximum at 279 nm. The specific absorbance at
the maximum is 79 to 85.
C. Examine by infrared absorption spectrophotometry (2.2.24) using noradrenaline base prepared as
described under identification test A and comparing with the spectrum obtained with noradrenaline
base prepared by the same method from a suitable amount of noradrenaline tartrate CRS. Examine the
substances prepared as discs.
D. Dissolve about 5 mg in 5 ml of water R. To 1 ml of the solution add 10 ml of buffer solution pH
3.6 R and 1 ml of 0.05M iodine. Allow to stand for 5 min. Add 2 ml of 0.1M sodium thiosulphate. A
faint red colour is produced.
E. To 1 ml of the solution prepared for identification test D add 1 ml of a 1 per cent V/V solution of
diethoxytetra-hydrofuran R in glacial acetic acid R. Heat at 80C for 2 min, cool in iced water and add
3 ml of a 20 g/l solution of dimethylaminobenzaldehyde R in a mixture of 1 volume of hydrochloric
acid R and 19 volumes of glacial acetic acid R. Mix and allow to stand for 2 min. The solution shows
an intense pink colour.
F. 0.2 ml of the filtrate obtained in identification test A gives reaction (b) of tartrates (2.3.1).
TESTS
Appearance of solution Dissolve 0.2 g in water R and dilute to 10 ml with the same solvent.
Examine the solution immediately. The solution is clear (2.2.1) and not more intensely coloured than
reference solution BY5 (Method II, 2.2.2).
28-46
Noradrenalone Dissolve 50.0 mg in 0.01M hydrochloric acid and dilute to 25.0 ml with the same
acid. The absorbance (2.2.25) of the solution measured at 310 nm is not greater than 0.20.
Adrenaline Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating
substance.
Test solution. Dissolve 0.25 g of the substance to be examined in water R and dilute to 10 ml with the
same solvent. Prepare immediately before use.
Reference solution (a). Dissolve 12.5 mg of adrenaline tartrate CRS in water R and dilute to 10 ml with
the same solvent. Prepare immediately before use.
Reference solution (c). Mix 2 ml of the test solution with 2 ml of reference solution (b).
Apply separately to the plate as bands 20 mm by 2 mm 6 l of the test solution, 6 l of reference
solution (a), 6 l of reference solution (b) and 12 l of reference solution (c). Allow to dry and spray
the bands with a saturated solution of sodium hydrogen carbonate R. Allow the plate to dry in air and
spray the bands twice with acetic anhydride R, drying between the two sprayings. Heat the plate at
50C for 90 min. Develop over a path of 15 cm using a mixture of 0.5 volumes of anhydrous formic
acid R, 50 volumes of acetone R and 50 volumes of methylene chloride R Allow the plate to dry in air
and spray with a solution freshly prepared by mixing 2 volumes of ethylenediamine R and 8 volumes of
methanol R and adding 2 volumes of a 5 g/l solution of potassium ferricyanide R. Dry the plate at 60C
for 10 min and examine in ultraviolet light at 254 nm and 365 nm. In the chromatogram obtained
with the test solution, any zone situated immediately above the most intense zone is not more intense
than the corresponding zone in the chromatogram obtained with reference solution (b) (1.0 per
cent). The test is not valid unless the chromatogram obtained with reference solution (c) shows
above the most intense zone a clearly separated zone corresponding to the most intense zone in the
ASSAY
Dissolve 0.300 g in 50 ml of anhydrous acetic acid R, heating gently if necessary. Titrate with 0.1M
perchloric acid using 0.1 ml of crystal violet solution R as indicator, until a bluish-green colour is
obtained.
STORAGE
Store in an airtight container, or preferably in a sealed tube under vacuum or under an inert gas,
__________________________________________________________________________________________________________ Ph Eur
28-47
Noradrenaline Hydrochloride / Norepinephrine Hydrochloride

revised 1/01
together when the substance is stated as the active ingredient in a formulated preparation (see Supplementary
H
HO
OH
NH2
,HCl
HO
C8H11NO3,HCl
205.6
329-56-6
Noradrenaline Hydrochloride complies with the requirements of the 3rd edition of the European
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Noradrenaline hydrochloride contains not less than 98.5 per cent and not more than the equivalent
of 101.0 per cent of (R)-2-amino-1-(3,4-dihydroxyphenyl)ethanol hydrochloride, calculated with
CHARACTERS
A white or brownish-white, crystalline powder, very soluble in water, slightly soluble in alcohol. It
becomes coloured on exposure to air and light.
IDENTIFICATION
First identification: A, D, F.
Second identification: A, B, C, E, F.
C. Dissolve 50.0 mg in 0.01M hydrochloric acid and dilute to 100.0 ml with the same acid. Dilute
D. Dissolve 2 g in 20 ml of a 5 g/l solution of sodium metabisulphite R and make alkaline by addition
of ammonia R. Keep in iced water for 1 h and filter. Wash the precipitate with three quantities, each
of 2 ml, of water R, with 5 ml of alcohol R and finally with 5 ml of ether R and dry in vacuo for 3 h.
Examine by infrared absorption spectrophotometry (2.2.24) the noradrenaline base thus prepared,
comparing with the spectrum obtained with noradrenaline base prepared by the same method from a
suitable amount of noradrenaline tartrate CRS. Examine the substances prepared as discs.
E. To 1 ml of a 1 g/l solution of the substance to be examined add 1 ml of a 1 per cent V/V solution
of diethoxytetrahydrofuran R in glacial acetic acid R. Heat at 80C for 2 min, cool in iced water and add
3 ml of a 20 g/l solution of dimethylaminobenzaldehyde R in a mixture of 1 volume of hydrochloric
acid R and 19 volumes of glacial acetic acid R. Mix and allow to stand for 2 min. An intense pink
colour develops.
F. 0.2 ml of solution S (see Tests) gives reaction (a) of chlorides (2.3.1).
TESTS
solvent.
Appearance of solution Dissolve 0.2 g in carbon dioxide-free water R and dilute to 10 ml with the
same solvent. Examine the solution immediately. The solution is clear (2.2.1) and not more intensely
coloured than a mixture of 0.2 ml of blue primary solution, 0.4 ml of yellow primary solution, 0.4 ml
of red primary solution and 9 ml of hydrochloric acid (10 g/l HCl) (Method II, 2.2.2).
28-48
Noradrenalone Dissolve 30.0 mg in 0.01M hydrochloric acid and dilute to 25.0 ml with the same
acid. The absorbance (2.2.25) of the solution measured at 310 nm is not greater than 0.20 (0.12 per
cent).
Adrenaline Examine by thin-layer chromatography (2.2.27), using a TLC silica gel G plate R.
Reference solution (a). Dissolve 12.5 mg of adrenaline tartrate CRS in water R and dilute to 10 ml with
Reference solution (c). Mix 2 ml of the test solution and 2 ml of reference solution (b).
Apply to the plate as bands 20 mm by 2 mm 6 l of the test solution, 6 l of reference solution (a),
6 l of reference solution (b) and 12 l of reference solution (c). Allow to dry in air and spray the
bands with a saturated solution of sodium hydrogen carbonate R. Allow the plate to dry in air and spray
the bands twice with acetic anhydride R, drying between the 2 sprayings. Heat the plate at 50C for
90 min. Develop over a path of 15 cm using a mixture of 0.5 volumes of anhydrous formic acid R, 50
volumes of acetone R and 50 volumes of methylene chloride R. Allow the plate to dry in air and spray
with a solution freshly prepared by mixing 2 volumes of ethylenediamine R and 8 volumes of
methanol R and adding 2 volumes of a 5 g/l solution of potassium ferricyanide R. Dry the plate at 60C
for 10 min and examine in ultraviolet light at 254 nm and 365 nm. In the chromatogram obtained
with the test solution, any zone situated immediately above the most intense zone is not more intense
than the corresponding zone in the chromatogram obtained with reference solution (b) (1.7 per
cent). The test is not valid unless the chromatogram obtained with reference solution (c) shows
above the most intense zone a clearly separated zone corresponding to the most intense zone in the
ASSAY
Dissolve 0.180 g in 50 ml of acetic anhydride R and add 10 ml of anhydrous formic acid R. Titrate with
STORAGE
Store in an airtight container, or preferably in a sealed tube under vacuum or under an inert gas,
__________________________________________________________________________________________________________ Ph Eur
28-49
Norethisterone
Me
OH
C
CH
H
H
O
C20H26O2
298.4
68-22-4
Norethisterone complies with the requirements of the 3rd edition of the European Pharmacopoeia [0234].
Preparation
Norethisterone Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Norethisterone contains not less than 98.0 per cent and not more than the equivalent of 102.0 per
cent of 17-hydroxy-19-nor-17-pregn-4-en-20-yn-3-one, calculated with reference to the dried
substance.
CHARACTERS
A white or yellowish-white, crystalline powder, practically insoluble in water, slightly soluble in
alcohol.
IDENTIFICATION
obtained with norethisterone CRS. Examine the substances prepared in the form of discs. If the spectra
obtained with the substance to be examined and the reference substance show differences, dissolve
the substances in chloroform R, evaporate to dryness on a water-bath and then record the spectra.
B. Examine by thin-layer chromatography (2.2.27), using kieselguhr G R as the coating substance.
Impregnate the plate by placing it in a tank containing the necessary quantity of a mixture of 10
volumes of formamide R and 90 volumes of acetone R so that the plate dips about 5 mm into the
liquid. When the front of the impregnation mixture has risen at least 1 cm above the level prescribed
for the mobile phase, remove the plate and allow it to stand at room temperature until the solvent has
completely evaporated (about 2 min to 5 min). Use the impregnated plate within 2 h and carry out
the chromatography in the same direction as the impregnation.
Test solution. Dissolve 10 mg of the substance to be examined in chloroform R and dilute to 10 ml with
the same solvent.
Reference solution. Dissolve 10 mg of norethisterone CRS in chloroform R and dilute to 10 ml with the
same solvent.
20 volumes of dioxan R and 80 volumes of hexane R. Heat the plate at 120C for 15 min and spray
with alcoholic solution of sulphuric acid R. Heat at 120C for 10 min to 15 min or until the spots
appear. Allow to cool and examine in daylight and in ultraviolet light at 365 nm. The principal spot
in the chromatogram obtained with the test solution is similar in position, colour, fluorescence and
size to the principal spot in the chromatogram obtained with the reference solution.
C. Dissolve about 2 mg in 2 ml of alcohol R, add 1 ml of ammoniacal silver nitrate solution R and heat
on a water-bath. The solution becomes turbid and a white precipitate is formed which becomes grey
when heated. A silver mirror is deposited on the walls of the tube.
D. Dissolve about 2 mg in a cooled mixture of 2 ml of ethanol R and 2 ml of sulphuric acid R and heat
to 70C. The resulting solution is dichroic, appearing blue-violet in transmitted light and red in
reflected light. The solution shows a bright-red fluorescence in ultraviolet light at 365 nm.
E. Dissolve about 2 mg in 2 ml of alcohol R, add 1 ml of a 10 g/l solution of butylhydroxytoluene R in
alcohol R and 2 ml of 1M sodium hydroxide. Heat in a water-bath at 80C for 30 min and cool to room
temperature. A yellowish-pink colour is produced.
28-50
TESTS
Solution S Dissolve 0.200 g in dioxan R and dilute to 10.0 ml with the same solvent.
solution Y6 (Method I, 2.2.2).
Specific optical rotation (2.2.7). Dilute 5.0 ml of solution S to 10.0 ml with dioxan R. The specific
optical rotation is 33 to 37, calculated with reference to the dried substance.
Absorbance (2.2.25). Dissolve 10.0 mg in alcohol R and dilute to 100.0 ml with the same solvent.
Dilute 10.0 ml of this solution to 100.0 ml with alcohol R. The solution shows an absorption
maximum at 240 nm. The specific absorbance at the maximum is 550 to 590, calculated with reference to the dried substance.
Related substances Examine by thin-layer chromatography (2.2.27), using a suitable silica gel as
the coating substance.
Reference solution (a). Dilute 1.0 ml of the test solution to 200 ml with a mixture of 1 volume of
Reference solution (b). Dissolve 25 mg of ethisterone CRS in a mixture of 1 volume of methanol R and 9
volumes of chloroform R, add 5 ml of the test solution and dilute to 100 ml with the same mixture of
solvents.
Apply separately to the plate, as two applications of 5 l, 10 l of each solution. Develop over a path
of 15 cm using a mixture of 10 volumes of acetone R and 90 volumes of chloroform R. Allow the plate
to dry in air, spray with alcoholic solution of sulphuric acid R and heat at 100C to 105C for 5 min.
Examine in ultraviolet light at 365 nm.
more intense than the spot in the chromatogram obtained with reference solution (a) (0.5 per cent).
The test is not valid unless the chromatogram obtained with reference solution (b) shows two clearly
separated spots of approximately equal intensity.
100C to 105C for 3 h.
ASSAY
Dissolve 0.200 g in 40 ml of tetrahydrofuran R. Add 10 ml of a 100 g/l solution of silver nitrate R.
Using 2 ml of bromocresol green solution R as indicator, titrate with 0.1M sodium hydroxide until a violet
colour is obtained. Carry out a blank titration.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
28-51
Norethisterone Acetate
Me
OAc
C
CH
H
H
O
C22H28O3
340.5
51-98-9
Norethisterone Acetate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Norethisterone acetate contains not less than 98.0 per cent and not more than the equivalent of
101.0 per cent of 17-hydroxy-19-nor-17-pregn-4-en-20-yn-3-one 17-acetate, calculated with reference to the dried substance.
CHARACTERS
A white or yellowish-white, crystalline powder, practically insoluble in water, soluble in alcohol and
in ether.
IDENTIFICATION
First identification: B, C.
Second identification: A, D, E, F.
obtained with norethisterone acetate CRS. Examine the substances prepared as discs.
Reference solution (a). Dissolve 10 mg of norethisterone acetate CRS in a mixture of 1 volume of
solvents.
Reference solution (b). Dissolve 10 mg of deoxycortone acetate CRS and 10 mg of norethisterone acetate
CRS in a mixture of 1 volume of methanol R and 9 volumes of methylene chloride R and dilute to 50 ml
with the same mixture of solvents.
10 volumes of acetone R and 90 volumes of methylene chloride R. Allow the plate to dry in air and
solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a). Spray the plate with alcoholic solution of sulphuric acid R. Heat at 120C for 10 min
or until the spots appear. Allow to cool. Examine in daylight and in ultraviolet light at 365 nm. The
principal spot in the chromatogram obtained with the test solution is similar in position, colour in
daylight, fluorescence in ultraviolet light at 365 nm and size to the principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained
with reference solution (b) shows two clearly separated spots of approximately equal intensity.
15 ml glass tube with a ground-glass stopper or a polytetrafluoro-ethylene cap. Add 10 ml of saturated
methanolic potassium hydrogen carbonate solution R and immediately pass a current of nitrogen R briskly
for 4 h. Allow to cool.
28-52
Reference solution (a). Dissolve 25 mg of norethisterone acetate CRS in methanol R and dilute to 5 ml
with the same solvent. This solution is also used to prepare reference solution (b). Dilute 2 ml of the
saturated methanolic potassium hydrogen carbonate solution R and immediately pass a current of
nitrogen R briskly through the solution for 5 min. Stopper the tube. Heat in a water-bath at 60C,
protected from light, for 4 h. Allow to cool.
volumes of methylene chloride R. Develop over a path of 15 cm. Allow the plate to dry in air and then
expose it to ultraviolet light at 254 nm for about 10 min. The principal spot in each of the chromatograms obtained with the test solutions is similar in position and size to the principal spot in the
chromatogram obtained with the corresponding reference solution. Spray the plate with alcoholic
solution of sulphuric acid R. Heat at 120C for 10 min or until the spots appear. Allow to cool.
Examine in daylight and in ultraviolet light at 365 nm. The principal spot in each of the chromatograms obtained with the test solutions is similar in position, colour in daylight, fluorescence in ultraviolet light at 365 nm and size to the principal spot in the chromatogram obtained with the corresponding reference solution. The principal spots in the chromatograms obtained with test solution (b)
and reference solution (b) have an Rf value distinctly lower than that of the principal spots in the
chromatograms obtained with test solution (a) and reference solution (a).
E. Dissolve about 2 mg in 2 ml of alcohol R, add 1 ml of ammoniacal silver nitrate solution R and heat
on a water-bath. The solution becomes turbid and a white precipitate is formed which becomes grey
when heated. A silver mirror is deposited on the walls of the tube.
F. About 10 mg gives the reaction of acetyl (2.3.1).
TESTS
solvent. The specific optical rotation is 32 to 38, calculated with reference to the dried substance.
Reference solution (a). Dissolve 2 mg of deoxycortone acetate CRS and 2 mg of norethisterone acetate CRS
as mobile phase at a flow rate of 1 ml per minute a mixture prepared as follows: carefully mix
350 ml of water R and 600 ml of acetonitrile R and allow to equilibrate; dilute to 1000 ml with
water R and mix again,
Adjust the sensitivity so that the height of the principal peak in the chromatogram obtained with
reference solution (b) is 70 per cent to 90 per cent of the full scale of the recorder.
conditions, the retention times are: deoxycortone acetate, about 7.5 min; norethisterone acetate,
about 9 min. The test is not valid unless the resolution between the peaks corresponding to
deoxycortone acetate and norethisterone acetate is at least 3.5; if necessary, adjust the concentration
of acetonitrile in the mobile phase.
chromatography for twice the retention time of the principal peak. In the chromatogram obtained
with the test solution: the area of any peak, apart from the principal peak, is not greater than half the
area of the principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent);
the sum of the areas of all the peaks, apart from the principal peak, is not greater than the area of the
any peak with an area less than 0.025 times the area of the principal peak in the chromatogram
obtained with reference solution (b).
100C to 105C.
ASSAY
Dissolve 0.200 g in 40 ml of tetrahydrofuran R. Add 10 ml of a 100 g/l solution of silver nitrate R and
28-53
titrate with 0.1M sodium hydroxide, determining the end-point potentiometrically (2.2.20). Carry out a
blank titration.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
28-54
Norfloxacin
HN
Et
N
COOH
O
C16H18FN3O3
319.3
70458-96-7
Norfloxacin complies with the requirements of the 3rd edition of the European Pharmacopoeia [1248]. These
Preparations
Norfloxacin Eye Drops
Norfloxacin Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Norfloxacin contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of 1-ethyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid, calculated with
CHARACTERS
A white or pale-yellow, hygroscopic, photosensitive, crystalline powder, very slightly soluble in water,
slightly soluble in acetone and in alcohol.
IDENTIFICATION
with norfloxacin CRS. Examine the substances prepared as discs.
TESTS
Appearance of solution Dissolve 0.5 g in a previously filtered 4 g/l solution of sodium hydroxide R in
methanol R and dilute to 50 ml with the same solution. The solution is not more opalescent than
reference suspension II (2.2.1) and not more intensely coloured than reference solution B7 (Method
II, 2.2.2).
plate R, previously washed with methanol R and dried in air.
Test solution (a). Dissolve 40 mg of the substance to be examined in a mixture of equal volumes of
Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with a mixture of equal volumes of
Reference solution (a). Dilute 1 ml of test solution (b) to 50 ml with a mixture of equal volumes of
Reference solution (b). Dissolve 4.0 mg of norfloxacin impurity A CRS in a mixture of equal volumes of
methanol R and methylene chloride R and dilute to 5 ml with the same mixture of solvents. Dilute 1 ml
of this solution to 2 ml with test solution (b).
Apply to the plate 5 l of test solution (a) and 5 l of each reference solution. Develop over a path of
18 cm using a mixture of 8 volumes of water R, 14 volumes of diethylamine R, 20 volumes of
toluene R, 40 volumes of chloroform R and 40 volumes of methanol R. Dry the plate in a current of air
and examine in ultraviolet light at 254 nm and then 365 nm. Any spot in the chromatogram obtained
with test solution (a), apart from the principal spot, is not more intense than the principal spot in the
chromatogram obtained with reference solution (a) (0.2 per cent) and there are no more than three
such spots. The test is not valid unless, in the chromatogram obtained with reference solution (b),
the ratio of the Rf value of impurity A to the Rf value of norfloxacin is at least 1.2.
100C to 105C under high vacuum for 2 h.
28-55
ASSAY
1 ml of 0.1M perchloric acid is equivalent to 31.93 mg of C16H18FN3O3.
STORAGE
IMPURITIES
Et
N
COOH
F
O
A. R = Cl: 7-chloro-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid,

B. R = NH-CH2-CH2-NH2: 7-[(2-aminoethyl)amino]-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline3-carboxylic acid.
__________________________________________________________________________________________________________ Ph Eur
28-56
Norgestrel
Et
CH
OH
H
H
O
and enantiomer
C21H28O2
312.5
6533-00-2
Norgestrel complies with the requirements of the 3rd edition of the European Pharmacopoeia [0940]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Norgestrel contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent of
rac-13-ethyl-17-hydroxy-18,19-dinor-17-pregn-4-en-20-yn-3-one, calculated with reference to
CHARACTERS
methylene chloride, slightly soluble in alcohol.
IDENTIFICATION
A. Dissolve 0.5 g in methylene chloride R and dilute to 10.0 ml with the same solvent. The angle of
optical rotation (2.2.7) is +0.05 to 0.05.
obtained with norgestrel CRS.
TESTS
coating substance.
Test solution. Dissolve 0.2 g of the substance to be examined in methylene chloride R and dilute to
Reference solution (a). Dilute 1 ml of the test solution to 10 ml with methylene chloride R. Dilute 1 ml
of this solution to 20 ml with methylene chloride R.
Reference solution (b). Dilute 4 ml of reference solution (a) to 10 ml with methylene chloride R.
Reference solution (c). Dissolve 5 mg of norgestrel CRS and 5 mg of ethinylestradiol CRS in methylene
chloride R and dilute to 50 ml with the same solvent.
20 volumes of ethyl acetate R and 80 volumes of methylene chloride R. Allow the plate to dry in air,
spray with a 100 g/l solution of phosphomolybdic acid R in alcohol R, heat at 100C to 105C for
15 min and examine immediately. Any spot in the chromatogram obtained with the test solution,
apart from the principal spot, is not more intense than the principal spot in the chromatogram
obtained with reference solution (a) (0.5 per cent) and at most two such spots are more intense than
the spot in the chromatogram obtained with reference solution (b) (0.2 per cent). The test is not
spots.
100C to 105C.
ASSAY
Dissolve 0.200 g in 45 ml of tetrahydrofuran R. Add 10 ml of a 100 g/l solution of silver nitrate R.
After 1 min, titrate with 0.1M sodium hydroxide determining the end-point potentiometrically (2.2.20).
Carry out a blank titration.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
28-57
Nortriptyline Hydrochloride
,HCl
NHMe
C19H21N,HCl
299.8
894-71-3
Nortriptyline Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparations
Nortriptyline Capsules
Nortriptyline Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Nortriptyline hydrochloride contains not less than 98.0 per cent and not more than the equivalent of
101.0 per cent of 3-(10,11-dihydro-5H-dibenzo[a,d][7]annulen-5-ylidene)-N-methylpropan-1-amine
hydrochloride, calculated with reference to the dried substance.
CHARACTERS
A white or almost white powder, sparingly soluble in water, soluble in alcohol and in methylene
chloride.
IDENTIFICATION
First identification: C, E.
B. Dissolve 20.0 mg in methanol R and dilute to 100.0 ml with the same solvent. Dilute 5.0 ml of this
solution to 100.0 ml with methanol R. Examined between 230 nm and 350 nm (2.2.25), the solution
shows an absorption maximum at 239 nm. The specific absorbance at the maximum is 465 to 495.
spectrum of nortriptyline hydrochloride.
D. Dissolve 50 mg in 3 ml of warm water R, cool and add 0.05 ml of a 25 g/l solution of
quinhydrone R in methanol R. A red colour develops slowly.
E. 50 mg gives reaction (b) of chlorides (2.3.1).
TESTS
Appearance of solution Dissolve 0.5 g in water R with gentle heating and dilute to 25 ml with the
same solvent. The solution is clear (2.2.1) and not more intensely coloured than reference solution B7
(Method II, 2.2.2).
Acidity or alkalinity Dissolve 0.2 g with gentle heating in carbon dioxide-free water R and dilute to
10 ml with the same solvent. Add 0.1 ml of methyl red solution R and 0.2 ml of 0.01M sodium
hydroxide. The solution is yellow. Add 0.4 ml of 0.01M hydrochloric acid. The solution is red.
Related substances Prepare the solutions in subdued light and develop the chromatograms protected from
light. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel plate R.
Test solution (a). Dissolve 0.20 g of the substance to be examined in alcohol R and dilute to 10 ml
Reference solution (a). Dissolve 10 mg of dibenzosuberone CRS in alcohol R and dilute to 10 ml with the
same solvent. Dilute 1 ml of the solution to 100 ml with alcohol R.
Reference solution (b). Dissolve 10 mg of norcyclobenzaprine CRS in alcohol R and dilute to 10 ml with
the same solvent. Dilute 1 ml of the solution to 100 ml with alcohol R.
Reference solution (c). To 0.1 ml of test solution (b) add 10 ml of reference solution (b).
Apply to the plate 5 l of each solution. Develop over a path of 15 cm in an unsaturated tank using a
mixture of 3 volumes of diethylamine R, 15 volumes of ethyl acetate R and 85 volumes of
28-58
cyclohexane R. Allow the plate to dry in air and spray with a freshly prepared mixture of 4 volumes of
formaldehyde solution R and 96 volumes of sulphuric acid R. Examine immediately in ultraviolet light at
365 nm and then at 254 nm. In the chromatogram obtained with test solution (a): any spot corresponding to dibenzosuberone, is not more intense than the spot in the chromatogram obtained with
reference solution (a) (0.05 per cent); and any spot in the chromatogram obtained with test solution
(b), apart from the principal spot and any spot corresponding to dibenzosuberone, is not more
intense than the spot in the chromatogram obtained with reference solution (b) (0.1 per cent). The
test is not valid unless the chromatogram obtained with reference solution (c) shows two clearly
separated spots.
100C to 105C for 2 h.
ASSAY
Dissolve 0.250 g in 30 ml of alcohol R. Add 1.0 ml of 0.1M hydrochloric acid. Carry out a potentiometric titration (2.2.20), using 0.1M sodium hydroxide. Read the volume added between the two
1 ml of 0.1M sodium hydroxide is equivalent to 29.98 mg of C19H22ClN.
STORAGE
IMPURITIES
A. 10,11-dihydro-5H-dibenzo[a,d][7]annulen-5-one(dibenzosuberone),
NHMe
B. 3-(5H-dibenzo[a,d][7]annulen-5-ylidene)-N-methylpropan-1-amine (norcyclobenzaprine),
HO
*
NHMe
C. 10,11-dihydro-5-[3-(methylamino)propylidene]-5H-dibenzo[a,d][7]annulen-10-ol.
__________________________________________________________________________________________________________ Ph Eur
28-59
Noscapine
O
NMe
O
MeO
H
H
O
MeO
MeO
C22H23NO7
O
413.4
128-62-1
Noscapine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0516]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Noscapine contains not less than 98.5 per cent and not more than the equivalent of 100.5 per cent of
(S)-6,7-dimethoxy-3-[(R)-8-methoxy-2-methyl-6,7-methylenedioxy-1,2,3,4-tetrahydro-1isoquinolyl]phthalide, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder or colourless crystals, practically insoluble in water at 20C, very slightly
soluble in water at 100C, soluble in acetone, slightly soluble in alcohol and in ether. It dissolves in
strong acids; on dilution of the solution with water, the base may be precipitated.
IDENTIFICATION
First identification: D.
Second identification: A, B, C.
C. Dissolve 50 mg in methanol R and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml of the
shows two absorption maxima, at 291 nm and 310 nm. The ratio of the absorbance at the maximum
at 310 nm to that at the maximum at 291 nm is 1.2 to 1.3.
D. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum
obtained with noscapine CRS.
TESTS
Appearance of solution Dissolve 0.2 g in acetone R and dilute to 10 ml with the same solvent.
Examined immediately after preparation, the solution is clear (2.2.1) and not more intensely coloured
Specific optical rotation (2.2.7). Dissolve 0.500 g in 0.1M hydrochloric acid and dilute to 25.0 ml
with the same acid. The specific optical rotation is +42 to +48, calculated with reference to the
dried substance.
coating substance.
Test solution. Dissolve 0.25 g of the substance to be examined in acetone R and dilute to 10 ml with
the same solvent.
1 volume of concentrated ammonia R, 3 volumes of alcohol R, 20 volumes of acetone R and 20 volumes
of toluene R. Dry the plate in a current of air and spray with dilute potassium iodobismuthate solution R.
more intense than the spot in the chromatogram obtained with the reference solution (0.5 per cent).
100C to 105C.
28-60
ASSAY
Dissolve 0.350 g in 40 ml of anhydrous acetic acid R, warming gently. Titrate with 0.1M perchloric acid,
STORAGE
__________________________________________________________________________________________________________ Ph Eur
28-61
Noscapine Hydrochloride
revised 1/01
O
NMe
O
MeO
H
,HCl
H
O
MeO
MeO
C22H23NO7,HCl,H2O
467.9
912-60-7
Noscapine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Noscapine hydrochloride contains not less than 98.5 per cent and not more than the equivalent of
100.5 per cent of (S)-6,7-dimethoxy-3-[(R)-8-methoxy-2-methyl-6,7-methylenedioxy-1,2,3,4tetrahydro-1-isoquinolyl]phthalide hydrochloride, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder or colourless crystals, hygroscopic, freely soluble in water and in alcohol.
Aqueous solutions are faintly acid; the base may be precipitated when the solutions are allowed to
stand.
IDENTIFICATION
First identification: C, E.
B. Dissolve 50 mg in methanol R containing 17 ppm of NH3 and dilute to 100.0 ml with the same
solvent. Dilute 1.0 ml of the solution to 10.0 ml with methanol R containing 17 ppm of NH3.
Examined between 250 nm and 350 nm (2.2.25), the solution shows two absorption maxima, at
291 nm and 310 nm. The ratio of the absorbance measured at the maximum at 310 nm to that
measured at the maximum at 291 is 1.2 to 1.3.
C. Examine the precipitate obtained in identification test E by infrared absorption spectrophotometry
(2.2.24), comparing with the spectrum obtained with noscapine CRS.
D. The melting point (2.2.14) of the precipitate obtained in identification test E is 174C to 177C.
E. Dissolve about 40 mg in a mixture of 2 ml of water R and 3 ml of alcohol R and add 1 ml of dilute
ammonia R2. Heat until dissolution is complete. Allow to cool, scratching the wall of the tube with a
glass rod. Filter. The filtrate gives reaction (a) of chlorides (2.3.1). Wash the precipitate with water R,
dry at 100C to 105C and reserve for identification tests C and D.
TESTS
Appearance of solution Dissolve 0.5 g in water R, add 0.3 ml of 0.1M hydrochloric acid and dilute to
25 ml with water R. The solution is clear (2.2.1) and not more intensely coloured than reference
pH (2.2.3). Dissolve 0.2 g in 10 ml of carbon dioxide-free water R. The pH of the solution is not less
than 3.0.
with the same acid. The specific optical rotation is +38.5 to +44.0, calculated with reference to the
dried substance.
the same solvent.
28-62
of concentrated ammonia R, 3 volumes of alcohol R, 20 volumes of acetone R and 20 volumes of
toluene R. Dry the plate in a current of air and spray with dilute potassium iodobismuthate solution R.
more intense than the spot in the chromatogram obtained with the reference solution (0.5 per cent).
100C to 105C.
ASSAY
1 ml of 0.1M sodium hydroxide is equivalent to 44.99 mg of C22H24ClNO7.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
28-63
Nutmeg Oil
1/01
Nutmeg Oil complies with the requirements of the 3rd edition of the European Pharmacopoeia [1552]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Nutmeg oil is obtained by steam distillation of the dried and crushed kernels of Myristica fragrans
Houtt.
CHARACTERS
A colourless or pale yellow liquid, with a spicy odour.
IDENTIFICATION
Second identification: A.
A. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel plate R.
Test solution. Dissolve 1 ml of the substance to be examined in toluene R and dilute to 10 ml with the
same solvent.
Reference solution. Dissolve 20 l of myristicine R in 10 ml of toluene R.
Apply to the plate as bands 10 l of each solution. Develop over a path of 15 cm using a mixture of 5
volumes of ethyl acetate R and 95 volumes of toluene R. Allow the plate to dry in air and spray with
vanillin reagent R. Heat the plate at 100C to 105C for 10 min. Examine in daylight. The chromatogram obtained with the reference solution shows in the upper third a pink to reddish-brown zone
(myristicine). The chromatogram obtained with the test solution shows a series of zones of which one
is similar in position and colour to the zone in the chromatogram obtained with the reference
solution. Above this zone a brownish zone (safrole) and a violet zone (hydrocarbons) are present.
Below the myristicine zone, 5 blue zones of variable intensity are present.
B. Examine the chromatograms obtained in the test for chromatographic profile. The retention times
of the principal peaks in the chromatogram obtained with the test solution are similar to those in the
TESTS
Optical rotation (2.2.7): +8 to +18.
Chromatographic profile Examine by gas chromatography (2.2.28).
Test solution. The substance to be examined.
Reference solution. Dissolve 15 l of -pinene R, 15 l of -pinene R, 15 l of sabinene R, 5 l of car-3ene R, 5 l of limonene R, 5 l of -terpinene R, 5 l of terpinen-4-ol R, 5 l of safrole R and 10 l of
myristicine R in 1 ml of hexane R.
a fused-silica column 25 m to 60 m long and about 0.3 mm in internal diameter, coated with
macrogol 20,000 R as the bonded phase,
a split ratio of 1:100.
Time
(min)
Column
Temperature
(C)
0 10 50
10 75 50 180
75 130 180
Injection port
200 220
Detector
240 250
Rate (C
per min)
2
Comment
isothermal
linear gradient
isothermal
Inject 0.2 l of the reference solution. When the chromatogram is recorded in the prescribed
28-64
conditions, the components elute in the order indicated in the composition of the reference solution.
Record the retention times of these substances.
The test is not valid unless the resolution between the peaks corresponding to -pinene and
sabinene is at least 1.5.
Inject 0.2 l of the test solution. Using the retention times determined from the chromatogram
obtained with the reference solution, locate the components of the reference solution in the chromatogram obtained with the test solution. Determine the percentage content of each of these
components by the normalisation procedure.
The percentages are within the following ranges:
-pinene
15 per cent to 28 per cent
-pinene
sabinene
car-3-ene
0.5 per cent to 2.0 per cent
limonene
-terpinene
terpinen-4-ol 2.0 per cent to 6.0 per cent
safrole
less than 2.5 per cent
myristicine
STORAGE
Store in a well-filled, airtight container, protected from light and heat.
The following type chromatogram is given for information.
Volts
3 5
0.5
0.4
0.3
4
0.2
0.1
1. -pinene; 2. -pinene; 3. sabinene; 4. car-3-ene; 5. limonene;

6. -terpinene; 7. terpinen-4-ol; 8. safrole; 9. myristicine.
Fig. 15521 Chromatographic profile of nutmeg oil

__________________________________________________________________________________________________________ Ph Eur
130
125
120
115
110
105
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
0.0
28-65
Nystatin
corrected 1/01
1400-61-9
Nystatin complies with the requirements of the 3rd edition of the European Pharmacopoeia [0517]. These
Preparations
Nystatin Ointment
Nystatin Oral Suspension
Nystatin Pastilles
Nystatin Pessaries
Nystatin Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Nystatin is an antifungal substance produced by the growth of certain strains of Streptomyces noursei.
It contains mainly tetraenes, the principal component being nystatin A1. The potency is not less than
4400 I.U. per milligram, calculated with reference to the dried substance.
PRODUCTION
If intended for oral administration, the method of manufacture is validated to demonstrate that the
product if tested would comply with the following test.
Abnormal toxicity (2.6.9). Inject intraperitoneally into each mouse a quantity equivalent to not less
than 600 I.U. suspended in 0.5 ml of a 5 g/l solution of acacia R.
CHARACTERS
A yellow or slightly brownish powder, hygroscopic, very slightly soluble in water, freely soluble in
dimethylformamide, slightly soluble in methanol, practically insoluble in alcohol and in ether.
IDENTIFICATION
A. Examine the solution prepared in the test for absorbance between 220 nm and 350 nm (2.2.25).
The solution shows four absorption maxima at 230 nm, 291 nm, 305 nm and 319 nm, and a
shoulder at 280 nm. The ratios of the absorbances at the maxima at 291 nm and 319 nm to the
absorbance at the maximum at 305 nm are 0.61 to 0.73 and 0.83 to 0.96, respectively. The ratio of
the absorbance measured at the maximum at 230 nm to the absorbance measured at the shoulder at
280 nm is 0.83 to 1.25.
B. To about 2 mg add 0.1 ml of hydrochloric acid R. A brown colour develops.
C. To about 2 mg add 0.1 ml of sulphuric acid R. A brown colour develops that becomes violet on
standing.
TESTS
Absorbance (2.2.25). Dissolve 0.10 g in a mixture of 5.0 ml of glacial acetic acid R and 50 ml of
methanol R and dilute to 100.0 ml with methanol R. Dilute 1.0 ml of the solution to 100.0 ml with
methanol R. Determined at the maximum at 305 nm within 30 min of preparation of the solution, the
absorbance is not less than 0.60.
ASSAY
Protect the solutions from light throughout the assay. Carry out the microbiological assay of antibiotics
(2.7.2). Dissolve the substance to be examined and nystatin CRS in dimethylformamide R and dilute
with a mixture of 5 volumes of dimethylformamide R and 95 volumes of buffer solution pH 6.0.
STORAGE
Store in an airtight container, protected from light, at a temperature of 2C to 8C.
__________________________________________________________________________________________________________ Ph Eur
28-66
Octanoic Acid
corrected 1/01
Caprylic Acid
COOH
H3C
C8H16O2
144.2
124-07-2
Octanoic Acid complies with the requirements of the 3rd edition of the European Pharmacopoeia for Caprylic
Acid [1401]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Caprylic acid contains not less than 99.0 per cent and not more than the equivalent of 100.5 per cent
of octanoic acid, calculated with reference to the anhydrous substance.
CHARACTERS
A clear, colourless or slightly yellowish, oily liquid, very slightly soluble in water, very soluble in
acetone and in alcohol. It dissolves in dilute solutions of alkali hydroxides.
IDENTIFICATION
A. It complies with the test for relative density (see Tests).
B. Examine the chromatograms obtained in the test for related substances. The retention time and
size of the principal peak in the chromatogram obtained with the test solution are approximately the
same as those of the principal peak in the chromatogram obtained with reference solution (a).
TESTS
reference solution Y5 (Method II, 2.2.2).
Related substances Examine by gas chromatography (2.2.28).
Test solution. Dissolve 0.10 g of the substance to be examined in ethyl acetate R and dilute to 10.0 ml
Reference solution (a). Dissolve 0.10 g of caprylic acid CRS in ethyl acetate R and dilute to 10.0 ml with
the same solvent.
Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with ethyl acetate R. Dilute 5.0 ml
of the solution to 50.0 ml with ethyl acetate R.
a fused-silica column 30 m long and 0.25 mm in internal diameter coated with macrogol 20,000
2-nitroterephthalate R (film thickness 0.25 m),
a split ratio of 1:100,
Time
(min)
Column 0 1
1 25
25 35
Temperature
(oC)
Rate
(oC/min)
Comment
100
isothermal
linear gradient
100 220
220
Injection port
250
Detector
250
isothermal
Inject 1 l of reference solution (b). The test is not valid unless in the chromatogram obtained the
principal peak has a signal-to-noise ratio of at least 5.
Inject 1 l of the test solution and 1 l of reference solution (a). Calculate the percentage content of
related substances from the areas of the peaks in the chromatogram obtained with the test solution by
the normalisation procedure, disregarding any peaks with an area less than 0.5 times the area of the
peak in the chromatogram obtained with reference solution (b). The content of any related substance
is not greater than 0.3 per cent and the sum of the contents is not greater than 0.5 per cent.
28-67
Heavy metals (2.4.8). Dissolve 2.0 g in alcohol R and dilute to 20 ml with the same solvent. 12 ml of
the solution complies with limit test B for heavy metals (10 ppm). Prepare the standard using 1 ml of
lead standard solution (10 ppm Pb) R and 9 ml of alcohol R.
ASSAY
Dissolve 0.125 g in 25 ml of alcohol R. Titrate with 0.1M sodium hydroxide, determining the end-point
STORAGE
IMPURITIES
COOH
H3C
A. hexanoic acid,
H3C
COOH
B. heptanoic acid,
H3C
COOH
C. nonanoic acid,
COOH
H3C
D. decanoic acid,
E. valproic acid,
COOR
H3C
F. R = CH3: methyl octanoate,

G. R = C2H5: ethyl octanoate,
COOMe
H3C
H. methyl decanoate,
O
H3C
CH3
I. undecan-2-one,
O
H3C
O
and enantiomer
J. 5-butyltetrahydrofuran-2-one (-hydroxyoctanoic acid lactone).

__________________________________________________________________________________________________________ Ph Eur
28-68
Octoxinol 10
1/01
C14H21-[C2H4O]n-OH
Octoxinol 10 complies with the requirements of the 3rd edition of the European Pharmacopoeia [1553]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Octoxinol 10 contains not less than 97.0 per cent and not more than the equivalent of 105.0 per cent
of -[4-(1,1,3,3-tetramethylbutyl)phenyl]--hydroxydeca(oxyethylene). It is an anhydrous liquid
mixture consisting mainly of mono-octylphenyl ethers of macrogols, with the general formula
C14H21-[OCH2-CH2]n-OH where the value of n is 10 on average but may range between 5 and 15. It
may contain free macrogols.
CHARACTERS
A clear, colourless or light yellow, viscous liquid, miscible with water, with ethanol and with vegetable
oils.
IDENTIFICATION
obtained with octoxinol 10 CRS.
B. Examine the chromatograms obtained in the assay. The retention time of the principal peak in the
chromatogram obtained with the test solution is approximately the same as that of the principal peak
in the chromatogram obtained with the reference solution.
TESTS
Acidity and alkalinity Boil 1.0 g with 20 ml of carbon dioxide-free water R for 1 min, with constant
stirring. Cool and filter. To 10 ml of the filtrate, add 0.05 ml of bromothymol blue solution R1. Not
more than 0.5 ml of 0.01M hydrochloric acid or 0.01M sodium hydroxide is required to change the
Hydroxyl value (2.5.3, Method A): 85 to 101.
Cloud point 63C to 69C. Dissolve 1.0 g in 99 g of water R. Transfer about 30 ml of this solution
to a test-tube, place the tube in a water-bath and stir constantly using a glass rod until the solution
becomes cloudy. Remove the test-tube from the water-bath (so that the temperature does not
increase more than 2C), continue to shake. The cloud point is the temperature at which the solution
becomes sufficiently clear.
and not more than 10 ppm of residual dioxan. Use a correction factor of 1/5 in the calculation of the
dioxan content.
Heavy metals (2.4.8). Dissolve 10.0 g in distilled water R and dilute to 100.0 ml with the same
solvent. 12 ml of this solution complies with limit test A for heavy metals (10 ppm). Prepare the
standard using lead standard solution (1 ppm Pb) R.
of water.
ASSAY
Reference solution. Dissolve 0.250 g of octoxinol 10 CRS in the mobile phase and dilute to 10.0 ml
as mobile phase at a flow rate of 1 ml/min a mixture of 20 volumes of water R and 80 volumes of
methanol R,
Inject 10 l of the test solution and 10 l of the reference solution. The oligomers of octoxinol elute
28-69
10.948
as a principal peak, usually with shoulders and irregularities (Fig. 1553-1). Measure the area of the
peak corresponding to octoxinol 10, including the shoulders and irregularities. Determine the
percentage content of octoxinol 10 from the area of the peak obtained with the test solution and the
declared content of octoxinol 10 CRS.
mAU
400
300
8.927
100
14.233
200
0
2.5
7.5
10
12.5
15
17.5
min
Fig. 1553-1 Chromatogram for the assay

STORAGE
__________________________________________________________________________________________________________ Ph Eur
28-70
Octyl Gallate
COO[CH2] 7CH3
HO
HO
OH
C15H22O5
282.3
1034-01-1
Definition Octyl Gallate is octyl 3,4,5-trihydroxybenzoate.

Characteristics A white or creamy white powder; odourless or almost odourless.
Practically insoluble in water; very soluble in methanol; freely soluble in acetone, in ethanol (96%), in
ether and in propane-1,2-diol; sparingly soluble in arachis oil and in chloroform.
Identification
A. The light absorption, Appendix II B, in the range 230 to 350 nm of a 0.002% w/v solution in
methanol exhibits a maximum only at 275 nm. The absorbance at the maximum is about 0.75.
B. Carry out the method for gas chromatography, Appendix III B, using a solution prepared in the
following manner. Boil 0.5 g with 50 ml of 5M sodium hydroxide under a reflux condenser for 10
minutes, cool and extract with 50 ml of ether.
The chromatographic procedure may be carried out using a glass column (1.5 m 4 mm) packed
with acid-washed, silanised diatomaceous support (80 to 100 mesh) coated with 10% w/w of free fatty
acid phase (Supelco FFAP is suitable) and maintained at 180.
The principal peak in the chromatogram has the same retention time as octan-1-ol, examined under
the same conditions.
C. Dissolve 5 mg in a mixture of 25 ml of acetone and 25 ml of water and add 0.5 ml of iron(III)
chloride solution. A purplish black colour is produced which rapidly becomes bluish black.
Acidity Dissolve 0.4 g in 50 ml of acetone, add 50 ml of carbon dioxide-free water and titrate with 0.1M
sodium hydroxide VS, using bromocresol green solution as indicator, to the blue colour indicative of pH
5.0. Repeat the operation without the substance being examined. The difference between the
titrations does not exceed 0.1 ml.
Chloride Shake 0.50 g with 100 ml of water at 60 for 10 minutes, cool and filter. 15 ml of the
filtrate complies with the limit test for chlorides, Appendix VII (0.07%).
Sulphate Shake 0.25 g with 30 ml of water at 60 for 10 minutes, cool and filter. 15 ml of the filtrate
complies with the limit test for sulphates, Appendix VII (0.12%).
1 g.
Storage Octyl Gallate should be kept in a well-closed container and protected from light. Contact
with metals should be avoided.
Action and use Antioxidant.
28-71
Octyldodecanol
Octyldodecanol complies with the requirements of the 3rd edition of the European Pharmacopoeia [1136].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Octyldodecanol is a condensation product of saturated liquid fatty alcohols. It contains not less than
90 per cent of (RS)-2-octyldodecan-1-ol (C20H42O; Mr 298.6), the remainder consisting mainly of
related alcohols.
CHARACTERS
A clear, colourless to yellowish, oily liquid, practically insoluble in water, miscible with alcohol.
IDENTIFICATION
B. It complies with the test for refractive index (see Tests).
C. It complies with the test for hydroxyl value (see Tests).
substance.
Test solution. Dissolve 0.2 g of the substance to be examined in toluene R and dilute to 20 ml with the
same solvent.
Reference solution. Dissolve 0.2 g of octyldodecanol CRS in toluene R and dilute to 20 ml with the same
solvent.
volumes of ethyl acetate R and 95 volumes of toluene R. Allow the plate to dry in air. Spray with about
7 ml of a mixture of 1 volume of a 25 g/l solution of vanillin R in alcohol R and 4 volumes of sulphuric
acid R and heat at 130C for 5 min to 10 min. The principal spot in the chromatogram obtained with
TESTS
Acidity or alkalinity Mix 5.0 g thoroughly for 1 min with a mixture of 0.1 ml of bromothymol blue
solution R1, 2 ml of heptane R and 10 ml of water R. If the aqueous layer is blue, not more than
0.15 ml of 0.01M hydrochloric acid is required to change the colour of the indicator to yellow. If the
aqueous layer is yellow add 0.45 ml of 0.1M sodium hydroxide and shake vigorously. After standing to
ensure complete separation the aqueous layer is blue.
Hydroxyl value (Method A, 2.5.3): 175 to 190.
Peroxide value (2.5.5). Not more than 5.0.
Saponification value (2.5.6). Not more than 5, determined on 2.00 g.
of water.
ASSAY
Examine by gas chromatography (2.2.28).
Test solution. Dissolve 0.100 g in hexane R and dilute to 10.0 ml with the same solvent.
a fused-silica column 20 m long and 0.3 mm in internal diameter, coated with poly(dimethyl)(diphenyl)siloxane R or poly(dimethyl)(diphenyl)(divinyl)siloxane R (film thickness 0.25 m),
hydrogen for chromatography R or helium for chromatography R at a flow rate of 1.1 ml per minute
as the carrier gas,
28-72
maintaining the temperature of the column at 280C, that of the injection port at 290C and that of
the detector at 300C. Inject 2 l of the test solution.
Determine the percentage content of C20H42O from the area of the peak in the chromatogram
obtained with the test solution by the normalisation procedure.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
28-73
Conjugated Oestrogens
1/01
Me
H
O
S
NaO
Me
S
NaO
C18H21O5NaS + C18H19O5NaS
O
372.4 + 370.4
Conjugated Oestrogens complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Conjugated Estrogens [1512]. These requirements are reproduced after the heading Definition below.
Action and use Oestrogen.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Conjugated estrogens are a mixture of various conjugated forms of estrogens obtained from the urine
of pregnant mares or by synthesis, dispersed in a suitable powdered diluent.
The two principal components are 17-oxoestra-1,3,5(10)-trien-3-yl sodium sulphate (sodium
estrone sulphate) and 17-oxoestra-1,3,5(10),7-tetraen-3-yl sodium sulphate (sodium equilin
sulphate). Concomitants are sodium 17-estradiol sulphate, sodium 17-dihydroequilin sulphate
and sodium 17-dihydroequilin sulphate.
Conjugated estrogens contain not less than 52.5 per cent and not more than 61.5 per cent of
sodium estrone sulphate, not less than 22.5 per cent and not more than 30.5 per cent of sodium
equilin sulphate, not less than 2.5 per cent and not more than 9.5 per cent of sodium 17-estradiol
sulphate, not less than 13.5 per cent and not more than 19.5 per cent of sodium 17-dihydroequilin
sulphate, not less than 0.5 per cent and not more than 4.0 per cent of sodium 17-dihydroequilin
sulphate. The total of sodium estrone sulphate and sodium equilin sulphate is not less than 79.5 per
cent and not more than 88.0 per cent.
All percentages are related to the labelled content.
CHARACTERS
An almost white to brownish amorphous powder.
IDENTIFICATION
A. Examine the chromatograms obtained in the assay. The retention times and sizes of the 2
principal peaks corresponding to estrone and equilin in the chromatogram obtained with test solution
(a) are approximately the same as those of the 2 principal peaks in the chromatogram obtained with
B. Examine the chromatogram obtained with test solution (b) in the chromatographic profile. The
chromatogram exhibits additional peaks corresponding to 17-estradiol, 17-dihydroequilin and
17-dihydroequilin, at relative retentions with reference to 3-O-methylestrone (internal standard) of
about 0.24, 0.30 and 0.35 respectively.
TESTS
Chromatographic profile Carry out the test as prescribed in the assay with the following additional
information.
Test solution (b). Prepare the test solution as described in the assay, do not add the sulphatase and use
6.0 ml of the upper layer instead of 3.0 ml. Prepare a blank in the same manner.
Reference solution (b). Prepare the reference solution as described in the assay. Dilute tenfold with
ethanol R before adding the internal standard.
Inject 1 l of reference solution (a). Measure the areas of the peaks due to 17-dihydroequilin,
estrone and 3-O-methylestrone, with relative retentions with reference to 3-O-methylestrone of about
28-74
0.30, 0.80 and 1 respectively.
Inject 1 l of test solution (a). Locate the peaks with relative retentions with reference to 3-Omethylestrone of 1 and about 0.24, 0.29, 0.30, 0.35, 0.56, 0.64, 0.90 and 1.3 and measure their
areas.
Calculate the percentage content of the components occurring as sodium sulphate salts using
expression (1) below.
Inject 1 l of reference solution (b). Measure the areas of the peaks due to estrone and 3-Omethylestrone, with relative retentions with reference to 3-O-methylestrone of about 0.80 and 1
respectively.
Inject 1 l of test solution (b). Locate the peaks with relative retentions with reference to 3-Omethylestrone of about 0.30, 0.80 and 0.87 and measure the sum of the areas.
Calculate the percentage content of 17-dihydroequilin, estrone and equilin occurring as free
steroids using expression (2):
S A S1 mR 137.8 1000
S R S1 m LC
(1)
S1 mE 100 1000
S FS
S E S1 m LC
(2)
SI = area of the peak due to the internal standard in the chromatogram obtained with the
corresponding reference solution,
SI = area of the peak due to the internal standard in the chromatogram obtained with the
corresponding test solution,
SR = area of the peak due to the reference substance (Table 1512.-1) in the chromatogram
obtained with the corresponding reference solution,
SA = area of the peak due to the analyte in the chromatogram obtained with the corresponding
test solution,
mR = mass of the reference substance (Table 1512.-1) in the corresponding reference solution,
in milligrams,
m = mass of the substance to be examined in the corresponding test solution, in milligrams,
SFS = sum of the areas of the peaks due to 17-dihydroequilin, estrone and equilin in the
chromatogram obtained with the corresponding test solution,
SE = area of the peak due to estrone CRS in the chromatogram obtained with the corresponding
reference solution,
mE = mass of estrone CRS in the corresponding reference solution, in milligrams,
LC = labelled content, in milligrams per gram.
sodium 17-estradiol sulphate
2.5 to 9.5 per cent
sodium 17-dihydroequilin sulphate
13.5 to 19.5 per cent
sodium 17-dihydroequilin sulphate
0.5 to 4.0 per cent
sodium 17-estradiol sulphate
not more than 2.25 per cent
sodium 17-dihydroequilenin sulphate
sodium 17-dihydroequilenin sulphate
sodium 8,9-didehydroestrone sulphate
sodium equilenin sulphate
Total estrone, equilin and 17-dihydroequilin
Table 15121
Relative retention
(to 3-O-methylestrone)
Analyte
Quantified with
reference to CRS
Present as
0.24
17-estradiol
17-estradiol
17
-dihydroequilin CRS
estrone CRS
sodium sulphate
17-dihydroequilin
17-dihydroequilin
17
-dihydroequilin CRS
17
-dihydroequilin CRS
estrone CRS
estrone CRS
0.29
0.30
0.35
0.56
0.64
0.80
0.87
0.90
1
1.3
17-dihydroequilenin
17-dihydroequilenin
estrone
equilin
8,9-didehydroestrone
3-O-methylestrone
equilenin
estrone CRS
estrone CRS
estrone CRS
(internal standard)
estrone CRS
sodium sulphate
free steroid, sodium sulphate (assay)
sodium sulphate
sodium sulphate
sodium sulphate
sodium sulphate
sodium sulphate
28-75
ASSAY
Examine by gas chromatography (2.2.28), using 3-O-methylestrone R as the internal standard.
Internal standard solution. Dissolve 8 mg of 3-O-methylestrone R in 10.0 ml of ethanol R. Dilute 2.0 ml
of this solution to 10.0 ml with ethanol R.
Acetate buffer solution pH 5.2. Dissolve 10 g of sodium acetate R in 100 ml of water R and add 10 ml of
acetic acid dilute R. Dilute with water R to 500 ml and adjust to pH 5.2 0.1.
Test solution (a). Considering the labelled content, transfer an accurately weighed quantity corresponding to about 2 mg of conjugated estrogens to a 50 ml centrifuge tube containing 15 ml of
acetate buffer solution pH 5.2 and 1 g of barium chloride R. Cap the tube tightly and shake for
30 min. If necessary, adjust the pH of the solution to 5.0 0.5 with acetic acid R or a 120 g/l solution
of sodium acetate R. Sonicate for 30 s, then shake for 30 min. Add a suitable sulphatase preparation
equivalent to 2500 units and shake mechanically for 10 min in a water-bath at 50 1C. Swirl the
tube by hand, then shake mechanically for 10 min in the water-bath. Allow to cool. Add 15.0 ml of
ethylene chloride R to the mixture, immediately cap the tube tightly and shake for 15 min. Centrifuge
for 10 min or until the lower layer is clear. Draw out the organic layer to a screw cap tube and add
5 g of anhydrous sodium sulphate R and shake. Allow the solution to stand until clear. Protect the
solution from any loss due to evaporation.
Transfer 3.0 ml of the clear solution to a suitable centrifuge tube fitted with a screw cap. Add
1.0 ml of the internal standard solution. Evaporate the mixture to dryness with the aid of a stream of
nitrogen R maintaining the temperature below 50C. To the dry residue add 15 l of anhydrous
pyridine R and 65 l of N,O-bis(trimethylsilyl) trifluoroacetamide R containing 1 per cent chlorotrimethylsilane R. Immediately cap the tube tightly, mix thoroughly and allow to stand for 15 min. Add 0.5 ml
of toluene R and mix mechanically.
Reference solution (a). Dissolve separately 8 mg of estrone CRS, 7 mg of equilin CRS and 5 mg of 17dihydroequilin CRS in 10.0 ml of ethanol R. Dilute together 2.0 ml, 1.0 ml and 1.0 ml respectively of
these solutions to 10.0 ml with ethanol R. Transfer 1.0 ml of this solution and 1.0 ml of the internal
standard solution to a centrifuge tube fitted with a screw-cap. Evaporate the mixture to dryness with
the aid of a stream of nitrogen R, maintaining the temperature below 50C. To the dry residue add
15 l of anhydrous pyridine R and 65 l of N,O-bis(trimethylsilyl)trifluoroacetamide R containing 1 per
cent chlorotrimethylsilane R. Immediately cap the tube tightly, mix and allow to stand for 15 min. Add
0.5 ml of toluene R.
a fused-silica column 15 m long and 0.25 mm in internal diameter coated with
poly[(cyanopropyl)(methyl)][(phenyl)(methyl)]siloxane R (film thickness 0.25 m),
hydrogen for chromatography R as the carrier gas at a flow rate of 2 ml/min,
a split ratio of 20 to 30,
maintaining the temperature of the column at 220C and that of the injection port and of the
detector at 260C. Set the temperature and the flow rate of the carrier gas in such a manner that the
required resolution is achieved.
Inject 1 l of reference solution (a). The relative retentions with reference to 3-O-methylestrone are
about 0.30, 0.80, 0.87, and 1 for 17-dihydroequilin, estrone, equilin and 3-O-methylestrone,
respectively.
The test is not valid unless the resolution between estrone and equilin is at least 1.2. The relative
standard deviation of the ratio of the peak area due to estrone to that of the internal standard,
obtained from at least 6 injections, is not more than 2.0 per cent.
Inject 1 l of reference solution (a). Measure the areas of the peaks due to estrone or equilin and
O-methylestrone. Inject 1 l of test solution (a). Measure the areas of the peaks due to estrone,
equilin and 3-O-methylestrone.
Calculate the percentage content of sodium estrone sulphate and sodium equilin sulphate using
expression (1).
STORAGE
Do not freeze.
LABELLING
The label states:
the name of the substance,
the content of the substance,
the nature of the diluent.
28-76
IMPURITIES AND CONCOMITANTS
Me R2
R1
H
H
R3O
A. R1 = OH, R2 = H, R3 = SO3Na: 17-hydroxyestra-1,3,5(10)-trien-3-yl sodium sulphate (sodium

17-estradiol sulphate),
D. R1 = H, R2 = OH, R3 = SO3Na: 17-hydroxyestra-1,3,5(10)-trien-3-yl sodium sulphate (sodium
17-estradiol sulphate),
I. R1 + R2 = O, R3 = H: 3-hydroxyestra-1,3,5(10)-trien-17-one (estrone),
Me R2
R1
R3O
B. R1 = OH, R2 = H, R3 = SO3Na: 17-hydroxyestra-1,3,5(10),7-tetraen-3-yl sodium sulphate

(sodium 17-dihydroequilin sulphate),
C. R1 = H, R2 = OH, R3 = SO3Na: 17-hydroxyestra-1,3,5(10),7-tetraen-3-yl sodium sulphate
(sodium 17-dihydroequilin sulphate),
J. R1 + R2 = O, R3 = H: 3-hydroxyestra-1,3,5(10),7-tetraen-17-one (equilin),
K. R1 = OH, R2 = R3 = H: estra-1,3,5(10),7-tetraene-3,17-diol (17-dihydroequilin),
Me R2
R1
O
O
S
NaO
E. R1 = OH, R2 = H: 17-hydroxyestra-1,3,5(10),6,8-pentaen-3-yl sodium sulphate (sodium 17dihydroequilenin sulphate),

F. R1 = H, R2 = OH: 17-hydroxyestra-1,3,5(10),6,8-pentaen-3-yl sodium sulphate (sodium 17dihydroequilenin sulphate),
H. R1 + R2 = O: 17-oxoestra-1,3,5(10),6,8-pentaen-3-yl sodium sulphate (sodium equilenin
sulphate),
Me O
O
S
NaO
G. 17-oxoestra-1,3,5(10),8-tetraen-3-yl sodium sulphate (sodium 8,9-didehydroestrone sulphate).

__________________________________________________________________________________________________________ Ph Eur
28-77
Ofloxacin
H
MeN
O
N
Me
COOH
O
and enantiomer
C18H20FN3O4
361.4
82419-36-1
Ofloxacin complies with the requirements of the 3rd edition of the European Pharmacopoeia [1455]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ofloxacin contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of
(RS)-9-fluoro-3-methyl-10-(4-methylpiperazin-1-yl)-7-oxo-2,3-dihydro-7H-pyrido-[1,2,3de][1,4]benzoxazine-6-carboxylic acid, calculated with reference to the dried substance.
CHARACTERS
A pale yellow or bright yellow, crystalline powder, slightly soluble in water, soluble in glacial acetic
acid, slightly soluble to soluble in methylene chloride, slightly soluble in methanol.
IDENTIFICATION
with ofloxacin CRS. Examine the substances prepared as discs.
TESTS
Absorbance (2.2.25). Dissolve 0.5 g in 0.1M hydrochloric acid and dilute to 100 ml with the same
solvent. The absorbance of the solution measured at 440 nm is not greater than 0.25.
Optical rotation (2.2.7). Dissolve 0.300 g in a mixture of 10 volumes of methanol R and 40 volumes
of methylene chloride R and dilute to 10 ml with the same mixture of solvents. The angle of optical
rotation is 0.10 to +0.10.
Impurity A Examine by thin-layer chromatography (2.2.27), using a TLC silica gel GF254 plate R
(2 m to 10 m).
Test solution. Dissolve 0.250 g of the substance to be examined in a mixture of 10 volumes of
methanol R and 40 volumes of methylene chloride R and dilute to 5.0 ml with the same mixture of
solvents.
Reference solution. Dissolve 10 mg of ofloxacin impurity A CRS in a mixture of 10 volumes of
methanol R and 40 volumes of methylene chloride R and dilute to 100.0 ml with the same mixture of
solvents.
of glacial acetic acid R, 1 volume of water R and 2 volumes of ethyl acetate R. Allow the plate to dry in
air and examine in ultraviolet light at 254 nm. Any spot due to impurity A in the chromatogram
obtained with the test solution is not more intense than the spot in the chromatogram obtained with
the reference solution (0.2 per cent).
before use.
Test solution. Dissolve 10.0 mg of the substance to be examined in a mixture of 10 volumes of
acetonitrile R and 60 volumes of water R and dilute to 50.0 ml with the same mixture of solvents.
Reference solution (a). Dilute 1.0 ml of the test solution to 50.0 ml with a mixture of 10 volumes of
acetonitrile R and 60 volumes of water R. Dilute 1.0 ml of this solution to 10.0 ml with a mixture of
10 volumes of acetonitrile R and 60 volumes of water R.
Reference solution (b). Dissolve 10.0 mg of ofloxacin impurity E CRS in a mixture of 10 volumes of
28-78
Mix 10.0 ml of this solution with 5.0 ml of the test solution. Dilute to 50.0 ml with a mixture of 10
volumes of acetonitrile R and 60 volumes of water R. Dilute 1.0 ml of this solution to 50.0 ml with a
mixture of 10 volumes of acetonitrile R and 60 volumes of water R.
as mobile phase a mixture prepared as follows: dissolve 4.0 g of ammonium acetate R and 7.0 g of
sodium perchlorate R in 1300 ml of water R. Adjust to pH 2.2 with phosphoric acid R. Add 240 ml
of acetonitrile R. Adjust the flow rate of the mobile phase so that a retention time of about 20 min
is obtained for ofloxacin,
Inject 10 l of reference solution (b). Adjust the sensitivity of the system so that the heights of the
recorder. The test is not valid unless: in the chromatogram obtained, the resolution between the
peaks corresponding to impurity E and ofloxacin is at least 2.0. Inject 10 l of the test solution and
10 l of reference solution (a). Continue the chromatography for 2.5 times the retention time of the
principal peak. In the chromatogram obtained with the test solution, the area of any peak, apart from
with reference solution (a) (0.2 per cent); the sum of the areas of all the peaks is not greater than 2.5
times the area of the principal peak in the chromatogram obtained with reference solution (a)
(0.5 per cent). Disregard any peak with an area less than 0.1 times the area of the principal peak in
105C for 4 h.
ASSAY
Dissolve 0.300 g in 100 ml of anhydrous acetic acid R. Titrate with 0.1M perchloric acid determining
STORAGE
IMPURITIES
H
O
F
Me
and enantiomer
COOH
O
A. (RS)-9,10-difluoro-3-methyl-7-oxo-2,3-dihydro-7H-pyrido[1,2,3-de][1,4]benzoxazine-6carboxylic acid (FPA),

H
MeN
O
N
Me
and enantiomer
F
O
B. (RS)-9-fluoro-3-methyl-10-(4-methylpiperazin-1-yl)-2,3-dihydro-7H-pyrido[1,2,3de][1,4]benzoxazin-7-one,
H
MeN
O
N
Me
and enantiomer
COOH
O
C. (RS)-3-methyl-10-(4-methylpiperazin-1-yl)-7-oxo-2,3-dihydro-7H-pyrido[1,2,3de][1,4]benzoxazine-6-carboxylic acid,
28-79
H
O
F
Me
and enantiomer
COOH
O
MeN
D. (RS)-10-fluoro-3-methyl-9-(4-methylpiperazin-1-yl)-7-oxo-2,3-dihydro-7H-pyrido[1,2,3de][1,4]benzoxazine-6-carboxylic acid,
H
HN
O
N
Me
and enantiomer
COOH
O
E. (RS)-9-fluoro-3-methyl-10-(piperazin-1-yl)-7-oxo-2,3-dihydro-7H-pyrido[1,2,3de][1,4]benzoxazine-6-carboxylic acid,
O
MeN
O
N
Me
and enantiomer
COOH
O
F. (RS)-9-fluoro-3-methyl-10-(4-methyl-4-oxopiperazin-1-yl)-7-oxo-2,3-dihydro-7Hpyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic acid.
__________________________________________________________________________________________________________ Ph Eur
28-80
Oleic Acid
H
COOH
COOH
H
C18H34O2
282.5
112-80-1
Oleic Acid complies with the requirements of the 3rd edition of the European Pharmacopoeia [0799]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Oleic acid contains not less than 60.0 per cent of (Z)-octadec-9-enoic acid (C18H34O2; Mr 282.5)
together with varying amounts of saturated and other unsaturated fatty acids. It may contain a suitable antioxidant.
CHARACTERS
A clear, yellowish or brownish, oily liquid, practically insoluble in water, miscible with alcohol, with
ether and with methylene chloride.
IDENTIFICATION
A. It complies with the test for iodine value (see Tests).
B. Examine the chromatogram obtained in the assay. The principal peak in the chromatogram
obtained with the test solution corresponds to methyl oleate.
C. To 1 ml add 1 ml of alcohol R. The mixture is clear. Add 0.1 ml of methyl red solution R. The
solution is orange or red.
D. Into a test-tube with an inner diameter of about 12 mm introduce a mixture of 1 ml of nitric
acid R and 1 ml of water R. Deposit 1 ml of the substance to be examined on the surface of the
mixture. Introduce 0.5 g of copper R (wire or turnings not longer than 10 mm) into the lower layer.
Allow to stand under a hood for 4 h. The upper layer solidifies.
TESTS
Appearance of solution The substance to be examined is not more intensely coloured than reference solution Y1 or BY1 (Method I, 2.2.2).
Acid value (2.5.1): 195 to 202, determined on 0.5 g.
Iodine value (2.5.4): 87 to 97.
Fats and hydrocarbons Heat 1 ml to boiling with 5 ml of sodium carbonate solution R and 25 ml of
water R. The aqueous layer while still hot is not more opalescent than reference suspension II (2.2.1).
Water-soluble acids Shake 5 ml with 5 ml of water R for 2 min. Filter the aqueous layer through
moistened paper. To the filtrate add 0.05 ml of methyl orange solution R. The liquid does not become
red.
ASSAY
Carry out the test for foreign oils in fatty oils by gas chromatography (2.4.22), using a test solution
prepared as follows.
Test solution. Weigh 4.0 g in a 100 ml flask with a ground-glass neck fitted with a reflux condenser
and a device allowing the passage of a current of gas. Add 40 ml of anhydrous methanol R and 0.5 ml
of sulphuric acid R. Fit the condenser and pass a current of nitrogen R at a flow rate of about
100 ml per minute. Shake and heat under reflux for 30 min. Cool with running water and transfer to
a separating funnel. Rinse the flask with 20 ml of heptane R and transfer to the separating funnel. Add
about 40 ml of water R and shake gently. If an emulsion forms, add heptane R dropwise using a
pipette. Allow the layers to separate, remove the aqueous layer, shake a second time with 20 ml of
28-81
heptane R and allow to decant. Combine the organic layers. Wash with two quantities, each of 20 ml,
of water R, dry over anhydrous sodium sulphate R, filter through cotton into a 50 ml conical flask and
evaporate to a suitable volume on a water-bath whilst passing a current of nitrogen R.
The principal peak corresponds to methyl oleate. Calculate the percentage content of oleic acid by
normalisation.
STORAGE
Store in a well-closed container, protected from light, at a temperature of 8C to 15C.
LABELLING
The label states the name and concentration of any antioxidant.
__________________________________________________________________________________________________________ Ph Eur
28-82
Oleoyl Macrogolglycerides
Oleoyl Macrogolglycerides comply with the requirements of the 3rd edition of the European Pharmacopoeia
PhEur ___________________________________________________________________________________________________________
D EFINITION
Oleoyl macrogolglycerides are mixtures of monoesters, diesters and triesters of glycerol and monoesters and diesters of macrogols. They are obtained by partial alcoholysis of an unsaturated oil mainly
containing triglycerides of oleic acid using macrogol with a mean relative molecular mass between
300 and 400 or by esterification of glycerol and macrogol with unsaturated fatty acids or by mixing
glycerol esters and condensates of ethylene oxide with the fatty acids of this unsaturated oil.
CHARACTERS
Amber oily liquids, which may give rise to a deposit after prolonged periods at 20C, practically
insoluble but dispersible in water, freely soluble in methylene chloride.
The viscosity at 40C is about 35 mPas, the relative density at 20C is about 0.95 and the
refractive index at 20C is about 1.47.
IDENTIFICATION
substance.
Apply to the plate 10 l of the test solution. Develop over a path of 15 cm using a mixture of 30
volumes of hexane R and 70 volumes of ether R. Allow the plate to dry in air. Spray with a 0.1 g/l
solution of rhodamine B R in alcohol R and examine in ultraviolet light at 365 nm. The chromatogram
shows a spot corresponding to triglycerides with an Rf value of about 0.9 (Rst 1) and spots corresponding to 1,3-diglycerides (Rst 0.7), to 1,2-diglycerides (Rst 0.6), to monoglycerides (Rst 0.1) and to
esters of macrogol (Rst 0).
B. They comply with the test for hydroxyl value (see Tests).
C. They comply with the test for fatty acid composition (see Tests).
D. They comply with the test for saponification value (see Tests).
TESTS
Hydroxyl value (2.5.3). 45 to 65, determined on 1.0 g (Method A).
Iodine value (2.5.4). 75 to 95.
Peroxide value (2.5.5). Not more than 12.0, determined on 2.0 g.
Alkaline impurities Introduce 5.0 g into a test-tube and carefully add a mixture, neutralised if
necessary with 0.01M hydrochloric acid or with 0.01M sodium hydroxide, of 0.05 ml of a 0.4 g/l solution
of bromophenol blue R in alcohol R, 0.3 ml of water R and 10 ml of alcohol R. Shake and allow to stand.
Not more than 1.0 ml of 0.01M hydrochloric acid is required to change the colour of the upper layer to
yellow.
Free glycerol Not more than 3.0 per cent. Dissolve 1.20 g in 25.0 ml of methylene chloride R. Heat if
necessary. After cooling, add 100 ml of water R. Shake and add 25.0 ml of a 6 g/l solution of periodic
acid R. Shake and allow to stand for 30 min. Add 40 ml of a 75 g/l solution of potassium iodide R.
Allow to stand for 1 min. Add 1 ml of starch solution R. Titrate the iodine with 0.1M sodium
thiosulphate. Carry out a blank titration.
1 ml of 0.1M sodium thiosulphate is equivalent to 2.3 mg of glycerol.
Fatty acid composition Examine by gas chromatography (2.4.22). The fatty-acid fraction has the
following composition:
linoleic acid: 15.0 per cent to 35.0 per cent,
linolenic acid: not more than 2.0 per cent,
eicosenoic acid: not more than 2.0 per cent.
28-83
and 10 ppm of residual dioxan. When determining the dioxan content, apply a correction factor of
1/5 in the calculation.
of water. Use a mixture of 30 volumes of anhydrous methanol R and 70 volumes of methylene chloride R
as solvent.
STORAGE
Store in a well-closed container, protected from light and at room temperature.
LABELLING
The label states the type of macrogol used (mean relative molecular mass) or the number of units of
ethylene oxide per molecule (nominal value).
__________________________________________________________________________________________________________ Ph Eur
28-84
Refined Olive Oil

corrected 1/01
Refined Olive Oil complies with the requirements of the 3rd edition of the European Pharmacopoeia [1456].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Refined olive oil is the fatty oil obtained by refining of crude olive oil, obtained by cold expression or
other suitable mechanical means from the ripe drupes of Olea europaea L. A suitable antioxidant may
be added.
CHARACTERS
A clear, colourless or greenish-yellow, transparent liquid, practically insoluble in alcohol, miscible
with light petroleum (50C to 70C).
When cooled, it begins to become cloudy at 10C and becomes a butter-like mass at about 0C. It
has a relative density of about 0.913.
IDENTIFICATION
A. It complies with the test for absorbance (see Tests).
B. Carry out the test for identification of fatty oils by thin-layer chromatography (2.3.2). The
chromatogram obtained shows spots corresponding to those in the typical chromatogram for olive oil.
For certain types of refined olive oil, the difference in the size of spots E and F is less pronounced
than in the typical chromatogram.
TESTS
Peroxide value (2.5.5, Method A). Not more than 10.0. If intended for use in the manufacture of
parenteral dosage forms, not more than 5.0.
Unsaponifiable matter Not more than 1.5 per cent. Place 5.0 g (m g) in a 150 ml flask fitted with a
reflux condenser. Add 50 ml of 2M alcoholic potassium hydroxide R and heat on a water-bath for 1 h,
shaking frequently. Add 50 ml of water R through the top of the condenser, shake, allow to cool and
transfer the contents of the flask to a separating funnel. Rinse the flask with several portions to a total
of 50 ml of light petroleum R1 and add the rinsings to the separating funnel. Shake vigorously for
1 min. Allow to separate and transfer the aqueous layer to a second separating funnel. If an emulsion
forms, add small quantities of alcohol R or a concentrated solution of potassium hydroxide R. Shake the
aqueous layer with two quantities, each of 50 ml, of light petroleum R1. Combine the light petroleum
layers in a third separating funnel and wash with three quantities, each of 50 ml, of alcohol (50 per cent
V/V) R. Transfer the light petroleum layer to a tared 250 ml flask. Rinse the separating funnel with
small quantities of light petroleum R1 and add to the flask. Evaporate the light petroleum on a waterbath and dry the residue at 100C to 105C for 15 min, keeping the flask horizontal. Allow to cool in
a desiccator and weigh (a g). Repeat the drying for successive periods of 15 min until the loss of mass
between two successive weighings does not exceed 0.1 per cent. Dissolve the residue in 20 ml of
alcohol R, previously neutralised to 0.1 ml of bromophenol blue solution R. If necessary, titrate with 0.1M
hydrochloric acid (b ml).
Calculate the percentage content of unsaponifiable matter from the expression:
100 ( a 0.032b )
m
If 0.032 b is greater than 5 per cent of a, the test is invalid and must be repeated.
Absorbance (2.2.25). Dissolve 1.00 g in cyclohexane R and dilute to 100.0 ml with the same solvent.
The absorbance measured at the maximum at 270 nm is 0.20 to 1.20.
(2.4.22, Method A). The fatty-acid fraction of the oil has the following composition:
saturated fatty acids of chain length less than C16: not more than 0.1 per cent,
palmitoleic acid (equivalent chain length on polyethyleneglycol adipate 16.3): not more than
3.5 per cent,
28-85
oleic acid (equivalent chain length on polyethyleneglycol adipate 18.3): 56.0 per cent to 85.0 per
cent,
linoleic acid (equivalent chain length on polyethyleneglycol adipate 18.9): 3.5 per cent to
20.0 per cent,
linolenic acid (equivalent chain length on polyethyleneglycol adipate 19.7): not more than
1.2 per cent,
eicosenoic acid (equivalent chain length on polyethyleneglycol adipate 20.3): not more than
0.4 per cent,
lignoceric acid: not more than 0.2 per cent.
Sterols (2.4.23). The sterol fraction of the oil has the following composition:
sum of contents of -sitosterol, 5,23-stigmastadienol, clerosterol, sitostanol, 5-avenasterol and
5,24-stigmastadienol: not less than 93.0 per cent,
cholesterol: not more than 0.5 per cent,
7-stigmasterol: not more than 0.5 per cent,
campesterol: not more than 4.0 per cent,
and the content of 7-stigmasterol is not more than that of campesterol.
Sesame oil In a ground-glass-stoppered cylinder shake 10 ml for about 1 min with a mixture of
0.5 ml of a 0.35 per cent V/V solution of furfural R in acetic anhydride R and 4.5 ml of acetic
anhydride R. Filter through a filter paper impregnated with acetic anhydride R. To the filtrate add
0.2 ml of sulphuric acid R. No bluish-green colour develops.
0.1 per cent, determined on 5.0 g by the coulometric method. Use a mixture of equal volumes of
decanol R and anhydrous methanol R as solvent.
STORAGE
Store in a well-filled container, protected from light, at a temperature not exceeding 25C. If
intended for use in the manufacture of parenteral dosage forms, store under an inert gas.
LABELLING
The label states:
forms,
the name and concentration of any added antioxidant,
the name of the inert gas.
__________________________________________________________________________________________________________ Ph Eur
28-86
Virgin Olive Oil

Olive Oil
Virgin Olive Oil complies with the requirements of the 3rd edition of the European Pharmacopoeia [0518].
Preparation
Olive Oil Ear Drops
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Virgin olive oil is the fatty oil obtained by cold expression or other suitable mechanical means from
the ripe drupes of Olea europaea L.
CHARACTERS
A clear, yellow or greenish-yellow, transparent liquid with a characteristic odour, practically insoluble
in alcohol, miscible with light petroleum (50C to 70C).
When cooled, it begins to become cloudy at 10C and becomes a butter-like mass at about 0C. It
has a relative density of about 0.913.
IDENTIFICATION
Carry out the test for identification of fatty oils by thin-layer chromatography (2.3.2). The chromatogram obtained shows spots corresponding to those in the typical chromatogram for olive oil. For
certain types of olive oil, the difference in the size of spots E and F is less pronounced than in the
typical chromatogram.
TESTS
Peroxide value (2.5.5, Method A). Not more than 20.0.
Unsaponifiable matter Not more than 1.5 per cent. Place 5.0 g (m g) in a 150 ml flask fitted with a
reflux condenser. Add 50 ml of 2M alcoholic potassium hydroxide R and heat on a water-bath for 1 h,
shaking frequently. Add 50 ml of water R through the top of the condenser, shake, allow to cool and
transfer the contents of the flask to a separating funnel. Rinse the flask with several portions to a total
of 50 ml of light petroleum R1 and add the rinsings to the separating funnel. Shake vigorously for
1 min. Allow to separate and transfer the aqueous layer to a second separating funnel. If an emulsion
forms, add small quantities of alcohol R or a concentrated solution of potassium hydroxide R. Shake the
aqueous layer with two quantities, each of 50 ml, of light petroleum R1. Combine the light petroleum
layers in a third separating funnel and wash with three quantities, each of 50 ml, of alcohol (50 per cent
V/V) R. Transfer the light petroleum layer to a tared 250 ml flask. Rinse the separating funnel with
small quantities of light petroleum R1 and add to the flask. Evaporate the light petroleum on a waterbath and dry the residue at 100C to 105C for 15 min, keeping the flask horizontal. Allow to cool in
a desiccator and weigh (a g). Repeat the drying for successive periods of 15 min until the loss of mass
between two successive weighings does not exceed 0.1 per cent. Dissolve the residue in 20 ml of
alcohol R, previously neutralised to 0.1 ml of bromophenol blue solution R. If necessary, titrate with
0.1M hydrochloric acid (b ml).
Calculate the percentage content of unsaponifiable matter from the expression:
100 (a 0.032b )
m
If 0.032b is greater than 5 per cent of a, the test is invalid and must be repeated.
Absorbance (2.2.25). Dissolve 1.00 g in cyclohexane R and dilute to 100.0 ml with the same solvent.
The absorbance measured at the maximum at 270 nm is not greater than 0.20. The ratio of the
absorbance at 232 nm to that at 270 nm is greater than 8.
(2.4.22, Method A). The fatty acid fraction of the oil has the following composition:
3.5 per cent,
cent,
20.0 per cent,
28-87
linolenic acid (equivalent chain length on polyethylene glycol adipate 19.7): not more than
1.2 per cent,
0.4 per cent,
lignoceric acid: not more than 0.2 per cent.
Sterols (2.4.23). The sterol fraction of the oil has the following composition:
sum of contents of -sitosterol, 5,23-stigmastadienol, clerosterol, sitostanol, 5-avenasterol and
5,24-stigmastadienol: not less than 93.0 per cent,
cholesterol: not more than 0.5 per cent,
7-stigmasterol: not more than 4.0 per cent,
campesterol: not more than 4.0 per cent,
and the content of 7-stigmasterol is not more than that of campesterol.
Sesame oil In a ground-glass-stoppered cylinder shake 10 ml for about 1 min with a mixture of
0.5 ml of a 0.35 per cent V/V solution of furfural R in acetic anhydride R and 4.5 ml of acetic
anhydride R. Filter through a filter paper impregnated with acetic anhydride R. To the filtrate add
0.2 ml of sulphuric acid R. No bluish-green colour develops.
STORAGE
Store in a well-filled container, protected form light, at a temperature not exceeding 25C.
__________________________________________________________________________________________________________ Ph Eur
28-88
Olsalazine Sodium
revised 1/01
HO
NaOOC
COONa
OH
C14H8N2Na2O6
346.2
6054-98-4
Olsalazine Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia [1457].
Action and use Anti-inflammatory; used in treatment of ulcerative colitis.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Olsalazine sodium contains not less than 98.0 per cent and not more than the equivalent of 102.0 per
cent of disodium 3,3-diazenediylbis(6-hydroxybenzoate), calculated with reference to the dried and
acetate-free substance.
CHARACTERS
A yellow, fine, crystalline powder, sparingly soluble in water, soluble in dimethyl sulphoxide, very
slightly soluble in methanol.
IDENTIFICATION
A. Dissolve 40.0 mg in 5 ml of 0.1M sodium hydroxide and dilute to 100.0 ml with a 7.8 g/l solution of
sodium dihydrogen phosphate R adjusted to pH 7.2 with strong sodium hydroxide solution R (buffer
solution). Dilute 2.0 ml of the solution to 100.0 ml with the buffer solution. Examined between
240 nm and 400 nm (2.2.25), the solution shows absorption maxima at 255 nm and 362 nm. The
ratio of the absorbance measured at the maximum at 255 nm to that measured at the maximum at
362 nm is 0.53 to 0.56.
obtained with olsalazine sodium CRS. If the spectra obtained in the solid state show differences,
dissolve the substance to be examined and the reference substance separately in methanol R, evaporate to dryness and record new spectra using the residues.
Test solution. Dissolve 10 mg of the substance to be examined in a mixture of 1 volume of dilute
ammonia R2 and 4 volumes of alcohol R and dilute to 10 ml with the same mixture of solvents.
Reference solution (a). Dissolve 10 mg of olsalazine sodium CRS in a mixture of 1 volume of dilute
ammonia R2 and 4 volumes of alcohol R and dilute to 10 ml with the same mixture of solvents.
Reference solution (b). Dissolve 5 mg of sulfasalazine CRS in reference solution (a) and dilute to 5 ml
of anhydrous formic acid R, 50 volumes of acetone R and 60 volumes of methylene chloride R. Allow the
with reference solution (b) shows two separated spots.
D. To 0.5 g of the substance to be examined add 2 ml of sulphuric acid R. Progressively heat to
ignition and continue heating until an almost white, or at most greyish, residue is obtained. Carry out
the ignition at a temperature up to 800C. Dissolve the residue in 10 ml of boiling water R and filter.
2 ml of the filtrate gives reaction (a) of sodium (2.3.1).
TESTS
Acetate Not more than 1.0 per cent, determined by liquid chromatography (2.2.29).
Test solution. Dissolve 0.125 g of the substance to be examined in 25.0 ml of water R and add 1.0 ml
of dilute hydrochloric acid R. Centrifuge and then filter the solution through a 0.45 m filter and also
28-89
through an appropriate filter for removal of chlorides.
Reference solution (a). Dissolve 0.140 g of sodium acetate R, 0.150 g of sodium formate R and 0.180 g of
potassium sulphate R in 100.0 ml of water R. Dilute 1.0 ml of this solution to 100.0 ml with water R.
Reference solution (b). Use suitable amounts of sodium acetate R to prepare not fewer than five
reference solutions containing 10 g/ml to 50 g/ml of acetate.
The ion-exclusion chromatographic procedure may be carried out using:
a separation column 0.25 m long and 6 mm in internal diameter packed with ion-exclusion resin
for chromatography R with a capacity of about 27 meq/column.
a suppressor column,
as mobile phase at a flow rate of 0.9 ml/min 0.0001M hydrochloric acid,
a conductivity detector set at 10 Scm-1.
Inject 0.1 ml of reference solution (a). The chromatogram shows three separated peaks. Inject
0.1 ml of the test solution and 0.1 ml of reference solution (b). Prepare a calibration curve from the
average of the readings obtained with the reference solutions and determine the concentration of
acetate in the test solution from the curve obtained. Measure the peak area for acetate. Calculate the
percentage content of acetate content from the following expression:
2.6c
m
c = concentration of acetate in the test solution (g/ml), determined by linear interpolation of
the standard curve for reference solution (b),
m = mass of sample (mg).
Methanesulphonic acid Liquid chromatography (2.2.29).

Test solution. Dissolve 0.25 g of the substance to be examined in 20 ml of water R, add 1.0 ml of dilute
hydrochloric acid R and dilute to 25.0 ml with water R. Centrifuge and then filter the solution through
a 0.45 m filter and also through an appropriate filter for removal of chloride.
Reference solution (a). Dissolve 0.25 g of methanesulphonic acid R in 50 ml of water R. Add 0.58 g of
sodium acetate R and 0.08 g of sodium chloride R and dilute to 100.0 ml with water R. Dilute 1.0 ml of
the solution to 100.0 ml with water R.
Reference solution (b). Dissolve 0.10 g of methanesulphonic acid R in water R and dilute to 100.0 ml
with water R. Dilute 3.0 ml of the solution to 100.0 ml with water R.
The reversed-phase ion chromatographic procedure may be carried out using:
a pre-column 0.035 m long and 4 mm in internal diameter packed with reversed-phase ion resin for
a separation column 0.25 m long and 4 mm in internal diameter packed with reversed-phase ion
resin for chromatography R (10 m),
as mobile phase at a flow rate of 1.0 ml/min, a mixture of 10 volumes of acetonitrile for chromatography R and 990 volumes of a solution containing 1.6 g/l of tetrabutylammonium hydroxide R and
0.053 g/l of anhydrous sodium carbonate R,
a conductivity detector set at 50 Scm1.
Inject 100 l of reference solution (a). The test is not valid unless the chromatogram shows 3
separated peaks. Inject 100 l each of the test solution and reference solution (b). In the chromatogram obtained with the test solution, the area of the peak corresponding to methanesulphonic acid is
not greater than the area of the corresponding peak in the chromatogram obtained with reference
solution (b) (0.3 per cent).
25.0 ml with mobile phase A.
Reference solution (a). Dilute 0.5 ml of the test solution to 100.0 ml with the mobile phase A.
Reference solution (b). Dissolve 20.0 mg of olsalazine sodium for performance test CRS in mobile phase A
and dilute to 25.0 ml with mobile phase A.
as mobile phase at a flow rate of 1 ml/min, the following linear gradient programme:
Mobile phase A. Dissolve 2.38 g of tetrabutylammonium hydrogen sulphate R and 3.6 g of disodium
hydrogen phosphate dihydrate R in 900 ml of water R. Adjust to pH 7.6 with dilute sodium hydroxide
solution R. Dilute to 1000.0 ml with water R. Mix 700 ml of this buffer solution with 300 ml of
methanol R,
Mobile phase B. Dissolve 4.75 g of tetrabutylammonium hydrogen sulphate R and 3.6 g of disodium
hydrogen phosphate dihydrate R in 900 ml of water R. Adjust to pH 7.6 with dilute sodium hydroxide
solution R. Dilute to 1000.0 ml with water R. Mix 350 ml of this buffer solution with 650 ml of
methanol R,
28-90
Time
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
0 15
15 45
45 50
55
45
55 0
0 55
45 100
100 45
50 65
55
45
isocratic
linear gradient
return to initial
composition
equilibration
(min)

Inject 20 l of reference solution (b). The test is not valid unless the chromatogram obtained is
similar to the chromatogram obtained with olsalazine sodium for performance test CRS. If necessary,
adjust the proportion of mobile phase A in the mobile phase (increasing the proportion of mobile
phase A increases the retention time).
Inject 20 l of the test solution. In the chromatogram obtained with the test solution: the area of
any peak, apart from the principal peak, is not greater than twice the area of the principal peak in the
chromatogram obtained with reference solution (a) (1 per cent), and not more than one such peak
has an area greater than the area of the principal peak in the chromatogram obtained with reference
solution (a) (0.5 per cent); the sum of the areas of all the peaks, apart from the principal peak, is not
greater than four times the area of the principal peak in the chromatogram obtained with reference
solution (a) (2 per cent). Disregard any peak with an area less than 0.05 times that of the principal
peak in the chromatogram obtained with reference solution (a) (0.025 per cent).
150C.
ASSAY
Dissolve 0.100 g in 15 ml of ethylene glycol R. Add 40 ml of dioxan R and 0.2 ml of a 224 g/l solution
of potassium chloride R. Titrate with 0.1M hydrochloric acid, determining the end-point potentiometrically (2.2.20). Carry out a blank titration.
Correct the volume consumed for the content of acetate, taking the molecular mass of acetate to be
59.0.
1 ml of 0.1M hydrochloric acid is equivalent to 17.31 mg of C14H8N2Na2O6.
IMPURITIES
R3
R2
COOH
R1
OH
A. R1 = H, R2 = CO2H, R3 = OCH3: 6-hydroxy-6-methoxy-3,3-diazenediyldibenzoic acid,

B. R1 = OH, R2 = CO2H, R3 = H: 2,6-dihydroxy-3,3-diazenediyldibenzoic acid,
C. R1 = R2 = H, R3 = OH: 2-hydroxy-5-[(4-hydroxyphenyl)diazenyl]benzoic acid,
D. R1 = H, R2 = CO2H, R3 = Cl: 6-chloro-6-hydroxy-3,3-diazenediyldibenzoic acid,
E. R1 = H, R2 = CO-CH2-SO3H, R3 = OH: 2-hydroxy-5-[[4-hydroxy-3(sulphoacetyl)phenyl]diazenyl]benzoic acid,
COOH
OH
HO
HOOC
COOH
OH
F. 2-[(3-carboxy-4-hydroxyphenyl)diazenyl]-4,5-dihydroxybiphenyl-3,4-dicarboxylic acid,
28-91
OH
HOOC
HO
N
HOOC
COOH
OH
G. 5-[(3-carboxy-4-hydroxyphenyl)diazenyl]-2,4-dihydroxybiphenyl-3,3-dicarboxylic acid,
OH
HOOC
HO
R
COOH
OH
H. R = CO2H: 3,3-[5-carboxy-4-hydroxy-1,3-phenylenebis(diazenediyl)]bis(6-hydroxybenzoic)
acid,
I. R = H: 3,3-[4-hydroxy-1,3-phenylenebis(diazenediyl)]bis(6-hydroxybenzoic) acid.
__________________________________________________________________________________________________________ Ph Eur
28-92
Omega-3-Acid Ethyl Esters

Omega-3-Acid Ethyl Esters comply with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Omega-3-acid ethyl esters are obtained by esterification of the body oil of fat fish species coming
from families such as Engaulidae, Carangidae, Clupeidae, Osmeridae, Salmonidae and Scrombroidae and
subsequent physico-chemical purification processes including urea fractionation followed by
molecular distillation. The omega-3-acid ethyl esters are defined as the ethyl esters of alpha-linolenic
acid (C18:3 n-3), moroctic acid (C18:4 n-3), C20:4 n-3, timnodonic (eicosapentaenoic) acid (C20:5
n-3; EPA), C21:5 n-3, clupanodonic acid (C22:5 n-3) and cervonic (docosahexaenoic) acid (C22:6
n-3; DHA). The content of the total omega-3-acid ethyl esters is not less than 90 per cent. The
content of the omega-3-acid ethyl esters EPA and DHA is not less than 80 per cent, with not less
than 40 per cent of EPA ethyl esters and not less than 34 per cent of DHA ethyl esters.
Tocopherol may be added as an antioxidant.
CHARACTERS
A light yellow liquid with a slight fish-like smell, practically insoluble in water, very soluble in
acetone, in ethanol, in heptane and in methanol.
IDENTIFICATION
Examine the chromatograms obtained in the assay for EPA and DHA ethyl esters. The retention
time and size of the peaks corresponding to eicosapentaenoic acid ethyl ester and to docosahexaenoic
acid ethyl ester in the chromatogram obtained with the test solution are approximately the same as
those of the corresponding peaks in the chromatogram obtained with the reference solution.
TESTS
Acid value (2.5.1). Dissolve 10 g in 50 ml of the prescribed mixture of solvents. The acid value is
not more than 2.0.
Anisidine value Not more than 20.0.
The anisidine value is defined as 100 times the absorbance measured in a 1 cm cell of a solution
containing 1 g of the substance to be examined in 100 ml of a mixture of solvents and reagents
according to the method described below.
Carry out the operations as rapidly as possible, avoiding exposure to actinic light.
Test solution (a). Dissolve 0.500 g of the substance to be examined in trimethylpentane R and dilute to
Test solution (b). To 5.0 ml of test solution (a) add 1.0 ml of a 2.5 g/l solution of p-anisidine R in
glacial acetic acid R, shake and protect from light.
Reference solution. To 5.0 ml of trimethylpentane R add 1.0 ml of a 2.5 g/l solution of p-anisidine R in
glacial acetic acid R, shake and protect from light.
Measure the absorbance of test solution (a) at 350 nm using trimethylpentane R1 as the compensation
liquid. Measure the absorbance of test solution (b) at 350 nm exactly 10 min after its preparation,
using the reference solution as the compensation liquid.
Calculate the anisidine value from the expression:
25 (12
. As Ab )
m
As = absorbance of test solution (b) at 350 nm,
Ab = absorbance of test solution (a) at 350 nm,
m = mass of the substance to be examined in test solution (a) in grams.
Oligomers Not more than 1.0 per cent. Examine by size-exclusion chromatography (2.2.30).
Test solution. Weigh 10.0 mg of the substance to be examined in a 10 ml volumetric flask, dissolve in
tetrahydrofuran R and dilute to 10.0 ml with the same solvent.
Reference solution. Weigh 15.0 mg of docosahexaenoic acid ethyl ester CRS and 15.0 mg of polystyrene
9001000 R, dissolve in tetrahydrofuran R and dilute to 20.0 ml with the same solvent.
28-93
a gel permeation column 0.3 m long and 7.8 mm in internal diameter packed with styrenedivinylbenzene copolymer R (pore size 10 nm, particle size 7 m) and two gel permeation columns
0.3 m long and 7.8 mm in internal diameter packed with styrene-divinylbenzene copolymer R (pore
size 50 nm, particle size 7 m) (these columns are placed closest to the injector),
as mobile phase at a flow rate of 0.8 ml per minute tetrahydrofuran R,
as detector a differential refractometer,
an integrator.
Inject 40 l of the test solution. Calculate the percentage content of oligomers using the following
expression:
B
100
A
A = sum of the areas of all the peaks in the chromatogram,
B = sum of the areas of the peaks with a retention time smaller than the retention time of the
ethyl ester peak(s).
The ethyl ester peak(s), which may be present in the form of a single peak or an unresolved double
peak, are identified as the major peak(s) in the chromatogram (see Figure 12501).
The test is not valid unless: the chromatogram obtained with the reference solution shows two
peaks corresponding to polystyrene and DHA ethyl ester representing not less than 90 per cent of the
sum of the areas of all the peaks in the chromatogram; if the method of standard addition to the test
solution is used, there is greater than 95 per cent recovery of the added eicosapentaenoic acid ethyl ester
CRS or docosahexaenoic acid ethyl ester CRS. Disregard any peak due to the solvent.
Fig. 12501 Oligomers
Conjugated dienes Not more than 1.5 per cent. Examine by ultraviolet absorption
spectrophotometry (2.2.25).
Carry out the operations as rapidly as possible, avoiding exposure to actinic light, oxidising agents, oxidation
catalysts (for example, copper and iron) and air.
Test solution. Dissolve 0.200 g to 0.800 g of the substance to be examined in trimethylpentane R1 and
Measure the absorbance at 233 nm using a spectro-photometer. Adjust the concentration of the
solution if necessary so that the absorbance is 0.2 to 0.8. Calculate the percentage content of
conjugated dienes using the following expression:
28-94
A
0.91 0.07
C
A = absorbance at 233 nm,

C = concentration of the test solution in grams per litre,
0.91 = conversion factor,
0.07 = correction factor for the absorbance of the ester group at 233 nm.
ASSAY
EPA and DHA ethyl esters Carry out the operations as rapidly as possible, avoiding exposure to actinic
light, oxidising agents, oxidation catalysts (for example copper and iron) and air.
The assay is carried out on the ethyl esters of all-cis-eicosapenta-5,8,11,14,17-enoic acid (EPA;
20:5 n-3) and all-cis-docosahexa-4,7,10,13,16,19-enoic acid (DHA; 22:6 n-3) in the substance to be
examined.
Internal standard. Methyl tricosanoate R.
Test solution. Introduce 0.17 g of the substance to be examined and about 70.0 mg of the internal
standard into a 10 ml volumetric flask, dissolve in trimethylpentane R containing 50 mg of
butylhydroxytoluene R per litre and dilute to 10.0 ml with the same solvent.
Reference solution. Introduce 55.0 mg of docosahexaenoic acid ethyl ester CRS, about 70.0 mg of the
internal standard and 88.0 mg of eicosapentaenoic acid ethyl ester CRS into a 10 ml volumetric flask.
Dissolve in trimethylpentane R containing 50 mg of butylhydroxytoluene R per litre and dilute to
a fused-silica capillary column at least 30 m long and 0.25 mm in internal diameter coated with
macrogol 20,000 R (film thickness 0.25 m),
hydrogen for chromatography R or helium for chromatography R as the carrier gas where oxygen
scrubber is applied,
a suitable integrator,
a split injector (1:200),
maintaining the temperature of the column at 170C for 0.5 min, then raising the temperature at a
rate of 10C per minute to 240C and maintaining at 240C for 22 min; maintaining the temperature
of the injection port at 250C and that of the detector at 280C.
Inject 1 l of each solution twice.
The test is not valid unless: the chromatogram obtained with the reference solution shows three
peaks corresponding to EPA ethyl ester, tricosanoic acid methyl ester and DHA ethyl ester (see
Figures 12502 and 12503); if the method of standard addition to the test solution is used there is
greater than 95 per cent recovery of the added eicosapentaenoic acid ethyl ester CRS or docosahexaenoic
acid ethyl ester CRS when due consideration has been given to correction by the internal standard.
Calculate both the percentage content of EPA and DHA ethyl esters using the following expression
and taking into account the assigned values of eicosapentaenoic acid ethyl ester CRS and docosahexaenoic
acid ethyl ester CRS:
Ax
A2 m1 mx,r
1
100
m3 A1 Ax,r m2
= Ax
1
1
m1
100
A1 Rf,x m2
where Rf,x is the response factor:
Rf,x =
Af,x m3
mx,r A2
m1 = mass of the internal standard in the test solution in milligrams,

m2 = mass of the substance to be examined in the test solution in milligrams,
m3 = mass of the internal standard in the reference solution in milligrams,
mx,r = mass of eicosapentaenoic acid ethyl ester CRS or docosahexaenoic acid ethyl ester CRS in the
reference solution in milligrams,
Ax = area of the peak corresponding to eicosapentaenoic acid ethyl ester or docosahexaenoic
acid ethyl ester in the test solution,
Ax,r = area of the peak corresponding to eicosapentaenoic acid ethyl ester or docosahexaenoic
acid ethyl ester in the reference solution,
A1 = area of the peak corresponding to tricosanoic acid methyl ester in the test solution,
A2 = area of the peak corresponding to tricosanoic acid methyl ester in the reference solution.
28-95
Fig. 12502 Assay of omega-3-acid ethyl esters
Fig. 12503 Assay of omega-3-acid ethyl esters
Total omega-3-acids ethyl esters From the assay for EPA and DHA ethyl esters, calculate
the percentage content of the total omega-3-acid ethyl esters using the following expression:
EPA + DHA +
An 3( EPA + DHA )
AEPA + ADHA
EPA = percentage content of EPA ethyl ester,

DHA = percentage content of DHA ethyl ester,
An-3 = sum of the areas of the peaks corresponding to C18:3 n-3, C18:4 n-3, C20:4 n-3, C21:5
n-3 and C22:5 n-3 ethyl esters in the chromatogram obtained with the test solution,
AEPA = area of the peak corresponding to EPA ethyl ester in the chromatogram obtained with the
test solution,
ADHA = area of the peak corresponding to DHA ethyl ester in the chromatogram obtained with the
test solution.
STORAGE
Store in a well-filled, airtight container, protected from light, under an inert gas.
LABELLING
The label states, where applicable, that the substance contains tocopherol as an antioxidant.
__________________________________________________________________________________________________________ Ph Eur
28-96
Omega-3-Marine Triglycerides
revised 1/01
Omega-3-Marine Triglycerides comply with the requirements of the 3rd edition of the European
Pharmacopoeia for Omega-3-Acid Triglycerides [1352]. These requirements are reproduced after the heading
Definition below.
Action and use Used in treatment of hypertriglyceridaemia.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Omega-3-acid triglycerides are a mixture of mono-, di- and triesters of omega-3 acids with glycerol.
They contain mainly triesters and are obtained either by esterification of concentrated and purified
omega-3 acids with glycerol or by transesterification of the omega-3 acid ethyl esters with glycerol.
The origin of the omega-3-acids is the body oil from fatty fish species coming from families like
Engraulidae, Carangidae, Clupeidae, Osmeridae, Salmonidae and Scrombroidae. The omega-3-acids are
identified as the following acids: alpha-linolenic acid (C18:3 n-3), moroctic acid (C18:4 n-3),
eicosatetraenoic acid (C20:4 n-3), timnodonic (eicosapentaenoic) acid (C20:5 n-3; EPA),
heneicosapentaenoic acid (C21:5 n-3), clupanodonic acid (C22:5 n-3) and cervonic
(docosahexaenoic) acid (C22:6 n-3; DHA). The concentration of the total amount of omega-3-acids,
expressed as triglycerides, is not less than 60.0 per cent. The sum of the concentrations of the omega3-acids EPA and DHA, expressed as triglycerides, is not less than 45.0 per cent.
Tocopherol may be added as an antioxidant.
CHARACTERS
A pale yellow liquid, practically insoluble in water, very soluble in acetone and in heptane, slightly
soluble in ethanol.
IDENTIFICATION
Examine the chromatograms obtained in the assay for EPA and DHA. The retention time and size of
the peaks corresponding to eicosapentaenoic acid methyl ester and to docosahexaenoic acid methyl
ester in the chromatogram obtained with test solution (b) are approximately the same as those of the
corresponding peaks in the chromatogram obtained with reference solution (a).
TESTS
Acid value (2.5.1). Not more than 3.0, determined on 10.0 g in 50 ml of the prescribed mixture of
solvents.
Anisidine value Not more than 30.0.
The anisidine value is defined as 100 times the absorbance measured in a 1 cm cell filled with a
solution containing 1 g of the substance to be examined in 100 ml of a mixture of solvents and
reagents according to the method described below.
Carry out the operations as rapidly as possible, avoiding exposure to actinic light.
Test solution (a). Dissolve 1.0 g of the substance to be examined in trimethylpentane R and dilute to
Test solution (b). To 5.0 ml of test solution (a) add 1.0 ml of a 2.5 g/l solution of p-anisidine R in
glacial acetic acid R, shake and store protected from light.
Reference solution. To 5.0 ml of trimethylpentane R add 1.0 ml of a 2.5 g/l solution of p-anisidine R in
glacial acetic acid R, shake and store protected from light.
Measure the absorbance (2.2.25) of test solution (a) at 350 nm using trimethylpentane R as the
compensation liquid. Measure the absorbance of test solution (b) at 350 nm exactly 10 min after its
preparation, using the reference solution as the compensation liquid.
Calculate the anisidine value from the expression:
25 (1.2 As Ab )
m
As = absorbance of test solution (b),
Ab = absorbance of test solution (a),
m = mass of the substance to be examined in test solution (a), in grams.
Oligomers and partial glycerides Not more than 3.0 per cent of oligomers and not more than
50.0 per cent of partial glycerides. Examine by size-exclusion chromatography (2.2.30).
Test solution. Dissolve 10.0 mg of the substance to be examined in tetrahydrofuran R and dilute to
28-97
a column 0.3 m long and 7.8 mm in internal diameter packed with styrene-divinylbenzene
copolymer R (pore size 10 nm, particle size 7 m) and two columns 0.3 m long and 7.8 mm in
internal diameter packed with styrene-divinylbenzene copolymer R (pore size 50 nm, particle size
7 m) (these columns are placed closest to the injector),
as mobile phase, at a flow rate of 0.8 ml/min, tetrahydrofuran R,
as detector a differential refractometer.
Inject 40 l of the test solution. Identify the peaks from the type chromatogram (Fig. 13521).
Calculate the percentage content of oligomers using the following expression:
B
100
A
A = sum of the areas of all the peaks in the chromatogram,
B = area of the peak with a retention time smaller than the retention time of the triglyceride
peak.
Calculate the percentage content of partial glycerides using the following expression:
C
100
A
C = (sum of the) area(s) of the peak(s) due to the mono- and diglycerides.
Fig. 13521 Type chromatogram for oligomers and partial glycerides

Conjugated dienes Not more than 2.7 per cent, determined by ultraviolet absorption
spectrophotometry (2.2.25). Carry out the operations as rapidly as possible.
Test solution. Dissolve 0.200 g to 0.800 g of the substance to be examined in trimethylpentane R1 and
Measure the absorbance at 233 nm. Adjust the concentration of the solution if necessary so that
the absorbance is 0.2 to 0.8. Calculate the percentage content of conjugated dienes using the following expression:
A
0.91 0.07
C
A = absorbance of the test solution,

C = concentration of the test solution, in grams per litre,
0. 91 = conversion factor,
0. 07 = correction factor for the absorbance of the ester group at 233 nm.
ASSAY
EPA and DHA Carry out the operations as rapidly as possible, avoiding exposure to actinic light, oxidising
agents, oxidation catalysts (for example, copper and iron) and air.
28-98
The assay is carried out on the methyl esters of all-cis-eicosa-5,8,11,14,17-pentaenoic acid (EPA;
20:5 n-3) and all-cis-docosa-4,7,10,13,16,19-hexaenoic acid (DHA; 22:6 n-3) in the substance to be
examined.
Internal standard. Methyl tricosanoate R.
Test solution (a). Dissolve 0.300 g and about 70.0 mg of the internal standard in a 0.05 g/l solution of
butylhydroxytoluene R in trimethylpentane R and dilute to 10.0 ml with the same solution. Introduce
2.0 ml of the solution obtained into a quartz tube and evaporate the solvent with a gentle current of
nitrogen R. Add 1.5 ml of a 20 g/l solution of sodium hydroxide R in methanol R, cover with nitrogen R,
cap tightly with a polytetrafluoroethylene-lined cap, mix and heat on a water-bath for 7 min. Allow to
cool, add 2 ml of boron trichloride-methanol solution R, cover with nitrogen R, cap tightly, mix and heat
on a water-bath for 30 min. Cool to 40C to 50C, add 1 ml of trimethylpentane R, cap and shake
vigorously for at least 30 s. Immediately add 5 ml of a saturated solution of sodium chloride R, cover
with nitrogen R, cap and shake thoroughly for at least 15 s. Transfer the upper layer to a separate
tube. Shake the methanol layer once more with 1 ml of trimethylpentane R. Wash the combined
trimethylpentane extracts with two quantities, each of 1 ml, of water R and dry over anhydrous sodium
sulphate R. Prepare two solutions for each sample.
Test solution (b). Dissolve 0.300 g in a 0.05 g/l solution of butylhydroxytoluene R in trimethylpentane R
and dilute to 10.0 ml with the same solution. Proceed as described for test solution (a).
Reference solution (a). Dissolve 60.0 mg of docosahexaenoic acid ethyl ester CRS, about 70.0 mg of the
internal standard and 90.0 mg of eicosapentaenoic acid ethyl ester CRS in a 0.05 g/l solution of
butylhydroxytoluene R in trimethylpentane R and dilute to 10.0 ml with the same solution. Proceed as
described for test solution (a).
Reference solution (b). Introduce 0.3 g of methyl palmitate R, 0.3 g of methyl stearate R, 0.3 g of methyl
arachidate R and 0.3 g of methyl behenate R into a 10 ml volumetric flask, dissolve in a 0.05 g/l
solution of butylhydroxytoluene R in trimethylpentane R and dilute to 10.0 ml with the same solution.
a fused-silica column at least 30 m long and 0.25 mm in internal diameter coated with macrogol
hydrogen for chromatography R or helium for chromatography R as the carrier gas where oxygen
scrubber is applied,
maintaining the temperature of the column at 170C for 0.5 min, then raising the temperature at a
rate of 10C per minute to 240C and maintaining at 240C for 22 min; maintaining the temperature
of the injection port at 250C and that of the detector at 280C.
Inject twice 1 l of each solution.
The assay is not valid unless:
the chromatogram obtained with reference solution (a) shows three peaks corresponding to
EPA-methyl ester, methyl tricosanoate and DHA-methyl ester,
the chromatogram obtained with reference solution (b) gives area per cent compositions
increasing in the following order: methyl palmitate, methyl stearate, methyl arachidate, methyl
behenate; the difference between the percentage area of methyl palmitate and that of methyl
behenate is less than two area per cent units,
experiments using the method of standard additions to test solution (a) show more than 95 per
cent recovery of the added eicosapentaenoic acid ethyl ester CRS and docosahexaenoic acid ethyl ester
CRS, when due consideration has been given to the correction by the internal standard.
Calculate the percentage content of EPA and DHA, expressed as triglycerides, using the following
expression and taking into account the assigned value of the reference substances:
Ax
m
A3
m1
1
x,r
0.955 100
m3 A1 ( A2 C ) Ax,r m2
(A2 C) is a correction term for any peak co-eluting with the internal standard:
C=
A4
A5
m1 = mass of the internal standard in test solution (a), in milligrams,

m2 = mass of the sample in test solution (a), in milligrams,
m3 = mass of the internal standard in reference solution (a), in milligrams,
mx,r = mass of eicosapentaenoic acid ethyl ester CRS or docosahexaenoic acid ethyl ester CRS in
reference solution (a), in milligrams,
Ax = area of the peak corresponding to eicosapentaenoic acid methyl ester or docosahexaenoic
acid methyl ester in the chromatogram obtained with test solution (a),
Ax,x = area of the peak corresponding to eicosapentaenoic acid methyl ester or docosahexaenoic
28-99
acid methyl ester in the chromatogram obtained with reference solution (a),
A1 = area of the peak corresponding to methyl tricosanoate in the chromatogram obtained with
test solution (a),
A2 = area of the peak corresponding to eicosapentaenoic acid methyl ester in the chromatogram obtained with test solution (a),
A3 = area of the peak corresponding to the internal standard in the chromatogram obtained
with reference solution (a),
A4 = area of the peak in the chromatogram obtained with test solution (b) with a retention time
corresponding to that of the peak due to the internal standard in the chromatograms
obtained with test solution (a) and reference solution (a),
A5 = area of the peak corresponding to eicosapentaenoic acid methyl ester in the chromatogram obtained with test solution (b).
Total omega-3-acids From the assay for EPA and DHA, calculate the percentage content of the
total omega-3-acids, as triglycerides, using the following expression and identifying the peaks from
the type chromatogram (Figs. 13522 and 13523):
EPA + DHA +
An 3( EPA + DHA)
AEPA + ADHA
EPA = percentage content of EPA obtained from the assay for EPA and DHA,
DHA = percentage content of DHA obtained from the assay for EPA and DHA,
An-3 = sum of the areas of the peaks corresponding to C18:3 n-3, C18:4 n-3, C20:4 n-3, C21:5
n-3 and C22:5 n-3 methyl esters in the chromatogram obtained with test solution (b),
AEPA = area of the peak corresponding to EPA methyl ester in the chromatogram obtained with
test solution (b),
ADHA = area of the peak corresponding to DHA methyl ester in the chromatogram obtained with
test solution (b).
Fig. 13522 Type chromatogram for the assay of total omega-3 acids
28-100
Fig. 13523 Type chromatogram for the assay of total omega-3 acids
STORAGE
Store in an airtight, well-filled container, protected from light, under an inert gas.
LABELLING
The label states the concentration of any added tocopherol.
__________________________________________________________________________________________________________ Ph Eur
29-1
Omeprazole
OMe
Me
Me
O
MeO
S
N
NH
C17H19N3O3S
345.4
73590-58-6
Omeprazole complies with the requirements of the 3rd edition of the European Pharmacopoeia [0942]. These
Action and use Treatment of peptic ulcer.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Omeprazole contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of (RS)-5-methoxy-2-[[(4-methoxy-3,5-dimethylpyridin-2-y1)methyl]sulphinyl]-1H-benzimidazole,
CHARACTERS
A white or almost white powder, very slightly soluble in water, soluble in methylene chloride,
sparingly soluble in alcohol and in methanol. It dissolves in dilute solutions of alkali hydroxides.
IDENTIFICATION
A. Dissolve 2.0 mg in 0.1M sodium hydroxide and dilute to 100.0 ml with the same solvent. Examined
obtained with omeprazole CRS.
C. Examine the chromatograms obtained in the test for omeprazole impurity C. The principal spot in
the chromatogram obtained with test solution (b) is similar in position and size to the principal spot
in the chromatogram obtained with reference solution (a). Place the plate in a tank saturated with
vapour from acetic acid R. The spots rapidly turn brown.
TESTS
Solution S Dissolve 0.50 g in methylene chloride R and dilute to 25 ml with the same solvent.
Absorbance (2.2.25). The absorbance of solution S measured at 440 nm is not more than 0.10.
(This limit corresponds to 0.035 per cent of omeprazole impurity F or omeprazole impurity G).
Omeprazole impurity C Examine by thin-layer chromatography (2.2.27), using a TLC silica gel
F254 plate R.
Test solution (a). Dissolve 0.10 g of the substance to be examined in 2.0 ml of a mixture of equal
volumes of methanol R and methylene chloride R.
Test solution (b). Dilute 1.0 ml of test solution (a) to 10 ml with methanol R.
Reference solution (a). Dissolve 10 mg of omeprazole CRS in 2.0 ml of methanol R.
Reference solution (b). Dilute 1 ml of test solution (a) to 10 ml with a mixture of equal volumes of
methanol R and methylene chloride R. Dilute 1 ml of this solution to 100 ml with a mixture of equal
volumes of methanol R and methylene chloride R.
20 volumes of 2-propanol R, 40 volumes of methylene chloride R previously shaken with concentrated
ammonia R (shake 100 ml of methylene chloride R with 30 ml of concentrated ammonia R in a separating
funnel; allow the layers to separate and use the lower layer) and 40 volumes of methylene chloride R.
Allow the plate to dry in air. Examine in ultraviolet light at 254 nm. Any spot in the chromatogram
obtained with test solution (a) with a higher Rf value than that of the spot corresponding to
omeprazole is not more intense than the spot in the chromatogram obtained with reference solution
(b) (0.1 per cent).
29-2
Reference solution (a). Dissolve 1.0 mg of omeprazole CRS and 1.0 mg of omeprazole impurity D CRS in
the mobile phase and dilute to 10.0 ml with the mobile phase.
a stainless steel column 0.15 m long and 4 mm in internal diameter packed with octylsilyl silica gel
for chromatography R (5 m),
volumes of a 1.4 g/l solution of disodium hydrogen phosphate R previously adjusted to pH 7.6 with
phosphoric acid R,
When the chromatograms are recorded under the prescribed conditions, the retention time of
omeprazole is about 9 min and the relative retention time of omeprazole impurity D is about 0.8.
Inject separately 40 l of each solution and continue the chromatography for three times the retention
time of omeprazole. Adjust the sensitivity of the system so that the height of the principal peak in the
chromatogram obtained with reference solution (b) is at least 15 per cent of the full scale of the
recorder. The test is not valid unless in the chromatogram obtained with reference solution (a), the
resolution between the peaks corresponding to omeprazole impurity D and omeprazole is greater
than 3. If necessary, adjust the pH of the mobile phase or the concentration of acetonitrile R; an
increase in the pH will improve the resolution. The area of any peak apart from the principal peak, in
the chromatogram obtained with the test solution is not greater than the area of the peak in the
chromatogram obtained with reference solution (b) (0.1 per cent).
Residual solvents Examine by head-space gas chromatography (2.2.28), using the standard
additions method. The content of chloroform is not more than 50 ppm, the content of methylene
chloride is not more than 100 ppm and the content of trichloroethylene is not more than 100 ppm.
a fused-silica column 30 m long and 0.32 mm in internal diameter coated with a 1.8 m film of
cross-linked poly[(cyanopropyl)(methyl)][(phenyl)(methyl)]siloxane R,
nitrogen for chromatography R as the carrier gas,
a suitable headspace sampler.
Place 0.50 g of the substance to be examined in a 10 ml vial. Add 4.0 ml of dimethylacetamide R and
stopper the vial. Equilibrate the vial at 80C for 1 h.
Loss on drying (2.2.32). Not more than 0.2 per cent, determined on 1.000 g by drying under high
vacuum at 60C for 4 h.
ASSAY
Dissolve 1.100 g in a mixture of 10 ml of water R and 40 ml of alcohol R. Titrate with 0.5M sodium
hydroxide, determining the end-point potentiometrically (2.2.20).
1 ml of 0.5M sodium hydroxide corresponds to 0.1727 g of C17H19N3O3S.
STORAGE
Store in an airtight container, protected from light, at a temperature between 2C and 8C.
IMPURITIES
NH
MeO
SH
A. 5-methoxy-1H-benzimidazole-2-thiol,
NH
MeO
Me
S
O
Me
B. 5-methoxy-2-[[(3,5-dimethylpyridin-2-yl)methyl]sulphinyl]-1H-benzimidazole,
29-3
NH
MeO
Me
OMe
S
N
Me
C. 5-methoxy-2-[[(4-methoxy-3,5-dimethylpyridin-2-yl)methyl]sulphanyl]-1H-benzimidazole
(ufiprazole),
Me
NH
eO
OMe
S
O O
Me
D. 5-methoxy-2-[[(4-methoxy-3,5-dimethylpyridin-2-yl)methyl]sulphonyl]-1H-benzimidazole
(omeprazole sulphone),
NH
MeO
Me
OMe
S
O
O
Me
E. 5-methoxy-2-[[(4-methoxy-3,5-dimethylpyridin-2-yl 1-oxide)methyl]sulphinyl]-1Hbenzimidazole,
N
Me
N
N
MeO
O
S
Me
F. 2,12-dihydro-1,3-dimethyl-8-methoxy-12-thioxobenzo[4,5]pyrido[1,2-c]imidazo[1,2a]imidazol-2-one,
MeO
Me
N
N
O
S
Me
G. 2,12-dihydro-1,3-dimethyl-9-methoxy-12-thioxobenzo[4,5]pyrido[1,2-c]imidazo[1,2a]imidazol-2-one.
__________________________________________________________________________________________________________ Ph Eur
29-4
Omeprazole Sodium
OMe
Me
Me
O
MeO
S
N
NNa
C17H18N3NaO3S,H2O
385.4
95510-70-6
Omeprazole Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Treatment of peptic ulcer.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Omeprazole sodium contains not less than 98.0 per cent and not more than the equivalent of
101.0 per cent of sodium (RS)-5-methoxy-2-[[(4-methoxy-3,5-dimethylpyridin-2yl)methyl]sulphinyl]-1H-benzimidazole, calculated with reference to the anhydrous substance.
CHARACTERS
A white or almost white powder, hygroscopic, freely soluble in water and in alcohol, soluble in
propylene glycol, very slightly soluble in methylene chloride.
IDENTIFICATION
A. Dissolve 2.0 mg in 0.1M sodium hydroxide and dilute to 100.0 ml with the same solvent. Examined
B. Examine the chromatograms obtained in the test for omeprazole impurity C. The principal spot in
the chromatogram obtained with test solution (b) is similar in position and size to the principal spot
in the chromatogram obtained with reference solution (a). Place the plate in a tank saturated with
vapour of acetic acid R. The spots rapidly turn brown.
C. Ignite 1 g and cool. Add 1 ml of water R to the residue and neutralise with hydrochloric acid R.
Filter and dilute the filtrate to 4 ml with water R. 0.1 ml of the solution gives reaction (b) of sodium
(2.3.1).
TESTS
Omeprazole impurity C Examine by thin-layer chromatography (2.2.27), using silica gel HF254 R as
Test solution (a). Dissolve 0.10 g of the substance to be examined in 2.0 ml of methanol R.
Reference solution (a). Dissolve 9 mg of omeprazole CRS in 2.0 ml of methanol R.
Reference solution (b). Dilute 1.0 ml of test solution (b) to 100 ml with methanol R.
20 volumes of 2-propanol R, 40 volumes of methylene chloride R previously shaken with concentrated
ammonia R (shake 100 ml of methylene chloride R with 30 ml of concentrated ammonia R in a separating
funnel, allow the layers to separate and use the lower layer) and 40 volumes of methylene chloride R.
Allow the plate to dry in air. Examine in ultraviolet light at 254 nm. Any spot in the chromatogram
obtained with test solution (a) with a higher Rf value than that of the spot corresponding to
omeprazole is not more intense than the spot in the chromatogram obtained with reference solution
(b) (0.1 per cent).
29-5
Reference solution (a). Dissolve 1.0 mg of omeprazole CRS and 1.0 mg of omeprazole impurity D CRS in
the mobile phase and dilute to 10.0 ml with the mobile phase.
volumes of a 1.4 g/l solution of disodium hydrogen phosphate R, previously adjusted to pH 7.6
with phosphoric acid R,
omeprazole is about 9 min and the relative retention time of omeprazole impurity D is about 0.8.
Inject separately 40 l of each solution and continue the chromatography for three times the retention
time of omeprazole. Adjust the sensitivity of the detector so that the height of the principal peak in
the chromatogram obtained with reference solution (b) is not less than 15 per cent of the full scale of
the recorder. The test is not valid unless in the chromatogram obtained with reference solution (a),
the resolution between the peaks corresponding to omeprazole impurity D and omeprazole is greater
than 3. If necessary adjust the pH of the mobile phase or the concentration of acetonitrile R, an
increase in the pH will improve the resolution. The area of any peak apart from the principal peak in
the chromatogram obtained with the test solution is not greater than the area of the peak in the
ASSAY
Dissolve 0.300 g in 50 ml of water R. Titrate with 0.1M hydrochloric acid, determining the end-point
1 ml of 0.1M hydrochloric acid corresponds to 36.74 mg of C17H18N3NaO3S.
STORAGE
IMPURITIES
NH
MeO
SH
A. 5-methoxy-1H-benzimidazole-2-thiol,
NH
MeO
Me
S
O
Me
B. 5-methoxy-2-[[(3,5-dimethylpyridin-2-yl)methyl]sulphinyl]-1H-benzimidazole,
NH
MeO
Me
OMe
S
N
Me
C. 5-methoxy-2-[[(4-methoxy-3,5-dimethylpyridin-2-yl)methyl]thio]-1H-benzimidazole
(ufiprazole),
NH
MeO
Me
OMe
S
O O
Me
D. 5-methoxy-2-[[(4-methoxy-3,5-dimethylpyridin-2-yl)methyl]sulphonyl]-1H-benzimidazole
(omeprazole sulphone),
29-6
NH
MeO
Me
OMe
S
O
N
O
Me
G 5-methoxy-2-[[(4-methoxy-3,5-dimethylpyridin-2-yl)methyl]-sulphinyl]-1H-benzimidazole
1-oxide.
__________________________________________________________________________________________________________ Ph Eur
29-7
Opium
Opium complies with the requirements of the 3rd edition of the European Pharmacopoeia for Raw Opium
Action and use Narcotic analgesic.
Preparation
Opium Tincture
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Raw opium is intended only as starting material for the manufacture of galenical preparations. It is not
dispensed as such.
Raw opium is the air-dried latex obtained by incision from the unripe capsules of Papaver somniferum
L. It contains not less than 10.0 per cent of morphine (C17H19NO3; Mr 285.3) and not less than
2.0 per cent of codeine (C18H21NO3; Mr 299.4), both calculated with reference to the drug dried at
100C to 105C.
CHARACTERS
Raw opium has a characteristic odour and a blackish-brown colour. It has the microscopic characters
described in identification test A. It consists of masses of various sizes, which tend to be soft and
shiny and, after drying, become hard and brittle.
IDENTIFICATION
Strip off any covering, cut the substance to be examined into thin slices, if necessary, dry at about 60C for
48 h and reduce to a powder (500).
A. Examined under a microscope, a suspension of raw opium in a 20 g/l solution of potassium
hydroxide R is seen to consist of granules of latex agglomerated in irregular masses, and of light-brown
elongated filaments. Some fragments of vessels and rather elongated, refringent crystals are also
visible, as well as a smaller number of round pollen grains and fragments of elongated fibres. Hairs of
various lengths with sharp points and a few grains of starch introduced during the handling of the
latex may be present. Fragments of epicarp consisting of polygonal cells with thick walls defining a
stellate lumen may also be present.
B. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance.
Test solution. Triturate 0.10 g of the powdered drug with 5 ml of alcohol (70 per cent V/V) R, add 3 ml
of alcohol (70 per cent V/V) R, transfer to a 25 ml conical flask and heat in a water-bath at 50C to
60C with stirring for 30 min. Cool, filter, wash the filter with alcohol (70 per cent V/V) R and dilute
the filtrate to 10 ml with the same solvent.
Reference solution. Dissolve 2.0 mg of papaverine hydrochloride R, 12.0 mg of codeine phosphate R,
12.0 mg of noscapine hydrochloride R and 25.0 mg of morphine hydrochloride R in alcohol (70 per cent
V/V) R and dilute to 25.0 ml with the same solvent.
15 cm using a freshly prepared mixture of 2 volumes of concentrated ammonia R, 6 volumes of
alcohol R, 40 volumes of acetone R and 40 volumes of toluene R. Dry the plate at 100C to 105C for
15 min, allow to cool and spray with potassium iodobismuthate solution R2 and then with a 4 g/l
solution of sulphuric acid R. The chromatogram obtained with the reference solution shows in the
lower part an orange-red or red zone (morphine), above it a similarly coloured zone (codeine) and in
the upper part an orange-red or red zone (papaverine) and above it a similarly coloured zone
(noscapine). The chromatogram obtained with the test solution shows orange-red or red zones
corresponding to those in the chromatogram obtained with the reference solution. The chromatogram obtained with the test solution may also show a dark red zone (thebaine) situated between those
due to codeine and to papaverine.
C. To 1.0 g of the powdered drug add 5 ml of water R, shake for 5 min and filter. To the filtrate add
0.25 ml of ferric chloride solution R2. A red colour develops which does not disappear on the addition
of 0.5 ml of dilute hydrochloric acid R.
TESTS
Thebaine Not more than 3.0 per cent, determined by liquid chromatography (2.2.29) and calculated
with reference to the dried drug.
Test solution. Prepare the test solution as described in the assay.
Reference solution. Dissolve 25.0 mg of thebaine R in the mobile phase and dilute to 25.0 ml with the
mobile phase. Dilute 10.0 ml of the solution to 100.0 ml with the mobile phase.
29-8
The chromatographic procedure is carried out as described in the assay. The test is not valid unless
the mass distribution ratio for thebaine is at least 3.0 and the number of theoretical plates is at least
3000. Calculate the percentage content of thebaine from the expression given in the assay.
Loss on drying (2.2.32). Not more than 15.0 per cent, determined on 1.000 g of raw opium cut
into thin slices, by drying in an oven at 100C to 105C for 4 h.
ASSAY
Test solution. Suspend 1.00 g of raw opium, cut into thin slices, in 50 ml of alcohol (50 per cent
V/V) R, mix with the aid of ultrasound for 1 h, allow to cool and dilute to 100.0 ml with the same
solvent. Allow to stand. To 10.0 ml of the supernatant liquid add 5 ml of ammonium chloride buffer
solution pH 9.5 R, dilute to 25.0 ml with water R and mix. Transfer 20.0 ml of the solution to a
chromatography column about 150 mm long and about 30 mm in internal diameter containing 15 g
of kieselguhr for chromatography R. Allow to stand for 15 min. Elute with two quantities, each of
40 ml, of a mixture of 15 volumes of 2-propanol R and 85 volumes of methylene chloride R. Evaporate
the eluate to dryness in vacuo at 40C. Transfer the residue to a volumetric flask with the aid of the
Reference solution. Dissolve 0.100 g of morphine hydrochloride R and 25.0 mg of codeine R in the mobile
phase and dilute to 25.0 ml with the mobile phase. Dilute 10.0 ml of the solution to 100.0 ml with
the mobile phase.
a column 0.25 m long and 4.6 mm in internal diameter packed with octylsilyl silica gel for chromatography R (5 m), equipped with a guard column 40 mm long and 4.6 mm in internal diameter
packed with octylsilyl silica gel for chromatography R (5 m),
as mobile phase at a flow rate of 1.5 ml per minute a solution prepared as follows: dissolve 1.0 g
of sodium heptanesulphonate monohydrate R in 420 ml of water R, adjust to pH 3.2 by addition of
phosphoric acid (4.9 g/l H3PO4) (about 5 ml) and add 180 ml of acetonitrile R,
a loop injector.
Inject suitable volumes of each solution.
The assay is not valid unless the resolution between the peaks corresponding to morphine and
codeine is at least 2.5. If necessary, adjust the volume of acetonitrile in the mobile phase. Inject the
reference solution six times. The assay is not valid unless the relative standard deviation of the peak
area for morphine is at most 1.0 per cent. Inject alternately the test solution and the reference
solution.
Calculate the percentage content of each alkaloid from the expression:
m1 A2 625
100
m2 A1 5 100 h
m1 = mass in grams of the alkaloid used to prepare the reference solution,
m2 = mass in grams of the substance to be examined used to prepare the test solution,
A1 = area of the peak corresponding to the alkaloid in the chromatogram obtained with the
reference solution,
A2 = area of the peak corresponding to the alkaloid in the chromatogram obtained with the test
solution,
h = percentage loss on drying.
For the calculation, 1 mg of morphine hydrochloride R is taken to be equivalent to 0.759 mg of
morphine and 1 mg of codeine R is taken to be equivalent to 0.943 mg of codeine.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
29-9
Bitter-Orange Flower Oil

Bitter-Orange Flower Oil complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Bitter-orange-flower oil is obtained by steam distillation from the fresh flowers of Citrus aurantium L.
subsp. aurantium (C. aurantium L. subsp. amara Engl.).
CHARACTERS
A clear, pale-yellow or dark-yellow liquid, with a characteristic odour reminiscent of bitter-orange
flowers, miscible with alcohol, with ether, with light petroleum, with fatty oils and with liquid
paraffin.
IDENTIFICATION
A. Examine in ultraviolet light at 365 nm the chromatograms obtained in the test for bergapten.
Before spraying with the reagent, the chromatogram obtained with the test solution shows a band
similar in position and fluorescence to that corresponding to methyl anthranilate in the chromatogram obtained with the reference solution. Other bands may be visible. Examine in ultraviolet light at
365 nm after spraying with the reagent. The chromatogram obtained with the reference solution
shows in the upper half a band of brownish-orange fluorescence corresponding to linalyl acetate, in
the lower half a band of brownish-orange fluorescence corresponding to linalol and immediately
below, a band of yellow-greenish fluorescence corresponding to bergapten. The chromatogram
obtained with the test solution shows two bands similar in position and fluorescence to the bands
corresponding to linalyl acetate and to linalol in the chromatogram obtained with the reference
solution. Other bands may also be present.
of the principal peaks in the chromatogram obtained with the test solution are approximately the
same as those of the peaks in the chromatogram obtained with the reference solution.
TESTS
Optical rotation (2.2.7). +1.5 to +11.5.
Acid value (2.5.1). Not more than 2.0.
Bergapten Examine by thin-layer chromatography (2.2.27), using a suitable silica gel as the coating
substance.
Test solution. Dissolve 0.1 g of the substance to be examined in alcohol R and dilute to 5.0 ml with the
same solvent.
Reference solution. Dissolve 5 l of methyl anthranilate R, 10 l of linalol R, 20 l of linalyl acetate R and
10 mg of bergapten R in alcohol R and dilute to 10.0 ml with the same solvent.
Apply separately to the plate, as bands, 10 l of each solution. Develop over a path of 15 cm using a
mixture of 15 volumes of ethyl acetate R and 85 volumes of toluene R. Allow the plate to dry in air and
examine in ultraviolet light at 365 nm. The chromatogram obtained with the reference solution
shows in the middle a band of blue fluorescence (methyl anthranilate) and below a band of greenishyellow fluorescence (bergapten). Spray with anisaldehyde solution R. Heat the plate at 100C to 105C
for 10 min. Examine in ultraviolet light at 365 nm. The chromatogram obtained with the test
solution does not show a band corresponding to that due to bergapten (essential oil of bitter-orange
peel) in the chromatogram obtained with the reference solution.
Reference solution. Dissolve 20 l of -pinene R, 5 l of sabinene R, 40 l of limonene R, 40 l of
linalol R, 20 l of linalyl acetate R, 5 l of -terpineol R, 5 l of neryl acetate R, 5 l of geranyl acetate R,
5 l of trans-nerolidol R and 5 l of methyl anthranilate R in 1 ml of hexane R.
a fused-silica capillary column 25 m to 60 m long and about 0.25 mm in internal diameter,
impregnated with macrogol 20,000 R as the bonded phase,
29-10
helium for chromatography R as the carrier gas at a flow rate of 1.5 ml per minute,
a split ratio of 1/100,
of 4C per minute to 230C and maintaining at 230C for 20 min, maintaining the temperature of
the injection port and of the detector at 270C.
Inject about 0.1 l of the reference solution. When the chromatograms are recorded in the
prescribed conditions, the components elute in the order indicated in the composition of the
reference solution. Record the retention times of these substances.
The test is not valid unless: the number of theoretical plates is not less than 30,000, calculated from
the limonene peak at 110C; the resolution between the peaks due to -pinene and to sabinene is not
less than 1.5.
Inject about 0.2 l of the substance to be examined. Using the retention times determined from the
chromatogram obtained with the reference solution, locate the components of the reference solution
on the chromatogram obtained with the test solution (disregard the peak due to hexane).
Determine the percentage content of each of these components by the normalisation procedure.
-Pinene
7.0 to 17.0 per cent.
Limonene
Linalol
Linalyl acetate
-Terpineol
Neryl acetate
Geranyl acetate 1.5 to 4.0 per cent.
trans-Nerolidol 1.0 to 9.0 per cent.
Methyl anthranilate
STORAGE
The type chromatogram is given for information and guidance only. It is not part of the mandatory requirements of the monograph.
Fig. 11751 Type chromatogram for bitter-orange-flower oil

1. -pinene
6. -terpineol
2. sabinene
7. neryl acetate
3. D-limonene
8. geranyl acetate
4. linalol
9. trans-nerolidol
5. linalyl acetate
10. methyl anthranilate
__________________________________________________________________________________________________________ Ph Eur
29-11
Orange Oil
Definition Orange Oil is obtained by mechanical means from the fresh peel of the sweet orange,
Citrus sinensis (L.) Osbeck.
Characteristics A yellow to yellowish brown liquid, visibly free from water; odour, that of orange.
Optical rotation +94 to +99, Appendix V F. On distillation, the first 10% of the distillate has an
optical rotation the same as, or only slightly lower than, the original oil.
Residue on evaporation 1.0 to 5.0% when determined by the method for residue on evaporation of
volatile oils, Appendix X M. Use 2 g and heat for 4 hours.
Solubility in ethanol Soluble at 20, in 7 parts of ethanol (90%), Appendix X M. A bright solution
is rarely obtained due to the presence of waxy non-volatile substances.
Content of aldehydes Not less than 1.0% w/w, calculated as decanal, C10H20O. Carry out the
method for the determination of aldehydes, Appendix X K, using 10 g, omitting the toluene and using a
volume, not less than 7 ml, of alcoholic hydroxylamine solution that exceeds by 1 to 2 ml the volume of
0.5M potassium hydroxide in ethanol (60%) VS required. Each ml of 0.5M potassium hydroxide in
ethanol (60%) VS is equivalent to 78.76 mg of C10H20O.
Storage Orange Oil should be kept in a well-filled, well-closed container, protected from light and
29-12
Terpeneless Orange Oil

Definition Terpeneless Orange Oil may be prepared by concentrating Orange Oil under reduced
pressure until most of the terpenes have been removed or by solvent partition.
Characteristics A clear, yellow or orange-yellow liquid, visibly free from water; odour and taste,
those of orange.
Optical rotation Not more than +60, Appendix V F.
Solubility in ethanol Soluble, at 20, in 1 part of ethanol (90%), Appendix X M.
Content of aldehydes Not less than 18% w/w, calculated as decanal, C10H20O. Carry out the
method for the determination of aldehydes, Appendix X K, using 1.5 g, omitting the toluene and using a
volume, not less than 7 ml, of alcoholic hydroxylamine solution that exceeds by 1 to 2 ml the volume of
0.5M potassium hydroxide in ethanol (60%) VS required. Each ml of 0.5M potassium hydroxide in ethanol
(60%) VS is equivalent to 78.76 mg of C10H20O.
Storage Terpeneless Orange Oil should be kept in a well-filled, well-closed container, protected from
light and stored at a temperature not exceeding 25.
Preparation
Compound Orange Spirit
29-13
Dried Bitter-Orange Peel

Definition Dried Bitter-Orange Peel is the dried outer part of the pericarp of the ripe, or nearly ripe,
fruit of Citrus aurantium L.
Characteristics Odour, aromatic; taste, aromatic and bitter.
Macroscopical In strips or pieces; outer surface dark orange-red and somewhat rough from the
presence of numerous minute pits, each corresponding to an oil gland; inner surface with only a small
remnant of white, spongy pericarp. Fracture, short.
Microscopical Epidermis of small polyhedral cells. Tissue subjacent to the epidermis parenchymatous,
many of the cells containing prismatic crystals of calcium oxalate. Numerous large oil glands and
small vascular strands embedded in the parenchyma.
Volatile oil Not less than 2.5% v/w, Appendix XI E, using 300 ml of water as the distillation liquid
but do not use xylene in the graduated tube. Use 20 g, reduced in size without loss of oil, for example
by crushing under water, and distil for 3 hours.
Preparations
Orange Peel Infusion
Orange Tincture
29-14
Orciprenaline Sulphate
H
OH
NHPri
HO
,H2SO4
OH
and enantiomer
(C11H17NO3)2,H2SO4
520.6
5874-97-5
Orciprenaline Sulphate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Beta-adrenoceptor agonist.
Preparations
Orciprenaline Oral Solution
Orciprenaline Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Orciprenaline sulphate contains not less than 98.0 per cent and not more than the equivalent of
102.0 per cent of di[(RS)-1-(3,5-dihydroxyphenyl)-2-[(1-methylethyl)amino]ethanol] sulphate,
calculated with reference to the anhydrous, solvent-free substance.
CHARACTERS
A white, crystalline powder, slightly hygroscopic, freely soluble in water and in alcohol, practically
insoluble in ether and in methylene chloride.
IDENTIFICATION
A. Dissolve 50.0 mg in a 0.04 per cent V/V solution of hydrochloric acid R and dilute to 50.0 ml with
the same acid solution. Dilute 5.0 ml of the solution to 50.0 ml with a 0.04 per cent V/V solution of
hydrochloric acid R. Examined between 240 nm and 350 nm (2.2.25), the solution shows an
absorption maximum at 278 nm. The specific absorbance at the maximum is 68.5 to 76.0, calculated
with reference to the anhydrous and solvent-free substance.
obtained with orciprenaline sulphate CRS. Examine the substances prepared as discs. If the spectra
obtained show differences, dissolve 50 mg of the substance to be examined and the reference
substance separately in the minimum volume of water R with heating. Add 10 ml of acetone R and
centrifuge. Dry the precipitate at 40C under reduced pressure for 3 h and record new spectra using
the residues.
Test solution. Dissolve 10 mg of the substance to be examined in alcohol R and dilute to 10 ml with
the same solvent.
Reference solution (a). Dissolve 10 mg of orciprenaline sulphate CRS in alcohol R and dilute to 10 ml
Reference solution (b). Dissolve 10 mg of orciprenaline sulphate CRS and 10 mg of salbutamol CRS in
alcohol R and dilute to 10 ml with the same solvent.
1.5 volumes of ammonia R, 10 volumes of water R and 90 volumes of aldehyde-free methanol R. Allow
the plate to dry in air and spray with a 10 g/l solution of potassium permanganate R. The principal spot
in the chromatogram obtained with the test solution is similar in position, colour and size to the
the chromatogram obtained with reference solution (b) shows two clearly separated principal spots.
D. Dissolve about 20 mg in 2 ml of alcohol R. Add 2 ml of a 1 g/l solution of dichloroquinonechlorimide R in alcohol R and 1 ml of sodium carbonate solution R. A violet colour is produced, turning
to brown.
E. It gives reaction (a) of sulphates (2.3.1).
29-15
TESTS
coating substance.
Test solution. Dissolve 0.20 g of the substance to be examined in a mixture of 1 volume of water R and
5 volumes of methanol R and dilute to 10 ml with the same mixture of solvents.
Reference solution (a). Dilute 1 ml of the test solution to 100 ml with a mixture of 1 volume of water R
and 5 volumes of methanol R.
water R and 5 volumes of methanol R.
Reference solution (c). Dilute 5 ml of reference solution (a) to 20 ml with a mixture of 1 volume of
water R and 5 volumes of methanol R.
4 volumes of concentrated ammonia R, 16 volumes of water R, 30 volumes of 2-propanol R and 50
volumes of ethyl acetate R. Allow the plate to dry in air. Expose the plate to iodine vapour. Any spot
intense than the principal spot in the chromatogram obtained with reference solution (a) (1.0 per
cent) and at most one such spot is more intense than the principal spot in the chromatogram
obtained with reference solution (b) (0.5 per cent). The test is not valid unless the spot in the
chromatogram obtained with reference solution (c) is clearly visible.
Methanol and 2-propanol Not more than 0.1 per cent m/m of methanol and 0.3 per cent m/m of
2-propanol, determined by gas chromatography (2.2.28), using ethanol R1 as internal standard.
Internal standard solution. Dilute 2 ml of ethanol R1 to 100 ml with water R. Dilute 1 ml of the
Test solution (a). Dissolve 1.0 g of the substance to be examined in water R and dilute to 10.0 ml with
the same solvent.
Test solution (b). Dissolve 1.0 g of the substance to be examined in water R, add 1.0 ml of the internal
standard solution and dilute to 10.0 ml with water R.
Reference solution. Dilute a mixture of 1.0 ml of methanol R and 3.0 ml of 2-propanol R to 100.0 ml
with water R. Dilute 1.0 ml of the solution to 10.0 ml with water R. To 1.0 ml of this solution, add
1.0 ml of the internal standard solution and dilute to 10.0 ml with water R.
a glass column 2 m long and 2 mm in internal diameter packed with ethylvinylbenzenedivinylbenzene co-polymer R (150 m to 180 m),
nitrogen for chromatography R as the carrier gas at a flow rate of 30 ml per minute,
detector at 180C.
Inject 1 l of each solution. In the chromatogram obtained with test solution (a), verify that there is
no peak with the same retention time as the internal standard. Calculate the content of methanol and
2-propanol taking their density at 20C to be 0.792 g per millilitre and 0.785 g per millilitre,
respectively.
Phenone Dissolve 0.50 g in a 0.04 per cent V/V solution of hydrochloric acid R and dilute to 25.0 ml
with the same acid solution. The absorbance (2.2.25) of the solution measured at 328 nm is not
greater than 0.16 (0.1 per cent).
Heavy metals 1.0 g complies with limit test C for heavy metals (20 ppm). Prepare the standard
using 2 ml of lead standard solution (10 ppm Pb) R.
Iron (2.4.9). The residue obtained in the test for sulphated ash complies with the limit test for iron
(20 ppm). Prepare the standard using iron standard solution (2 ppm Fe) R.
of water.
ASSAY
Dissolve 0.400 g in 30 ml of glacial acetic acid R. Titrate with 0.1M perchloric acid using 0.1 ml of
crystal violet solution R as indicator.
29-16
STORAGE
IMPURITIES
OH
NPri
HO
OH
A. 2-(1-methylethyl)-1,2,3,4-tetrahydroisoquinoline-4,6,8-triol,
OH
O
NHPri
HO
B. 1-(3,5-dihydroxyphenyl)-2-[(1-methylethyl)amino]ethanone.
__________________________________________________________________________________________________________ Ph Eur
29-17
Orphenadrine Hydrochloride
Me
NMe2
O
H
,HCl
and enantiomer
C18H23NO,HCl
305.9
341-69-5
Definition Orphenadrine Hydrochloride is (RS)-dimethyl[2-(2-methylbenzhydryloxy)ethyl]amine

hydrochloride. It contains not less than 98.5% and not more than 101.0% of C18H23NO,HCl,
Freely soluble in water, in chloroform and in ethanol (96%); practically insoluble in ether.
Identification
orphenadrine hydrochloride (RS 252).
Lead Not more than 10 ppm when determined by the following method. Heat 1.5 g with 7.5 ml of
nitric acid in a Kjeldahl flask until the first reaction has subsided, add 2.5 ml of sulphuric acid and
continue heating until the solution is colourless, adding, dropwise, more nitric acid if necessary. Cool,
add 10 ml of water, evaporate to low volume, cool and dilute to 25 ml with water (solution A). To
15 ml of the solution add 1 g of citric acid. Transfer the solution to a Nessler cylinder and make alkaline
with 5M ammonia. In a second Nessler cylinder mix 5 ml of solution A and 1 g of citric acid, make
alkaline with 5M ammonia and add 1 ml of lead standard solution (10 ppm Pb). Treat the contents of
each cylinder in the following manner. Add 1 ml of potassium cyanide solution PbT; the solutions
should not be more than faintly opalescent. If the colours of the solutions differ, equalise them by the
addition of a few drops of a highly diluted solution of burnt sugar or other non-reactive substance.
Dilute to 50 ml with water, add 0.1 ml of a filtered 10% w/v solution of sodium sulphide and mix
thoroughly. Compare the colours of the two solutions by a suitable method, such as by light reflected
from a white tile through the Nessler cylinders. The colour of the solution in the first cylinder is not
more intense than that of the solution in the second cylinder.
Quaternary ammonium salt Carry out the method for thin-layer chromatography, Appendix III A,
using silica gel G as the coating substance and a mixture of 50 volumes of propan-2-ol, 30 volumes of
butyl acetate, 15 volumes of water and 5 volumes of 13.5M ammonia as the mobile phase. Apply
separately to the plate 5 l of each of two solutions in methanol containing (1) 1.0% w/v of the
substance being examined and (2) 0.0050% w/v of ethyldimethyl[2-(2-methylbenzhydryloxy)ethyl]ammonium chloride BPCRS. After removal of the plate, allow it to dry in air and spray with dilute
potassium iodobismuthate solution. Any spot corresponding to ethyldimethyl[2-(2-methylbenzhydryloxy)ethyl]ammonium hydroxide in the chromatogram obtained with solution (1) is not more
Secondary amine Carry out the method for thin-layer chromatography, Appendix III A, using silica
gel GF254 as the coating substance and a mixture of 96 volumes of butan-1-ol and 4 volumes of 13.5M
ammonia as the mobile phase. Apply separately to the plate 10 l of each of two solutions in methanol
containing (1) 4.0% w/v of the substance being examined and (2) 0.020% w/v of methyl[2-(2-methylbenzhydryloxy)ethyl]amine hydrochloride BPCRS. After removal of the plate, allow it to dry in air and
examine under ultraviolet light (254 nm). Spray the plate with dilute potassium iodobismuthate solution
and examine again. Using each method of visualisation any spot corresponding to methyl[2-(2methylbenzhydryloxy)ethyl]amine in the chromatogram obtained with solution (1) is not more
1 g.
Assay Carry out Method I for non-aqueous titration, Appendix VIII A, using 1 g, 20 ml of mercury(II)
acetate solution and 1-naphtholbenzein solution as indicator. Each ml of 0.1M perchloric acid VS is
equivalent to 30.59 mg of C18H23NO,HCl.
Storage Orphenadrine Hydrochloride should be kept in a well-closed container and protected from
light.
29-18
Action and use Used in treatment of Parkinsons disease.
Preparation
Orphenadrine Hydrochloride Tablets
29-19
Ouabain
O
OH
Me
OH
OH
CH2
H
H
OH
O
OH
HO
Me
OH
OH
C29H44O12,8H2O
729
11018-89-6
Ouabain complies with the requirements of the 3rd edition of the European Pharmacopoeia [0048]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ouabain contains not less than 96.0 per cent and not more than the equivalent of 104.0 per cent of
3-[(6-deoxy--L-mannopyranosyl)oxy]-1,5,11,14,19-pentahydroxy-5,14-card-20(22)-enolide,
CHARACTERS
A white, crystalline powder or colourless crystals, sparingly soluble in water and in ethanol,
practically insoluble in ether and in ethyl acetate.
IDENTIFICATION
A. Examine the chromatograms obtained in the test for related substances. The principal spot in the
chromatogram obtained with the test solution is similar in position, colour and size to the spot in the
B. Dissolve 2 mg to 3 mg in 2 ml of sulphuric acid R; a pink colour develops which quickly changes to
red. The solution shows green fluorescence in ultraviolet light.
C. Dissolve about 1 mg in 1 ml of dinitrobenzene solution R and add 0.2 ml of dilute sodium hydroxide
solution R. An intense blue colour develops.
D. Dissolve 0.1 g in 5 ml of a 150 g/l solution of sulphuric acid R and boil for a few minutes. The
solution becomes yellow and turbid. Filter and add to the filtrate 5 ml of a 120 g/l solution of sodium
hydroxide R and 3 ml of cupri-tartaric solution R. Heat. A red precipitate is formed.
TESTS
Solution S Dissolve 0.20 g in 15 ml of water R, heating on a water-bath. Allow to cool and dilute to
coating substance.
Test solution. Dissolve a quantity of the substance to be examined corresponding to 20 mg of the
anhydrous substance in 1.0 ml of a mixture of 32 volumes of water R, 100 volumes of chloroform R
and 100 volumes of methanol R.
Reference solution (a). Dissolve a quantity of ouabain CRS corresponding to 20 mg of the anhydrous
substance in 1.0 ml of a mixture of 32 volumes of water R, 100 volumes of chloroform R and 100
volumes of methanol R.
Reference solution (b). Dissolve a quantity of ouabain CRS corresponding to 10 mg of the anhydrous
29-20
substance in a mixture of 32 volumes of water R, 100 volumes of chloroform R, 100 volumes of
methanol R and dilute to 25 ml with the same mixture of solvents.
Reference solution (c). Dilute 2.5 ml of reference solution (b) to 10 ml with a mixture of 32 volumes of
water R, 100 volumes of chloroform R and 100 volumes of methanol R.
Apply separately to the plate 5 l of each solution. Develop over a path of 13 cm using a
homogeneous mixture of 4 volumes of water R, 15 volumes of methanol R, 15 volumes of dimethyl
sulphoxide R and 70 volumes of chloroform R. Dry the plate immediately at 140C for 30 min in a
ventilated drying oven. Allow to cool, spray with alcoholic sulphuric acid solution R and heat at 140C
for 15 min. Any spot in the chromatogram obtained with the test solution, apart from the principal
(2.0 per cent). The test is not valid unless the principal spot in the chromatogram obtained with
reference solution (a) and the principal spot in the chromatogram obtained with the test solution
migrate over a distance sufficient to give unequivocal separation of the secondary spots and the spot
in the chromatogram obtained with reference solution (c) is clearly visible.
Alkaloids and strophanthin-K To 5.0 ml of solution S add 0.5 ml of a 100 g/l solution of tannic
acid R. No precipitate is formed.
ASSAY
solution to 100.0 ml with alcohol R. Prepare a reference solution in the same manner using 40.0 mg of
ouabain CRS. To 5.0 ml of each solution add 3.0 ml of alkaline sodium picrate solution R, allow to
stand protected from bright light for 30 min and measure the absorbance (2.2.25) of each solution at
the maximum at 495 nm using as the compensation liquid a mixture of 5.0 ml of alcohol R and 3.0 ml
of alkaline sodium picrate solution R prepared at the same time.
Calculate the content of C29H44O12 from the absorbances measured and the concentrations of the
solutions.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
29-21
Oxazepam
O
H
N
OH
H
N
Cl
and enantiomer
C15H11ClN2O2
286.7
604-75-1
Oxazepam complies with the requirements of the 3rd edition of the European Pharmacopoeia [0778]. These
Preparation
Oxazepam Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Oxazepam contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of
7-chloro-3-hydroxy-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one, calculated with reference to
CHARACTERS
A white or almost white, crystalline powder, practically insoluble in water, slightly soluble in alcohol
and in methylene chloride.
IDENTIFICATION
A. Prepare the solutions immediately before use, protected from light. Dissolve 20.0 mg in alcohol R and
dilute to 100.0 ml with the same solvent. Dilute 10.0 ml of the solution to 50.0 ml with alcohol R
(solution A). Dilute 10.0 ml of solution A to 100.0 ml with alcohol R (solution B). Examined between
300 nm and 350 nm (2.2.25), solution A shows an absorption maximum at 316 nm. Examined
between 220 nm and 250 nm, solution B shows an absorption maximum at 229 nm. The specific
absorbance at the maximum at 229 nm is 1220 to 1300.
B. Examine by infrared absorption spectrophotometry (2.2.24) comparing with the spectrum
obtained with oxazepam CRS. Examine the substances prepared as discs.
D. Dissolve about 20 mg in a mixture of 5 ml of hydrochloric acid R and 10 ml of water R. Heat to
boiling for 5 min and cool. Add 2 ml of a 1 g/l solution of sodium nitrite R and allow to stand for
1 min. Add 1 ml of a 5 g/l solution of sulphamic acid R, mix and allow to stand for 1 min. Add 1 ml of
a 1 g/l solution of naphthylethylenediamine dihydrochloride R. A red colour develops.
TESTS
(2.2.27), using as the coating substance a suitable silica gel with a fluorescent indicator having an
optimal intensity at 254 nm. Before use, wash the plate with methanol R until the solvent front has
migrated at least 17 cm. Allow the plate to dry in air and heat at 100C to 105C for 30 min.
Test solution(a). Dissolve 50 mg of the substance to be examined in acetone R and dilute to 10 ml with
the same solvent.
Reference solution (a). Dissolve 10 mg of oxazepam CRS in acetone R and dilute to 10 ml with the
same solvent.
29-22
Reference solution (b). Dissolve 10 mg of oxazepam CRS and 10 mg of bromazepam CRS in acetone R
Reference solution (c). Dilute 1 ml of test solution (b) to 100 ml with acetone R.
Reference solution (d). Dilute 5 ml of reference solution (c) to 10 ml with acetone R.
Apply separately to the plate 20 l of each solution. Develop over a path of 15 cm, in the same
direction as the washing with methanol R, using a mixture of 10 volumes of methanol R and 100
volumes of methylene chloride R. Allow the plate to dry in air and examine in ultraviolet light at
is not more intense than the spot in the chromatogram obtained with reference solution (c) (0.2 per
cent) and at most one such spot is more intense than the spot in the chromatogram obtained with
reference solution (d) (0.1 per cent). The test is not valid unless the chromatogram obtained with
reference solution (b) shows two clearly separated spots.
100C to 105C at a pressure not exceeding 0.7 kPa.
ASSAY
Dissolve 0.250 g in a mixture of 10 ml of anhydrous acetic acid R and 90 ml of acetic anhydride R.
Titrate with 0.1M perchloric acid determining the end-point potentiometrically (2.2.20).
STORAGE
Store in an well-closed container, protected from light.
__________________________________________________________________________________________________________ Ph Eur
29-23
Oxetacaine
Me
Me
O Me
Me
Me
N
HO
N
Me
C28H41N3O3
467.6
126-27-6
Definition Oxetacaine is 2,2-(2-hydroxyethylimino)bis[N-(,-dimethylphenethyl)-Nmethylacetamide]. It contains not less than 99.0% and not more than 100.5% of C28H41N3O3,
Practically insoluble in water; freely soluble in methanol; very soluble in chloroform; soluble in ethyl
acetate.
spectrum of oxetacaine (RS 254).
silica gel 60 precoated plate (Merck plates are suitable) and a mixture of 79 volumes of toluene, 20
volumes of absolute ethanol and 1 volume of 18M ammonia as the mobile phase. Apply separately to
the plate 5 l of each of three solutions of the substance being examined in ethyl acetate containing (1)
10.0% w/v, (2) 0.050% w/v and (3) 0.010% w/v. After removal of the plate, dry it in a current of
warm air and spray liberally with a solution containing 6% w/v of ammonium thiocyanate and 2% w/v
of cobalt(II) chloride. Carefully remove excess solution by applying filter paper to the plate and allow
the plate to dry in air for 10 minutes or until spots appear. Any secondary spot in the chromatogram
obtained with solution (1) is not more intense than the spot in the chromatogram obtained with
solution (2) and not more than one such spot is more intense than the spot in the chromatogram
Assay Dissolve 1 g in 50 ml of anhydrous acetic acid and carry out Method I for non-aqueous titration,
equivalent to 46.76 mg of C28H41N3O3.
When oxethazaine is prescribed or demanded, Oxetacaine shall be dispensed or supplied.
29-24
Oxolinic Acid
Et
O
COOH
O
C13H11NO5
261.2
14698-29-4
Oxolinic Acid complies with the requirements of the 3rd edition of the European Pharmacopoeia [1353].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Oxolinic acid contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent
of 5-ethyl-8-oxo-5,8-dihydro-1,3-dioxolo[4,5-g]quinoline-7-carboxylic acid, calculated with reference
CHARACTERS
An almost white or pale yellow, crystalline powder, practically insoluble in water, very slightly soluble
in methylene chloride, practically insoluble in alcohol. It dissolves in dilute solutions of alkali
hydroxides.
IDENTIFICATION
A. Dissolve 25.0 mg in 5 ml of 0.1M sodium hydroxide, heating on a water-bath. Allow to cool and
dilute to 100.0 ml with methanol R. Dilute 2.0 ml of the solution to 100.0 ml with 0.1M hydrochloric
acid. Examined between 220 nm and 350 nm (2.2.25), the solution shows three absorption maxima,
at 260 nm, 322 nm and 336 nm respectively. The ratio of the absorbance measured at the maximum
at 260 nm to that measured at the maximum at 336 nm is 4.9 to 5.2.
obtained with oxolinic acid CRS. Examine the substances prepared as discs.
C. Examine by thin-layer chromatography (2.2.27), using a suitable silica gel as the coating
substance.
Test solution. Dissolve 10 mg of the substance to be examined in 3 ml of dilute sodium hydroxide
solution R and dilute to 20 ml with alcohol R.
Reference solution (a). Dissolve 10 mg of oxolinic acid CRS in 3 ml of dilute sodium hydroxide solution R
and dilute to 20 ml with alcohol R.
Reference solution (b). Dissolve 5 mg of ciprofloxacin hydrochloride CRS in methanol R and dilute to
10 ml with the same solvent. Dilute 1 ml of the solution to 2 ml with reference solution (a).
Apply separately to the plate 10 l of each solution. At the bottom of a chromatographic tank, place
an evaporating disk containing 50 ml of concentrated ammonia R. Close the tank and expose the plate
to the ammonia vapour for 15 min. Withdraw the plate and transfer to a chromatographic tank and
develop over a path of 15 cm using a mixture of 10 volumes of acetonitrile R, 20 volumes of
concentrated ammonia R, 40 volumes of methanol R and 40 volumes of methylene chloride R. Allow the
plate to dry in air. Examine in ultraviolet light at 254 nm. The principal spot in the chromatogram
obtained with the test solution is similar in position, fluorescence and size to the principal spot in the
chromatogram obtained with reference solution (a). The identification is not valid unless the chromatogram obtained with reference solution (b) shows two clearly separated spots.
TESTS
Solution S Dissolve 0.6 g in 20 ml of a 40 g/l solution of sodium hydroxide R.
a suitable cellulose with a particle size of narrow distribution.
29-25
Test solution. Dissolve 50 mg in 3 ml of dilute sodium hydroxide solution R and dilute to 10 ml with
alcohol R.
Reference solution (a). Dilute 1 ml of the test solution to 50.0 ml with alcohol R. Dilute 1.0 ml of the
solution to 5.0 ml with alcohol R.
Reference solution (b). Dissolve 2 mg of oxolinic acid impurity B CRS in alcohol R and dilute to 10 ml
with the same solvent. Dilute 0.5 ml of the solution to 10 ml with alcohol R.
Reference solution (c). Dissolve 5 mg of the substance to be examined and 5 mg of oxolinic acid
impurity A CRS in 2 ml of dilute sodium hydroxide solution R and dilute to 40 ml with alcohol R.
Apply separately to the plate 5 l of each solution, in sufficiently small portions to obtain small spots.
Develop over a path of 6 cm (corresponding to two thirds of the plate height) with a mixture of 15
volumes of ammonia R, 30 volumes of water R and 55 volumes of propanol R. Allow the plate to dry
in air and examine in ultraviolet light at 254 nm. In the chromatogram obtained with the test
solution: any spot corresponding to oxolinic acid impurity B is not more intense than the spot in the
chromatogram obtained with reference solution (b) (0.2 per cent); any spot apart from the principal
spot and any spot corresponding to oxolinic acid impurity B is not more intense than the spot in the
chromatogram obtained with reference solution (a) (0.4 per cent). The test is not valid unless the
chromatogram obtained with reference solution (c) shows two clearly separated principal spots.
Loss on drying (2.2.32). Not more than 0.5 per cent determined on 1.000 g by heating in an oven
at 100C to 105C.
Sulphated ash (2.4.14). Not more than 0.1 per cent determined on 1.0 g.
ASSAY
Dissolve 0.200 g in 150 ml of dimethyformamide R. Titrate with 0.1M tetrabutylammonium hydroxide,
determining the end-point potentiometrically (2.2.20). Use a glass indicator electrode and a calomel
reference electrode containing, as the electrolyte, a saturated solution of potassium chloride R in
methanol R. Carry out a blank titration.
1 ml of 0.1M tetrabutylammonium hydroxide is equivalent to 26.12 mg of C13H11NO5.
STORAGE
IMPURITIES
O
COOH
OH
A. 8-hydroxy-1,3-dioxolo[4,5-g]quinoline-7-carboxylic acid,
Et
O
COOEt
O
B. ethyl 5-ethyl-8-oxo-5,8-dihydro-1,3-dioxolo[4,5-g]quinoline-7-carboxylate,
Me
O
COOH
O
C. 5-methyl-8-oxo-5,8-dihydro-1,3-dioxolo[4,5-g]quinoline-7-carboxylic acid.
__________________________________________________________________________________________________________ Ph Eur
29-26
Oxprenolol Hydrochloride
revised 1/01
H
OH
NHPri
,HCl
CH2
and enantiomer
C15H23NO3,HCl
301.8
6452-73-9
Oxprenolol Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Oxprenolol Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Oxprenolol hydrochloride contains not less than 98.5 per cent and not more than the equivalent of
101.5 per cent of (RS)-1-(isopropylamino)-3-[2-(2-propenyl)oxy]phenoxy-2-propanol hydrochloride,
CHARACTERS
A white or almost white, crystalline powder, very soluble in water, freely soluble in alcohol.
IDENTIFICATION
obtained with oxprenolol hydrochloride CRS. If the spectra obtained in the solid state show differences,
dissolve the substance to be examined and the reference substance separately in ethyl acetate R,
TESTS
solution GY6 (Method II, 2.2.2).
pH (2.2.3). The pH of freshly prepared solution S is 4.5 to 6.0.
Test solution (a). Dissolve 0.10 g of the substance to be examined in 2 ml of a mixture of 1 volume of
Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with a mixture of 1 volume of methanol R
and 9 volumes of chloroform R.
Reference solution (a). Dissolve 10 mg of oxprenolol hydrochloride CRS in 2 ml of a mixture of 1 volume
of methanol R and 9 volumes of chloroform R.
Reference solution (b). Dilute 0.4 ml of test solution (a) to 100 ml with a mixture of 1 volume of
Reference solution (c). Dilute 5 ml of reference solution (b) to 10 ml with a mixture of 1 volume of
Apply to the plate 2 l of each solution. Allow the spots to dry in air for 15 min. Develop over a path
of 13 cm using a mixture of 2 volumes of concentrated ammonia R, 12 volumes of methanol R and 88
volumes of chloroform R. Dry the plate in a current of warm air for 10 min, allow to cool and spray
29-27
with anisaldehyde solution R. Heat at 100C to 105C for 5 min to 10 min. Examine in daylight. Any
spot in the chromatogram obtained with test solution (a), apart from the principal spot, is not more
Lead Not more than 5 ppm of Pb, determined by atomic absorption spectrometry (Method II,
2.2.23).
Test solution. Dissolve 1.00 g in water R and dilute to 25.0 ml with the same solvent.
Reference solutions. Prepare the reference solutions using 0.5 ml and 1.0 ml of lead standard solution
(10 ppm Pb) R diluted to 25.0 ml with water R.
Measure the absorbance at 217.0 nm using a lead hollow-cathode lamp as a source of radiation.
60C for 6 h.
ASSAY
STORAGE
__________________________________________________________________________________________________________ Ph Eur
29-28
Oxybuprocaine Hydrochloride
O
O
NEt 2
,HCl
H2N
OBu n
C17H28N2O3,HCl
344.9
5987-82-6
Oxybuprocaine Hydrochloride complies with the requirements of the 3rd edition of the European
Preparation
Oxybuprocaine Eye Drops
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Oxybuprocaine hydrochloride contains not less than 98.5 per cent and not more than the equivalent
of 101.5 per cent of 2-(diethylamino)ethyl 4-amino-3-butoxybenzoate hydrochloride, calculated with
CHARACTERS
A white, crystalline powder or colourless crystals, very soluble in water, freely soluble in alcohol.
IDENTIFICATION
obtained with oxybuprocaine hydrochloride CRS. Examine the substances prepared as discs. If the
spectra obtained show differences, dissolve the substance to be examined and the reference substance
separately in methanol R, evaporate to dryness and record the spectra again using the residues.
the same solvent.
Reference solution (a). Dissolve 40 mg of oxybuprocaine hydrochloride CRS in methanol R and dilute to
Reference solution (b). Dissolve 20 mg of procaine hydrochloride R in reference solution (a) and dilute to
10 volumes of anhydrous formic acid R, 15 volumes of methanol R, 15 volumes of water R and 60
volumes of ethyl acetate R. Dry the plate in a current of hot air for 10 min and examine in ultraviolet
light at 254 nm. Spray with dimethylaminobenzaldehyde solution R7. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in
D. Dilute 0.2 ml of solution S (see Tests) to 2 ml with water R. The solution gives reaction (a) of
chlorides (2.3.1).
TESTS
29-29
Buffer solution pH 2.5. Add 6 ml of perchloric acid solution R and 12 ml of dilute phosphoric acid R to
950 ml of water R. Adjust the pH to 2.5 with a 40 g/l solution of sodium hydroxide R and dilute to
Reference solution (a). Dilute 1.0 ml of the test solution to 20.0 ml with the mobile phase and dilute
Reference solution (b). Mix 1.0 ml of the test solution with 1 ml of a 40 g/l solution of sodium
hydroxide R and allow to stand for 20 min. Add 1 ml of dilute phosphoric acid R and dilute to 100.0 ml
with the mobile phase. Dilute 25 ml to 100.0 ml with the mobile phase.
The chromatographic conditions may be carried out using:
silica gel for chromatography R1 (5 m),
volumes of buffer solution pH 2.5,
When the chromatograms are recorded in the prescribed conditions the retention time is about 9
min for oxybuprocaine hydrochloride. Adjust the sensitivity of the system so that the height of the
principal peak in the chromatogram obtained with reference solution (a) is at least 50 per cent of the
full scale of the recorder.
Inject 20 l of reference solution (b). The test is not valid unless: in the chromatogram obtained
with reference solution (b), the resolution between the main peaks corresponding to oxybuprocaine
and oxybuprocaine impurity B (hydrolysis product) is at least twelve.
for four times the retention time of the principal peak. In the chromatogram obtained with the test
solution: the area of any peak, apart from the principal peak, is not greater than 0.4 times the area of
the principal peak in the chromatogram obtained with reference solution (a) (0.1 per cent); the sum
of the areas of all the peaks apart from the principal peak is not greater than the area of the principal
peak in the chromatogram obtained with reference solution (a) (0.25 per cent). Disregard any peak
with an area less than 0.05 times that of the peak due to oxybuprocaine hydrochloride in the chromatogram obtained with reference solution (a).
Loss on drying (2.2.32). Not more than 0.5 per cent determined on 1.000 g by drying in an oven at
100C to 105C.
ASSAY
STORAGE
IMPURITIES
COOH
H2N
R
A. R = H: 4-aminobenzoic acid,
B. R = O-CH2-CH2-CH2-CH:
4-amino-3-butoxybenzoic acid,
C. R = OH: 4-amino-3-hydroxybenzoic acid.
__________________________________________________________________________________________________________ Ph Eur
29-30
Oxybutynin Hydrochloride
O
O
HO
C22H31NO3,HCl
NEt2
394.0
,HCl
1508-65-2
Oxybutynin Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Anticholinergic.
Preparation
Oxybutynin Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Oxybutynin hydrochloride contains not less than 99.0 per cent and not more than the equivalent of
102.0 per cent of 4-(diethylamino)but-2-ynyl (R,S)-2-cyclohexyl-2-hydroxy-2-phenylacetate hydrochloride, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, freely soluble in water and in alcohol, soluble in acetone,
slightly soluble in cyclohexane.
IDENTIFICATION
obtained with oxybutynin hydrochloride CRS. Examine the substances prepared as discs.
the same solvent.
Reference solution. Dissolve 10 mg of oxybutynin hydrochloride CRS in alcohol R and dilute to 2 ml with
the same solvent.
Apply to the plate 5 l of each solution. Develop over a path of 15 cm, using methanol R. Allow the
plate to dry in air, and expose the plate to iodine vapour for 30 min. The principal spot in the
TESTS
+0.10.
Reference solution (a). Dissolve 50.0 mg of oxybutynin hydrochloride CRS and 50.0 mg of oxybutynin
impurity A CRS in the mobile phase and dilute to 100.0 ml with the mobile phase. Dilute 10.0 ml of
the solution to 100.0 ml with the mobile phase.
29-31
gel for chromatography R2 (5 m),
as mobile phase at a flow rate of 1 ml/min a mixture of 49 volumes of a solution containing
3.4 g/l of potassium dihydrogen phosphate R and 4.36 g/l of dipotassium hydrogen phosphate R and
51 volumes of acetonitrile R,
conditions the retention times are: oxybutynin hydrochloride about 15 min and impurity A about
24 min. Adjust the sensitivity of the system so that the heights of the peaks in the chromatogram
obtained are about 20 per cent of the full scale of the recorder. The test is not valid unless the
resolution between the peaks due to oxybutynin hydrochloride and to impurity A is at least 11.0.
Continue the chromatography for about twice the retention time of the principal peak. In the
chromatogram obtained with the test solution: the area of any peak due to impurity A is not greater
than 1.5 times the area of the peak due to impurity A in the chromatogram obtained with reference
solution (a) (1.5 per cent); the sum of the areas of all the peaks, apart from the principal peak and the
peak due to impurity A, is not greater than the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.5 per cent). Disregard any peak with an area less than 0.05
Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R.
100C to 105C.
ASSAY
STORAGE
IMPURITIES
O
O
HO
NEt2
and enantiomer
A. 4-(diethylamino)but-2-ynyl (RS)-2-(cyclohex-3-enyl)-2-cyclohexyl-2-hydroxyacetate,
O
O
HO
NEt 2
B. 4-(diethylamino)but-2-ynyl (RS)-2-hydroxy-2,2-diphenylacetate (diphenyl analogue of

oxybutynin),
O
HO
Me
N
and enantiomer
Et
C. 4-(ethylmethylamino)but-2-ynyl (RS)-2-cyclohexyl-2-hydroxy-2-phenylacetate (methylethyl

analogue of oxybutynin),
29-32
COOH
HO
and enantiomer
D. (RS)-2-cyclohexyl-2-hydroxy-2-phenylacetic acid (phenylcyclohexylglycolic acid),

O
HO
Et
N
Pr n
and enantiomer
E. 4-(ethylpropylamino)but-2-ynyl (RS)-2-cyclohexyl-2-hydroxy-2-phenylacetate (ethylpropyl

analogue of oxybutynin).
__________________________________________________________________________________________________________ Ph Eur
29-33
Oxygen
O2
32.00
7782-44-7
Oxygen complies with the requirements of the 3rd edition of the European Pharmacopoeia [0417]. These
Oxygen should be kept in approved metal cylinders, the shoulders of which are painted white and the
remainder black. The cylinder should carry a label stating Oxygen. In addition, Oxygen or the
symbol O2 should be stencilled in paint on the shoulder of the cylinder.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Oxygen contains not less than 99.5 per cent V/V of O2.
CHARACTERS
A colourless, odourless gas. At 20C and at a pressure of 101 kPa, 1 volume dissolves in about 32
volumes of water.
PRODUCTION
Carbon dioxide Not more than 300 ppm V/V, determined using an infrared analyser (2.5.24).
Reference gas (a). Use oxygen R.
Reference gas (b). Use a mixture containing 300 ppm V/V of carbon dioxide R1 in nitrogen R1.
of carbon dioxide in the gas to be examined.
Carbon monoxide Not more than 5 ppm V/V, determined using an infrared analyser (2.5.25).
Reference gas (a). Use oxygen R.
Reference gas (b). Use a mixture containing 5 ppm V/V of carbon monoxide R in nitrogen R1.
of carbon monoxide in the gas to be examined.
Assay Determine the concentration of oxygen using a paramagnetic analyser (2.5.27).
IDENTIFICATION
Second identification: A, B.
A. Place a glowing splinter of wood in the substance to be examined. The splinter bursts into flame.
B. Shake with alkaline pyrogallol solution R. The substance to be examined is absorbed and the
solution becomes dark brown.
C. It complies with the limits of the assay.
TESTS
(2.1.6).
Carbon monoxide Not more than 5 ppm V/V, determined using a carbon monoxide detector tube
(2.1.6).
STORAGE
Store as a compressed gas or liquid in appropriate containers, complying with the legal regulations.
Taps and valves are not to be greased or oiled.
IMPURITIES
A. carbon dioxide.
B. carbon monoxide,
C. water.
__________________________________________________________________________________________________________ Ph Eur
29-34
Oxymetazoline Hydrochloride
Me
HO
N
,HCl
But
C16H24N2O,HCl
HN
Me
296.8
2315-02-8
Oxymetazoline Hydrochloride complies with the requirements of the 3rd edition of the European
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Oxymetazoline hydrochloride contains not less than 99.0 per cent and not more than the equivalent
of 101.0 per cent of 3-[(4,5-dihydro-1H-imidazol-2-yl)methyl]-6-(1,1-dimethylethyl)-2,4-dimethylphenol hydrochloride, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, freely soluble in water and in alcohol, practically
insoluble in ether.
IDENTIFICATION
First identification: A, D.
A. Examine by infrared absorption spectrophotometry (2.2.24) comparing with the spectrum
obtained with oxymetazoline hydrochloride CRS.
C. To a solution of about 2 mg in 1 ml of water R add 0.2 ml of a 50 g/l solution of sodium
nitroprusside R and 0.2 ml of dilute sodium hydroxide R. Allow to stand for 10 min. Add 2 ml of sodium
hydrogen carbonate solution R. A violet colour develops.
TESTS
solution BY7 (Method II, 2.2.2)
Acidity or alkalinity Dissolve 0.25 g in water R and dilute to 25 ml with the same solvent. Add
0.1 ml of methyl red solution R and 0.2 ml of 0.01M hydrochloric acid. The solution is red. Not more
than 0.4 ml of 0.01M sodium hydroxide is required to change the colour of the indicator to yellow.
coating substance.
ethyl acetate R and methanol R and dilute to 10 ml with the same mixture of solvents.
Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with a mixture of equal volumes of ethyl
acetate R and methanol R.
Reference solution (a). Dissolve 40 mg of oxymetazoline hydrochloride CRS in a mixture of equal
volumes of ethyl acetate R and methanol R and dilute to 10 ml with the same mixture of solvents.
Reference solution (b). Dilute 1 ml of test solution (b) to 20 ml with a mixture of equal volumes of
ethyl acetate R and methanol R.
Reference solution (c). Dilute 1 ml of test solution (b) to 40 ml with a mixture of equal volumes of ethyl
acetate R and methanol R.
volumes of diethylamine R, 15 volumes of cyclohexane R and 79 volumes of ethanol R. Dry the plate in
a current of warm air for 5 min. Allow to cool and spray with a freshly prepared 5.0 g/l solution of
29-35
potassium ferricyanide R in ferric chloride solution R2. Examine in daylight. Any spot in the chromatogram obtained with test solution (a), apart from the principal spot, is not more intense than the spot
in the chromatogram obtained with reference solution (b) (0.5 per cent) and at most one such spot is
100C to 105C.
Sulphated ash (2.2.14.). Not more than 0.1 per cent, determined on 1.0 g.
ASSAY
1 ml of 0.1M perchloric acid is equivalent to 29.68 mg of C16H25ClN2O.
STORAGE
IMPURITIES
OH
But
Me
Me
N
H
NHAc
A. N-(2-aminoethyl)-2-[4-(1,1-dimethylethyl)-3-hydroxy-2,6-dimethylphenyl)]acetamide.
__________________________________________________________________________________________________________ Ph Eur
29-36
Oxymetholone
Me Me
OH
HO
Me
HC
H
O
H
C21H32O3
332.5
434-07-1
Definition Oxymetholone is 17-hydroxy-2-hydroxymethylene-17-methyl-5-androstan-3-one. It

contains not less than 97.0% and not more than 103.0% of C21H32O3, calculated with reference to
Characteristics A white to creamy white crystalline powder; odourless or almost odourless. It
exhibits polymorphism.
Practically insoluble in water; freely soluble in chloroform; soluble in ethanol (96%); slightly soluble
in ether.
Identification
ethanolic sodium hydroxide exhibits a maximum only at 315 nm. The absorbance at 315 nm is about
1.1.
ethanolic hydrochloric acid exhibits a maximum only at 277 nm. The absorbance at 277 nm is about
1.0.
C. The infrared absorption spectrum, Appendix II A, is concordant with the reference spectrum of
oxymetholone (RS 256). If the spectra are not concordant, dissolve the substance in the minimum of
chloroform, evaporate to dryness, dry the residue over phosphorus pentoxide at a pressure not exceeding
0.7 kPa and prepare a new spectrum.
D. Complies with the test for identification of steroids, Appendix III A, using impregnating solvent II and
mobile phase D.
gel G as the coating substance and a mixture of 98 volumes of toluene and 2 volumes of absolute
ethanol as the mobile phase. Apply separately to the plate 10 l of each of two solutions of the
substance being examined in a mixture of equal volumes of chloroform and ethanol (96%) containing
(1) 1.0% w/v and (2) 0.0050% w/v. After removal of the plate, allow it to dry in air and spray with
vanillinethanolic sulphuric acid reagent. Any secondary spot in the chromatogram obtained with
solution (1) is not more intense than the spot in the chromatogram obtained with solution (2).
1 g.
Assay Dissolve 0.1 g in sufficient 0.01M ethanolic sodium hydroxide to produce 200 ml, dilute 5 ml to
250 ml with 0.01M ethanolic sodium hydroxide and measure the absorbance of the resulting solution at
the maximum at 315 nm, Appendix II B. Calculate the content of C21H32O3 taking 547 as the value
of A(1%, 1 cm) at the maximum at 315 nm.
Storage Oxymetholone should be kept free from contact with ferrous metals and protected from
light.
Preparation
Oxymetholone Tablets
29-37
Oxyphenbutazone
OH
O
N
Bun
H
N
O
and enantiomer
C19H20N2O3,H2O
342.4
129-20-4
Oxyphenbutazone complies with the requirements of the 3rd edition of the European Pharmacopoeia [0418].
Preparation
Oxyphenbutazone Eye Ointment
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Oxyphenbutazone contains not less than 99.0 per cent and not more than the equivalent of 101.0 per
cent of 4-butyl-1-(4-hydroxyphenyl)-2-phenylpyrazolidine-3,5-dione, calculated with reference to the
CHARACTERS
A white, crystalline powder, practically insoluble in water, freely soluble in alcohol and soluble in
ether. It dissolves in dilute solutions of alkali hydroxides.
IDENTIFICATION
A. Dissolve 10.0 mg in 0.01M sodium hydroxide and dilute to 100.0 ml with the same solvent. Dilute
10.0 ml of the solution to 100.0 ml with 0.01M sodium hydroxide. Examined between 230 nm and
obtained with oxyphenbutazone CRS. Examine the substances as 60 g/l solutions in methylene
chloride R, using a 0.1 mm cell.
C. Dissolve about 20 mg in 2 ml of alcohol R. Add 2 ml of a 1 g/l solution of dichloroquinonechlorimide R in alcohol R and 1 ml of sodium carbonate solution R. An intense green colour is produced.
D. To 0.1 g add 1 ml of glacial acetic acid R and 2 ml of hydrochloric acid R. Boil under a reflux
condenser for 30 min. Cool, add 10 ml of water R and filter. To the filtrate add 3 ml of a 7 g/l
solution of sodium nitrite R. A yellow colour develops. To 1 ml of this solution add a solution of
10 mg of -naphthol R in 5 ml of sodium carbonate solution R. An orange or orange-red precipitate is
formed.
TESTS
Solution S To 0.50 g add a mixture of 8 ml of a 75 g/l solution of glycine R and 12 ml of 1M sodium
hydroxide. Shake for 1 min, keep the solution at 25C for exactly 60 min and use immediately.
Absorbance (2.2.25). The absorbance of solution S, measured at 420 nm in a 4 cm cell, is not
greater than 0.40.
coating substance.
Test solution. Dissolve 0.1 g of the substance to be examined in ethanol R containing 0.2 g/l of
butylhydroxytoluene R and dilute to 5 ml with the same solvent. Prepare immediately before use.
Reference solution. Dilute 1.0 ml of the test solution to 200 ml with ethanol R containing 0.2 g/l of
butylhydroxytoluene R. Prepare immediately before use.
29-38
Place the plate in the chromatographic tank containing the mobile phase consisting of a mixture of 20
volumes of glacial acetic acid R and 80 volumes of chloroform R, the mixture containing 0.2 g/l of
butylhydroxytoluene R. Allow the solvent to migrate over a path of about 4 cm. Remove the plate and
dry it for 1 min in a current of cold air. Apply immediately and separately to the plate under a current
of nitrogen R 5 l of each solution. Develop immediately over a path of 10 cm using the mobile phase
described above. Dry the plate in a current of cold air for 15 min and examine in ultraviolet light at
254 nm. Any spot in the chromatogram obtained with the test solution, apart from the principal spot,
is not more intense than the spot in the chromatogram obtained with the reference solution (0.5 per
cent).
ASSAY
Dissolve 0.300 g in 25 ml of acetone R and add 0.1 ml of bromothymol blue solution R1. Titrate with
0.1M sodium hydroxide until a blue colour appears which persists for 15 s. Carry out a blank titration.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
29-39
Oxytetracycline
Oxytetracycline Dihydrate
OH
OH
O
OH
CONH2
OH
Me
H
OH
C22H24N2O9
OH H
NMe2
460.4
79-57-2
Oxytetracycline complies with the requirements of the 3rd edition of the European Pharmacopoeia [0199].
Preparation
Oxytetracycline Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Oxytetracycline is (4S,4aR,5S,5aR,6S,12aS)-4-dimethylamino-1,4,4a, 5,5a,6,11,12a-octahydro3,5,6,10,12,12a-hexahydroxy-6-methyl-1,11-dioxonaphthacene-2-carboxamide, a substance
produced by the growth of certain strains of Streptomyces rimosus or obtained by any other means. It
contains a variable amount of water. It contains not less than 95.0 per cent and not more than the
equivalent of 100.5 per cent of oxytetracycline, calculated with reference to the anhydrous substance.
CHARACTERS
A yellow, crystalline powder, very slightly soluble in water. It dissolves in dilute acid and alkaline
solutions.
IDENTIFICATION
A. Examine by thin-layer chromatography (2.2.27), using silica gel H R as the coating substance.
Adjust the pH of a 100 g/l solution of sodium edetate R to 7.0 with strong sodium hydroxide solution R
and spray the solution evenly onto the plate (about 10 ml for a plate 100 mm 200 mm). Allow the
plate to dry in a horizontal position for at least 1 h. Before use, dry the plate in an oven at 110C for
1 h.
the same solvent.
Reference solution (a). Dissolve 5 mg of oxytetracycline CRS in methanol R and dilute to 10 ml with the
same solvent.
Reference solution (b). Dissolve 5 mg of oxytetracycline CRS and 5 mg of demeclocycline hydrochloride
CRS in methanol R and dilute to 10 ml with the same solvent.
volumes of water R, 35 volumes of methanol R and 59 volumes of methylene chloride R. Dry the plate
in a current of air and examine in ultraviolet light at 365 nm. The principal spot in the chromatogram
chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram
obtained with reference solution (b) shows two clearly separated spots.
B. To about 2 mg add 5 ml of sulphuric acid R. A deep red colour develops. Add the solution to
2.5 ml of water R. The colour becomes yellow.
C. Dissolve about 10 mg in a mixture of 1 ml of dilute nitric acid R and 5 ml of water R. Shake and
add 1 ml of silver nitrate solution R2. Any opalescence in the solution is not more intense than that in a
mixture of 1 ml of dilute nitric acid R, 5 ml of a 0.021 g/l solution of potassium chloride R and 1 ml of
silver nitrate solution R2.
TESTS
pH (2.2.3). Suspend 0.1 g in 10 ml of carbon dioxide-free water R. The pH of the suspension is 4.5 to
7.5.
29-40
with the same acid. The specific optical rotation is 203 to 216, calculated with reference to the
Absorbance (2.2.25). Dissolve 20.0 mg in buffer solution pH 2.0 R and dilute to 100.0 ml with the
same buffer solution. Dilute 10.0 ml of the solution to 100.0 ml with buffer solution pH 2.0 R. The
specific absorbance determined at 353 nm is 290 to 310, calculated with reference to the anhydrous
substance.
Light-absorbing impurities Dissolve 20.0 mg in a mixture of 1 volume of 1M hydrochloric acid and
99 volumes of methanol R and dilute to 10.0 ml with the same mixture of solvents. The absorbance
(2.2.25), determined at 430 nm and calculated with reference to the anhydrous substance, is not
greater than 0.25.
Dissolve 0.100 g in a mixture of 1 volume of 1M hydrochloric acid and 99 volumes of methanol R
and dilute to 10.0 ml with the same mixture of solvents. The absorbance, determined at 490 nm and
calculated with reference to the anhydrous substance, is not greater than 0.20.
Carry out the measurements within 1 h of preparation of the solutions.
Related substances Examine by liquid chromatography (2.2.29), as described under Assay. Inject
the test solution and reference solution (e). In the chromatogram obtained with the test solution; the
area of any peak corresponding to 4-epioxytetracycline or tetracycline is not greater than the area of
the corresponding peak in the chromatogram obtained with reference solution (e) (0.5 per cent and
2.0 per cent respectively); the area of any peak appearing on the tail of the main peak is not greater
than 4.0 times the area of the peak corresponding to 4-epioxytetracycline in the chromatogram
obtained with reference solution (e) (2.0 per cent).
ASSAY
Test solution. Dissolve 20.0 mg of the substance to be examined in 0.01M hydrochloric acid and dilute
to 25.0 ml with the same acid.
Reference solution (a). Dissolve 20.0 mg of oxytetracycline CRS in 0.01M hydrochloric acid and dilute to
Reference solution (b). Dissolve 20.0 mg of 4-epioxytetracycline CRS in 0.01M hydrochloric acid and
dilute to 25.0 ml with the same acid.
Reference solution (c). Dissolve 20.0 mg of tetracycline hydrochloride CRS in 0.01M hydrochloric acid and
Reference solution (d). Mix 1.5 ml of reference solution (a), 1.0 ml of reference solution (b) and 3.0 ml
of reference solution (c) and dilute to 25.0 ml with 0.01M hydrochloric acid.
Reference solution (e). Mix 1.0 ml of reference solution (b) and 4.0 ml of reference solution (c) and
dilute to 200.0 ml with 0.01M hydrochloric acid.
copolymer R (8 m to 10 m) and maintained at 60C,
as mobile phase at a flow rate of 1.0 ml per minute a mixture prepared as follows: weigh 60.0 g
of 2-methyl-2-propanol R and transfer to a 1000 ml volumetric flask with the aid of 200 ml of
water R. Add 60 ml of 0.33M phosphate buffer solution pH 7.5 R, 50 ml of a 10 g/l solution of
tetrabutylammonium hydrogen sulphate R adjusted to pH 7.5 with dilute sodium hydroxide solution R
and 10 ml of a 0.4 g/l solution of sodium edetate R adjusted to pH 7.5 with dilute sodium hydroxide
solution R; dilute to 1000 ml with water R,
a 20 l fixed loop injector.
Inject reference solution (d). Adjust the sensitivity of the detector to obtain peaks with a height
corresponding to at least half of the full scale of the recorder. The test is not valid unless the
resolution between the first peak (4-epioxytetracycline) and the second peak (oxytetracycline) is at
least 4.0 and the resolution between the second peak (oxytetracycline) and the third peak
(tetracycline) is at least 5.0. Adjust the 2-methyl-2-propanol content in the mobile phase if necessary.
The symmetry factor for the second peak is not more than 1.25. Inject reference solution (a) six
times. The assay is not valid unless the relative standard deviation for the peak area of oxytetracycline
is at most 1.0 per cent. If necessary adjust the integrator parameters. Inject alternately the test
solution and reference solution (a).
29-41
Calculate the percentage content of oxytetracycline.
STORAGE
IMPURITIES
OH
OH O
OH
H
H
HO Me OH NMe2
CONH2
OH
A. 4-epioxytetracycline,
OH
H
HO Me
OH O
OH
CONH2
OH
NMe2
B. tetracycline,
OH
OH O
OH
H
H
HO Me OH NMe2
Ac
OH
C. 2-acetyl-2-decarboxamido-oxytetracycline.
__________________________________________________________________________________________________________ Ph Eur
29-42
Oxytetracycline Calcium
O
OH
OH
CONH2
Ca 2+
OH
Me
H
OH
(C22H23N2O9)2Ca
OH H
NMe2
958.9
15251-48-6
Definition Oxytetracycline Calcium is the calcium salt of (4S,4aR,5S,5aR,6S,12aS)-4dimethylamino-1,4,4a,5, 5a,6,11,12a-octahydro-3,5,6,10,12,12a-hexahydroxy-6-methyl-1,11dioxonaphthacene-2-carboxamide, a substance produced by the growth of certain strains of
Streptomyces rimosus or obtained by any other means. It contains not less than 90.0% and not more
than 100.5% of (C22H23N2O9)2Ca, calculated with reference to the anhydrous substance.
Characteristics A pale yellow to greenish fawn, crystalline powder.
Practically insoluble in water; soluble in dilute acids. It dissolves slowly in 5M ammonia.
Identification
A. Carry out the method for thin-layer chromatography, Appendix III A, using a silica gel precoated
plate (Merck silica gel 60 plates are suitable) and a mixture of 6 volumes of water, 35 volumes of
methanol and 59 volumes of dichloromethane as the mobile phase. Adjust the pH of a 10% w/v solution
of disodium edetate to 7.0 with 10M sodium hydroxide and spray the solution evenly onto the plate
(about 10 ml for a plate 100 mm 200 mm). Allow the plate to dry in a horizontal position for at
least 1 hour. Before use, dry the plate at 110 for 1 hour. Apply separately to the plate 1 l of each of
three solutions in 0.01M methanolic hydrochloric acid containing (1) 0.05% w/v of the substance being
examined, (2) 0.05% w/v of oxytetracycline EPCRS and (3) 0.05% w/v of each of oxytetracycline
EPCRS and demeclocycline hydrochloride EPCRS. After removal of the plate, dry it in a current of air
and examine under ultraviolet light (365 nm). The principal spot in the chromatogram obtained with
solution (1) is similar in position, colour and size to that in the chromatogram obtained with solution
(2). The test is not valid unless the chromatogram obtained with solution (3) shows two clearly
separated spots.
B. To 2 mg add 5 ml of sulphuric acid; a deep red colour is produced. Add the solution to 2.5 ml of
water; the colour changes to yellow.
C. Yields reaction B characteristic of calcium salts, Appendix VI.
Acidity or alkalinity pH of a 2.5% w/v suspension, 6.0 to 7.5, Appendix V L.
Light absorption Absorbance of a 0.002% w/v solution in 0.1M chloride buffer pH 2.0 at the
maximum at 353 nm, 0.56 to 0.61, calculated with reference to the anhydrous substance, Appendix
II B.
Specific optical rotation In a 1% w/v solution in 0.1M hydrochloric acid, 194 to 210, calculated
with reference to the anhydrous substance, Appendix V F. Allow the solution to stand protected from
light for 30 minutes before measurement.
Light-absorbing impurities
A. Dissolve 0.2 g in 6 ml of 1M hydrochloric acid and add sufficient methanol to produce 100 ml. The
absorbance at 430 nm, when measured within 1 hour of preparing the solution, is not more than 0.30,
B. Dissolve 1 g in 6 ml of 1M hydrochloric acid and add sufficient methanol to produce 100 ml. The
absorbance at 490 nm, when measured within 1 hour of preparing the solution, is not more than 0.20,
Calcium 3.90 to 4.20%, calculated with reference to the anhydrous substance, when determined by
the following method. Transfer about 1 g, accurately weighed, to a Kjeldahl flask, cautiously add
10 ml of nitric acid and mix. Allow to stand for 5 minutes, add a glass bead and heat on a water bath
for 5 minutes. Remove from the water bath, cautiously add 5 ml of 9M perchloric acid and heat,
adding further 5-ml quantities of the perchloric acid at intervals until the liquid is almost colourless.
Add 0.1 ml of nitric acid and allow any further reaction to subside. Do not allow the volume of the
liquid in the flask to be reduced below 3 ml at any stage in the oxidation. Wash the walls of the flask
with 40 ml of water, collecting the washings in the flask, and boil for 3 to 4 minutes to expel chlorine.
Cool, transfer the contents of the flask to a conical flask with the aid of water and dilute to about
200 ml with water. Adjust to pH 9 with 5M sodium hydroxide and then add 100 ml of water followed
by 12 ml of 10M sodium hydroxide and mix. Add about 15 mg of calconcarboxylic acid triturate and
29-43
titrate with 0.05M disodium edetate VS until the colour changes from violet to full blue. Each ml of
0.05M disodium edetate VS is equivalent to 2.004 mg of Ca.
For solution (1) dilute 1 volume of a 0.1% w/v solution of the substance being examined in 0.1M
hydrochloric acid to 20 volumes with the mobile phase. For solution (2) dilute 1 volume of a 0.1% w/v
solution of oxytetracycline EPCRS in 0.1M hydrochloric acid to 20 volumes with the mobile phase.
Solution (3) contains 0.1% w/v of 4-epioxytetracycline EPCRS in 0.1M hydrochloric acid. Solution (4)
contains 0.1% w/v of tetracycline hydrochloride EPCRS in 0.1M hydrochloric acid. For solution (5) dilute
a mixture containing 1.5 ml of a 0.1% w/v solution of oxytetracycline EPCRS in 0.1M hydrochloric acid,
1 ml of solution (3) and 3 ml of solution (4) to 25 ml with the mobile phase.
The chromatographic procedure may be carried out using (a) a column (25 cm 4.6 mm) packed
with styrene-divinylbenzene copolymer (8 to 10 m) (Polymer Laboratories, PLRP-S 1000A, is suitable)
and maintained at 60, (b) as the mobile phase with a flow rate of 1 ml per minute a solution
prepared as described below and (c) a detection wavelength of 254 nm. To 50.0 g of 2-methylpropan2-ol add 200 ml of water, 60 ml of 0.33M phosphate buffer pH 7.5, 50 ml of a 1.0% w/v solution of
tetrabutylammonium hydrogen sulphate previously adjusted to pH 7.5 with 2M sodium hydroxide and
10 ml of a 0.04% w/v solution of disodium edetate previously adjusted to pH 7.5 with 2M sodium
hydroxide and dilute to 1 litre with water.
Inject solution (5). The assay is not valid unless (a) the resolution factor between the first peak
(4-epioxytetracycline) and the second peak (oxytetracycline) is at least 4.0, (b) the resolution factor
between the second peak and the third peak (tetracycline) is at least 5.0 (if necessary reduce the
content of 2-methylpropan-2-ol in the mobile phase to increase the resolution) and (c) the symmetry
factor of the peak due to oxytetracycline is not more than 1.25.
Calculate the content of (C22H23N2O9)2Ca from the declared content of C22H24N2O9 in
oxytetracycline EPCRS. Each mg of C22H24N2O9 is equivalent to 1.041 mg of (C22H23N2O9)2Ca.
Storage Oxytetracycline Calcium should be kept in a well-closed container, protected from light and
stored at a temperature of 2 to 8.
Labelling The label states (1) the date after which the material is not intended to be used; (2) the
conditions under which it should be stored.
29-44
Oxytetracycline Hydrochloride
OH
OH
O
OH
CONH2
,HCl
OH
Me
H
OH
C22H24N2O9,HCl
OH H
NMe2
496.9
2058-46-0
Oxytetracycline Hydrochloride complies with the requirements of the 3rd edition of the European
Preparation
Oxytetracycline Capsules
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Oxytetracycline hydrochloride is the hydrochloride of (4S,4aR,5S,5aR,6S,12aS)-4-dimethylamino1,4,4a,5,5a,6,11,12a-octahydro-3,5,6,10,12,12a-hexahydroxy-6-methyl-1,11-dioxonaphthacene-2carboxamide, a substance produced by the growth of certain strains of Streptomyces rimosus or
obtained by any other means. It contains not less than 95.0 per cent and not more than the equivalent of 100.5 per cent of oxytetracycline hydrochloride, calculated with reference to the anhydrous
substance.
CHARACTERS
A yellow, crystalline powder, hygroscopic, freely soluble in water, sparingly soluble in alcohol.
Solutions in water become turbid on standing, owing to the precipitation of oxytetracycline.
IDENTIFICATION
1 h.
the same solvent.
Reference solution (a). Dissolve 5 mg of oxytetracycline hydrochloride CRS in methanol R and dilute to
Reference solution (b). Dissolve 5 mg of oxytetracycline hydrochloride CRS and 5 mg of demeclocycline
hydrochloride CRS in methanol R and dilute to 10 ml with the same solvent.
B. To about 2 mg add 5 ml of sulphuric acid R. A deep red colour develops. Add the solution to
TESTS
pH (2.2.3). Dissolve 0.1 g in 10 ml of carbon dioxide-free water R. The pH of the solution is 2.3 to
2.9.
Absorbance (2.2.25). Dissolve 20.0 mg in buffer solution pH 2.0 R and dilute to 100.0 ml with the
same buffer solution. Dilute 10.0 ml of the solution to 100.0 ml with buffer solution pH 2.0 R. The
29-45
specific absorbance determined at 353 nm is 270 to 290, calculated with reference to the anhydrous
substance.
Light-absorbing impurities Dissolve 20.0 mg in a mixture of 1 volume of 1M hydrochloric acid and
99 volumes of methanol R and dilute to 10.0 ml with the same mixture of solvents. The absorbance
(2.2.25), determined at 430 nm and calculated with reference to the anhydrous substance, is not
greater than 0.50.
Dissolve 0.100 g in a mixture of 1 volume of 1M hydrochloric acid and 99 volumes of methanol R
and dilute to 10.0 ml with the same mixture of solvents. The absorbance, determined at 490 nm and
calculated with reference to the anhydrous substance, is not greater than 0.20.
Carry out the measurements within 1 h of preparation of the solutions.
Related substances Examine by liquid chromatography (2.2.29), as described under Assay. Inject
the test solution and reference solution (g). In the chromatogram obtained with the test solution: the
area of any peak corresponding to 4-epioxytetracycline or tetracycline is not greater than the area of
the corresponding peak in the chromatogram obtained with reference solution (g) (0.5 and 2.0 per
cent respectively); the total area of the peaks corresponding to -apo-oxytetracycline and -apooxytetracycline and any peak between the latter two is not greater than the area of the peak corresponding to -apo-oxytetracycline in the chromatogram obtained with reference solution (g) (2.0 per
cent); the area of any peak appearing on the tail of the main peak is not greater than 4.0 times the
area of the peak corresponding to 4-epioxytetracyline in the chromatogram obtained with reference
solution (g) (2.0 per cent).
without a further appropriate procedure for the removal of bacterial endotoxins, not more than 0.4
I.U. of endotoxin per milligram.
ASSAY
Reference solution (a). Dissolve 20.0 mg of oxytetracycline CRS in 0.01M hydrochloric acid and dilute to
Reference solution (b). Dissolve 20.0 mg of 4-epioxytetracycline CRS in 0.01M hydrochloric acid and
Reference solution (c). Dissolve 20.0 mg of tetracycline hydrochloride CRS in 0.01M hydrochloric acid and
Reference solution (d). Dissolve 8.0 mg of -apo-oxytetracycline CRS in 5 ml of 0.01M sodium hydroxide
and dilute to 100.0 ml with 0.01M hydrochloric acid.
Reference solution (e). Dissolve 8.0 mg of -apo-oxytetracycline CRS in 5 ml of 0.01M sodium hydroxide
and dilute to 100.0 ml with 0.01M hydrochloric acid.
Reference solution (f). Mix 1.5 ml of reference solution (a), 1.0 ml of reference solution (b), 3.0 ml of
reference solution (c), 3.0 ml of reference solution (d) and 3.0 ml of reference solution (e) and dilute
to 25.0 ml with 0.01M hydrochloric acid.
Reference solution (g). Mix 1.0 ml of reference solution (b), 4.0 ml of reference solution (c) and
40.0 ml of reference solution (e) and dilute to 200.0 ml with 0.01M hydrochloric acid.
copolymer R (8 - 10 m) maintained at 60C,
as mobile phase at a flow rate of 1.0 ml per minute a mixture prepared as follows: weigh 30.0 g
(for mobile phase A) or 100.0 g (for mobile phase B) of 2-methyl-2-propanol R and transfer
separately to 1000 ml volumetric flasks with the aid of 200 ml of water R. To each flask add
60 ml of 0.33M phosphate buffer solution pH 7.5 R, 50 ml of a 10 g/l solution of tetrabutylammonium hydrogen sulphate R adjusted to pH 7.5 with dilute sodium hydroxide solution R and
10 ml of a 0.4 g/l solution of sodium edetate R adjusted to pH 7.5 with dilute sodium hydroxide
solution R. Dilute each solution to 1000 ml with water R,
29-46
a 20 l fixed loop injector,
a pump allowing gradient elution.
Programme a one-step gradient elution as follows: pump a mixture containing 30 volumes of
mobile phase B and 70 volumes of mobile phase A for 15 min, then pump a mixture containing 30
volumes of mobile phase A and 70 volumes of mobile phase B for 15 min, and finally equilibrate with
the first mixture.
Inject reference solution (f). Adjust the sensitivity of the detector to obtain peaks with a height
corresponding to at least half- to full-scale deflection. The test is not valid unless the resolution for
the first peak (4-epioxytetracycline) and the second peak (oxytetracycline) is not less than 4.0, the
resolution for the second peak (oxytetracycline) and the third peak (tetracycline) is not less than 5.0
and the resolution for the fourth peak (-apo-oxy-tetracycline) and the fifth peak (-apo-oxytetracycline) is not less than 3.5. The symmetry factor for the second peak is not more than 1.25. If necessary
adapt the ratio mobile phase A: mobile phase B used to produce the one-step gradient elution. Adjust
the time programme for the one-step gradient elution if necessary. Inject reference solution (a) six
times. The test is not valid unless the relative standard deviation for the peak area of oxytetracycline is
not greater than 1.0 per cent. If necessary adjust the integrator parameters. Inject alternately the test
solution and reference solution (a).
Calculate the percentage content of oxytetracycline hydrochloride taking 1 mg of oxytetracycline as
equivalent to 1.079 mg of oxytetracycline hydrochloride.
STORAGE
LABELLING
The label states:
__________________________________________________________________________________________________________ Ph Eur
29-47
Oxytocin
revised 1/01
Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly
C43H66N12O12S2
1007
50-56-6
Oxytocin complies with the requirements of the 3rd edition of the European Pharmacopoeia [0780]. These
Action and use Oxytocic.
Preparations
Ergometrine and Oxytocin Injection
Oxytocin Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Oxytocin is a cyclic nonapeptide having the structure of the hormone produced by the posterior lobe
of the pituitary gland that stimulates contraction of the uterus and milk ejection in receptive
mammals. It is obtained by chemical synthesis and is available in the freeze-dried form as an acetate.
It contains not less than 93.0 per cent and not more than the equivalent of 102.0 per cent of the
peptide C43H66N12O12S2, calculated with reference to the anhydrous, acetic acid-free substance.
By convention, for the purpose of labelling oxytocin preparations, 1 mg of oxytocin peptide
(C43H66N12O12S2) is equivalent to 600 I.U. by biological activity.
CHARACTERS
A white or almost white powder, hygroscopic, very soluble in water and in dilute solutions of acetic
acid and of ethanol.
IDENTIFICATION
Examine the chromatograms obtained in the assay. The retention time of the principal peak in the
chromatogram obtained with the test solution is approximately the same as that of the principal peak
TESTS
pH (2.2.3). Dissolve 0.200 g in carbon dioxide-free water R and dilute to 10.0 ml with the same
solvent. The pH of the solution is 3.0 to 6.0.
Amino acids Examine by means of an amino-acid analyser. Standardise the apparatus with a
mixture containing equimolar amounts of ammonia, glycine and the L-form of the following amino
acids:
Lysine
Threonine
Alanine
Leucine
Histidine
Serine
Valine
Tyrosine
Arginine
Glutamic acid Methionine
Phenylalanine
Aspartic acid
Proline
Isoleucine
together with half the equimolar amount of L-cystine. For the validation of the method, an
appropriate internal standard, such as DL-norleucine R, is used.
Test solution. Place 1.0 mg of the substance to be examined in a rigorously cleaned hard-glass tube
100 mm long and 6 mm in internal diameter. Add a suitable amount of a 50 per cent V/V solution of
hydrochloric acid R. Immerse the tube in a freezing mixture at 5C, reduce the pressure to below
133 Pa and seal. Heat at 110C to 115C for 16 h. Cool, open the tube, transfer the contents to a
10 ml flask with the aid of five quantities, each of 0.2 ml, of water R and evaporate to dryness over
potassium hydroxide R under reduced pressure. Take up the residue in water R and evaporate to
dryness over potassium hydroxide R under reduced pressure; repeat these operations once. Take up the
residue in a buffer solution suitable for the amino-acid analyser used and dilute to a suitable volume
with the same buffer solution. Apply a suitable volume to the amino-acid analyser.
Express the content of each amino acid in moles. Calculate the relative proportions of the amino
acids, taking one-sixth of the sum of the number of moles of aspartic acid, glutamic acid, proline,
glycine, isoleucine and leucine as equal to one. The values fall within the following limits: aspartic
acid 0.95 to 1.05; glutamic acid 0.95 to 1.05; proline 0.95 to 1.05; glycine 0.95 to 1.05; leucine 0.90
to 1.10; isoleucine 0.90 to 1.10; tyrosine 0.7 to 1.05; half-cystine 1.4 to 2.1; not more than traces of
other amino acids are present.
29-48
Related peptides Examine by liquid chromatography (2.2.29) as described under Assay.
Inject 50 l of the test solution. In the chromatogram obtained the area of any peak, apart from the
principal peak, is not greater than 1.5 per cent of the total area of the peaks; the sum of the areas of
all the peaks, apart from the principal peak, is not greater than 5 per cent of the total area of the
peaks. Disregard any peak due to the solvent and any peak with an area less than 0.1 per cent of that
of the principal peak.
Acetic acid (2.5.34). 6.0 per cent to 10.0 per cent.
Test solution. Dissolve 15.0 mg of the substance to be examined in a mixture of 5 volumes of mobile
phase B and 95 volumes of mobile phase A and dilute to 10.0 ml with the same mixture of solvents.
Water (2.5.12). Not more than 5.0 per cent, determined on at least 50 mg by the semi-micro
determination of water.
300 I.U. of endotoxin per milligram of oxytocin.
ASSAY
Test solution. Prepare a 0.25 mg/ml solution of the substance to be examined in a 15.6 g/l solution of
sodium dihydrogen phosphate R.
Reference solution. Dissolve the contents of a vial of oxytocin CRS in a 15.6 g/l solution of sodium
dihydrogen phosphate R and dilute to 20.0 ml with the same solution.
Resolution solution. Dissolve the contents of a vial of oxytocin/desmopressin validation mixture CRS in
500 l of a 15.6 g/l solution of sodium dihydrogen phosphate R.
Mobile phase A. A 15.6 g/l solution of sodium dihydrogen phosphate R,
Mobile phase B. Mix 1 volume of acetonitrile for chromatography R with 1 volume of water R,
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
0 30
70 40
30 60
30 30.1
40 70
60 30
Linear gradient
Switch to initial
eluent composition
30.1 45
70
30
Re-equilibration

Equilibrate the column with a mixture of 30 volumes of mobile phase B and 70 volumes of mobile
phase A.
Inject 25 l of the resolution solution. When the chromatograms are recorded in the prescribed
conditions, the retention times are: oxytocin about 7.5 min and desmopressin about 10 min. The test
is not valid unless the resolution between the peaks corresponding to desmopressin and oxytocin is at
least 5.0.
Inject 25 l of the test solution and 25 l of the reference solution.
Calculate the content of oxytocin (C43H66N12O12S2) from the peak areas in the chromatograms
obtained with the test solution and the reference solution and the declared content of
C43H66N12O12S2 in oxytocin CRS.
STORAGE
Store in an airtight container, protected from light, at a temperature of 2C to 8C. If the substance
is sterile, store in a sterile, airtight, tamper-proof container.
LABELLING
The label states:
the oxytocin peptide content (C43H66N12O12S2),
__________________________________________________________________________________________________________ Ph Eur
29-49
Oxytocin Bulk Solution

Oxytocin Concentrated Solution
Oxytocin Bulk Solution complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Oxytocic.
Preparations
Ergometrine and Oxytocin Injection
Oxytocin Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Oxytocin bulk solution is a solution of oxytocin, a cyclic nonapeptide having the structure of the
hormone produced by the posterior lobe of the pituitary gland that stimulates contraction of the
uterus and milk ejection in receptive mammals. It is obtained by chemical synthesis. It is available as
a bulk solution with a stated concentration of not less than 0.25 mg of oxytocin per millilitre, in a
solvent that may contain an appropriate antimicrobial preservative. It contains not less than 95.0 per
cent and not more than 105.0 per cent of the amount of the peptide C43H66N12O12S2 stated per
millilitre1.
CHARACTERS
A clear, colourless liquid.
IDENTIFICATION
Examine the chromatograms obtained under Assay. The retention time of the principal peak in the
chromatogram obtained with the test solution is similar to that of the principal peak in the chromatogram obtained with the reference solution.
TESTS
pH (2.2.3). The pH of the preparation to be examined is 3.0 to 5.0.
acids:
lysine
threonine
alanine
leucine
histidine
serine
valine
tyrosine
arginine
glutamic acid methionine phenylalanine
aspartic acid proline
isoleucine
together with half the equimolar amount of L-cystine. For the validation of the method, an
appropriate internal standard, such as DL-norleucine R, is used.
Test solution. Place a volume of the preparation to be examined containing 0.25 mg of peptide in a
rigorously cleaned hard-glass tube 100 mm long and 6 mm in internal diameter. Evaporate to
dryness. Add a suitable amount of a 50 per cent V/V solution of hydrochloric acid R. Immerse the tube
in a freezing mixture at 5C, reduce the pressure to below 133 Pa and seal. Heat at 110C to 115C
for 16 h. Cool, open the tube, transfer the contents to a 10 ml flask with the aid of five quantities,
each of 0.2 ml, of water R and evaporate to dryness over potassium hydroxide R under reduced
pressure. Take up the residue in water R and evaporate to dryness over potassium hydroxide R under
reduced pressure; repeat these operations once. Take up the residue in a buffer solution suitable for
the amino-acid analyser used and dilute to a suitable volume with the same buffer solution.
Apply to the amino-acid analyser a suitable, accurately measured volume of the test solution such
that the peak given by the amino acid present in the largest amount occupies most of the available
chart height of the recorder.
acids taking one sixth of the sum of the number of moles of aspartic acid, glutamic acid, proline,
glycine, isoleucine and leucine as equal to 1. The values fall within the following limits: aspartic acid
0.95 to 1.05; glutamic acid 0.95 to 1.05; proline 0.95 to 1.05; glycine 0.95 to 1.05; leucine 0.90 to
1.10; isoleucine 0.90 to 1.10; tyrosine 0.7 to 1.05; half cystine 1.4 to 2.1; not more than traces of
other amino acids are present.
Inject 50 l of the test solution. In the chromatogram obtained, the area of any peak apart from the
principal peak is not greater than 1.5 per cent of the total area of the peaks; the sum of the areas of all
peaks, apart from the principal peak, is not greater than 5 per cent of the total area of the peaks.
Disregard any peak due to the solvent or to the anti-microbial preservative and any peak with an area
29-50
less than 0.1 per cent of that of the principal peak.
300 I.U. of endotoxin per milligram of oxytocin.
ASSAY
Test solution. Use the preparation to be examined.
Reference solution. Dissolve the contents of a vial of oxytocin CRS in a 15.6 g/l solution of sodium
dihydrogen phosphate R and dilute to 20.0 ml with the same solvent.
Resolution solution. Dissolve the contents of a vial of oxytocin/desmopressin validation mixture CRS with
500 l of a 15.6 g/l solution of sodium dihydrogen phosphate R.
as mobile phases at a flow rate of 1 ml/min:
Mobile phase A. A 15.6 g/l solution of sodium dihydrogen phosphate R,
Mobile phase B. Mix 1 volume of acetonitrile for chromatography R with 1 volume of water R,
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
0 30
70 40
30 60
linear gradient
30 30.1
40 70
60 30
30.1 45
70
30
return to initial
conditions
re-equilibration

phase A.
Inject 25 l of the resolution solution. When the chromatograms are recorded in the prescribed
conditions, the retention times are: oxytocin about 7.5 min and desmopressin about 10 min. The test
is not valid unless the resolution between the desmopressin peak and the oxytocin peak is at least 5.0.
Inject 25 l of the reference solution and 25 l of the test solution. Calculate the content of
oxytocin (C43H66N12O12S2) from the peak areas in the chromatograms obtained with the test
solution and the reference solution and the declared content of C43H66N12O12S2 in oxytocin CRS.
STORAGE
Store at a temperature between 2C and 8C, protected from light. If the substance is sterile, store in
a sterile, airtight, tamper-proof container.
LABELLING
The label states:
the oxytocin peptide content in milligrams of C43H66N12O12S2 per millilitre,
where applicable, that the preparation is sterile,
where applicable, that the preparation is free from bacterial endotoxins,
the name of any added antimicrobial preservative.
11
mg of oxytocin peptide (C43H66N12O12S2) is equivalent to 600 I.U.
__________________________________________________________________________________________________________ Ph Eur
29-51
Fractionated Palm Kernel Oil

The standards of this monograph encompass several different suppository bases. The selection of a basis for a
particular suppository formulation should be appropriate to the product concerned and it may be necessary to
apply more restrictive standards for a particular application.
Definition Fractionated Palm Kernel Oil is obtained by expression of the natural oil from the kernels
of Elaeis guineensis Jacq. followed by selective solvent fractionation and hydrogenation.
Characteristics A white, solid, brittle fat; odourless or almost odourless.
Practically insoluble in water; miscible with chloroform, with ether and with petroleum spirit (boiling
range, 40 to 60); practically insoluble in ethanol (96%).
Acid value Not more than 0.2, Appendix X B.
Iodine value Not more than 6.0 (iodine bromide method), Appendix X E.
Melting point 31 to 36, Appendix V A, Method IV. Prepare the substance being examined in the
following manner. Melt about 30 g in an oven at a temperature of 55 to 60 and filter through a
suitable dry filter paper, maintaining the temperature between 53 and 60. Cool with occasional
stirring until the temperature falls to between 32 and 34, stir continuously with a mechanical stirrer
until the first signs of cloudiness appear and continue to stir by hand until the substance has the
consistence of a paste. Immediately transfer to a vessel previously kept at a temperature of 15 to 22
and allow to stand at this temperature for 24 hours before carrying out the test.
Refractive index At 50, 1.445 to 1.447, Appendix V E.
Saponification value 246 to 250, Appendix X G.
Peroxides Dissolve 5 g in 15 ml of chloroform, add 20 ml of glacial acetic acid and 0.5 ml of a
saturated solution of potassium iodide, mix well, allow to stand in the dark for exactly 1 minute, add
30 ml of water and titrate with 0.01M sodium thiosulphate VS using starch mucilage as indicator. Not
more than 0.5 ml of 0.01M sodium thiosulphate VS is required.
Storage Fractionated Palm Kernel Oil should be stored at a temperature not exceeding 25.
Action and use Suppository basis.
29-52
Pancreatic Extract
Pancreatic Extract complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Pancreas Powder [0350]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Pancreas powder is prepared from the fresh or frozen pancreases of mammals. It contains various
enzymes having proteolytic, lipolytic and amylolytic activities.
1 milligram of pancreas powder contains not less than 1.0 European Pharmacopoeia Unit (Ph.
Eur. U.) of total proteolytic activity, 15 European Pharmacopoeia Units of lipolytic activity and 12
European Pharmacopoeia Units of amylolytic activity.
Pancreas powder is prepared in conditions designed to minimise the degree of microbial
contamination.
CHARACTERS
A slightly brown, amorphous powder, partly soluble in water, practically insoluble in alcohol and in
ether.
IDENTIFICATION
A. Triturate 0.5 g with 10 ml of water R and adjust to pH 8 by the addition of 0.1M sodium hydroxide,
using 0.1 ml of cresol red solution R as indicator. Divide the suspension into two equal parts
(suspension (a) and suspension (b)). Boil suspension (a). To each suspension add a few particles of
fibrin congo red R, heat to 38C to 40C and maintain at this temperature for 1 h. Suspension (a) is
colourless or slightly pink and suspension (b) is distinctly more red.
B. Triturate 0.25 g with 10 ml of water R and adjust to pH 8 by the addition of 0.1M sodium
hydroxide, using 0.1 ml of cresol red solution R as indicator. Divide the suspension into two equal parts
(suspension (a) and suspension (b)). Boil suspension (a). Dissolve 0.1 g of soluble starch R in 100 ml
of boiling water R, boil for 2 min, cool and dilute to 150 ml with water R. To 75 ml of the starch
solution add suspension (a) and to the remaining 75 ml add suspension (b). Heat each mixture to
38C to 40C and maintain at this temperature for 5 min.
To 1 ml of each mixture add 10 ml of iodine solution R2. The mixture obtained with suspension (a)
has an intense blue-violet colour; the mixture obtained with suspension (b) has the colour of the
iodine solution.
TESTS
Fat content In an extraction apparatus, treat 1.0 g with light petroleum R1 for 3 h. Evaporate the
solvent and dry the residue at 100C to 105C for 2 h. The residue weighs not more than 50 mg
(5.0 per cent).
Loss on drying (2.2.32). Not more than 5.0 per cent, determined on 0.50 g by drying at 60C at a
pressure not exceeding 670 Pa for 4 h.
Microbial contamination Total viable aerobic count (2.6.12) not more than 104 microorganisms per gram, determined by plate count. It complies with the tests for Escherichia coli and
Salmonella (2.6.13).
ASSAY
Total proteolytic activity The total proteolytic activity of pancreas powder is determined by
comparing the quantity of peptides non-precipitable by a 50 g/l solution of trichloroacetic acid R
released per minute from a substrate of casein solution with the quantity of such peptides released by
pancreas powder (protease) BRP from the same substrate in the same conditions.
Casein solution. Suspend a quantity of casein BRP equivalent to 1.25 g of dried substance in 5 ml of
water R, add 10 ml of 0.1M sodium hydroxide and stir for 1 min. (Determine the water content of
casein BRP prior to the test by heating at 60C in vacuo for 4 h.) Add 60 ml of water R and stir with a
magnetic stirrer until the solution is practically clear. Adjust to pH 8.0 with 0.1M sodium hydroxide or
0.1M hydrochloric acid. Dilute to 100.0 ml with water R. Use the solution on the day of preparation.
Enterokinase solution. Dissolve 50 mg of enterokinase BRP in 0.02M calcium chloride and dilute to
50.0 ml with the same solvent. Use the solution on the day of preparation.
For the test suspension and the reference suspension, prepare the suspension and carry out the dilution at 0C
to 4C.
Test suspension. Triturate 0.100 g of the substance to be examined for 5 min adding gradually 25 ml
of 0.02M calcium chloride. Transfer completely to a volumetric flask and dilute to 100.0 ml with 0.02M
calcium chloride. To 10.0 ml of this suspension add 10.0 ml of enterokinase solution and heat in a
29-53
water-bath at 35 0.5C for 15 min. Cool and dilute with borate buffer solution pH 7.5 R at 5 3C
to a final concentration of about 0.065 Ph. Eur. U. of total proteolytic activity per millilitre calculated
on the basis of the stated activity.
Reference suspension. Prepare a suspension of pancreas powder (protease) BRP as described for the test
suspension but without the addition of enterokinase so as to obtain a known final concentration of
about 0.065 Ph. Eur. U. per millilitre calculated on the basis of the stated activity.
Designate tubes in duplicate T, Tb, S1, S1b, S2, S2b, S3, S3b; designate a tube B.
Add borate buffer solution pH 7.5 R to the tubes as follows:
B: 3.0 ml,
S1 and S1b: 2.0 ml,
S2, S2b, T and Tb: 1.0 ml.
Add the reference suspension to the tubes as follows:
S1 and S1b: 1.0 ml,
S2 and S2b: 2.0 ml,
S3 and S3b: 3.0 ml.
Add 2.0 ml of the test suspension to tubes T and Tb.
Add 5.0 ml of a 50 g/l solution of trichloroacetic acid R to tubes B, S1b, S2b, S3b and Tb. Mix by
shaking.
Place the tubes and the casein solution in a water-bath at 35 0.5C. Place a glass rod in each
tube. When temperature equilibrium is reached, add 2.0 ml of the casein solution to tubes B, S1b,
S2b, S3b and Tb. Mix. At time zero, add 2.0 ml of casein solution successively and at intervals of 30 s
to tubes S1, S2, S3 and T. Mix immediately after each addition. Exactly 30 min after addition of the
casein solution, taking into account the regular interval adopted, add 5.0 ml of a 50 g/l solution of
trichloroacetic acid R to tubes S1, S2, S3 and T. Mix. Withdraw the tubes from the water-bath and
allow to stand at room temperature for 20 min.
Filter the contents of each tube twice through the same suitable filter paper previously washed with
a 50 g/l solution of trichloroacetic acid R, then with water R and dried.
A suitable filter paper complies with the following test: filter 5 ml of a 50 g/l of trichloroacetic acid R
on a 7 cm disc of white filter paper; the absorbance (2.2.25) of the filtrate, measured at 275 nm using
unfiltered trichloroacetic acid solution as the compensation liquid, is less than 0.04.
A schematic presentation of the above operations is shown in the Table 350-1.
Measure the absorbance (2.2.25) of the filtrates at 275 nm using the filtrate obtained from tube B
as the compensation liquid.
Correct the average absorbance values for the filtrates obtained from tubes S1, S2 and S3 by
subtracting the average values obtained for the filtrates from tubes S1b, S2b and S3b respectively. Draw
a calibration curve of the corrected values against volume of reference suspension used.
Determine the activity of the substance to be examined using the corrected absorbance for the test
suspension (TTb) and the calibration curve and taking into account the dilution factors.
The test is not valid unless the corrected absorbance values are between 0.15 and 0.60.
Table 3501
Tubes
S1 S1b S2 S2b S3 S3b T Tb B
Buffer solution
Reference solution
Suspension test
Trichloroacetic acid solution
Mix
Water-bath 35C
Casein solution
Mix
Casein solution
Mix
Water-bath 35C
Trichloroacetic acid solution
Mix
Room temperature 20 min
Filter
2
1
2
1
1
2
1
2
5
5
+
+
+ + + +
2
2
+
+
2
2
+
+
+ + + +
5
5
+
+
+ + + +
+ + + +
1
3
2 2
5
5
+
+
+ + + +
2
2
+
+
2
2
+
+
+ + + +
5
5
+
+
+ + + +
+ + + +
5
+
+
2
+
+
+
Lipolytic activity The lipolytic activity is determined by comparing the rate at which a suspension
of pancreas powder hydrolyses a substrate of olive oil emulsion with the rate at which a suspension of
pancreas powder (amylase and lipase) BRP hydrolyses the same substrate under the same conditions.
The test is carried out under nitrogen.
29-54
Olive oil stock emulsion. In an 800 ml beaker 9 cm in diameter, place 40 ml of olive oil R, 330 ml of
acacia solution R and 30 ml of water R. Place an electric mixer at the bottom of the beaker. Place the
beaker in a vessel containing alcohol R and a sufficient quantity of ice as a cooling mixture. Emulsify
using the mixer at an average speed of 1000 r/min to 2000 r/min. Cool to 5C to 10C. Increase the
mixing speed to 8000 r/min. Mix for 30 min keeping the temperature below 25C by the continuous
addition of crushed ice into the cooling mixture. (A mixture of calcium chloride and crushed ice is
also suitable). Store the stock emulsion in a refrigerator and use within 14 days. The emulsion must
not separate into two distinct layers. Check the diameter of the globules of the emulsion under a
microscope. At least 90 per cent have a diameter below 3 m and none has a diameter greater than
10 m. Shake the emulsion thoroughly before preparing the emulsion substrate.
Olive oil emulsion. For ten determinations, mix the following solutions in the order indicated: 100 ml
of stock emulsion, 80 ml of tris(hydroxymethyl)aminomethane solution R1, 20 ml of a freshly prepared
80 g/l of sodium taurocholate BRP and 95 ml of water R. Use on the day of preparation.
Apparatus. Use a reaction vessel of about 50 ml capacity provided with:
a device that will maintain a temperature of
37 0.5C,
a magnetic stirrer,
a lid with holes for the insertion of electrodes, the tip of a burette, a tube for the admission of
nitrogen and the introduction of reagents.
An automatic or manual titration apparatus may be used. In the latter case, the burette is
graduated in 0.005 ml and the pH-meter is provided with a wide reading scale and glass-calomel
electrodes. After each test the reaction vessel is evacuated by suction and washed several times with
water R, the washings being removed each time by suction.
Test suspension. In a small mortar cooled to 0C to 4C, triturate carefully a quantity of the substance
to be examined equivalent to about 2500 Ph. Eur. U. of lipolytic activity with 1 ml of cooled maleate
buffer solution pH 7.0 R (lipase solvent) until a very fine suspension is obtained. Dilute the suspension
with cold maleate buffer solution pH 7.0 R, transfer quantitatively to a volumetric flask and dilute to
100.0 ml with the cold buffer solution. Keep the flask containing the test suspension in iced water
during the titration.
Reference suspension. To avoid absorption of water formed by condensation, allow the reference preparation to
reach room temperature before opening the container.
Prepare a suspension of pancreas powder (amylase and lipase) BRP as described for the test
suspension using a quantity equivalent to about 2500 Ph. Eur. U.
Carry out the titrations immediately after preparation of the test suspension and the reference
suspension. Place 29.5 ml of olive oil emulsion in the reaction vessel equilibrated at 37 0.5C. Fit
the vessel with the electrodes, a stirrer and the burette (the tip being immersed in the olive oil
emulsion).
Put the lid in place and switch on the apparatus. Carefully add 0.1M sodium hydroxide with stirring.
Adjust to pH 9.2. Using a rapid-flow graduated pipette transfer about 0.5 ml of the previously
homogenised reference suspension, start the chronometer and add continuously 0.1M sodium
hydroxide to maintain the pH at 9.0. After exactly 1 min, note the volume of 0.1M sodium hydroxide
used. Carry out the measurement a further four times. Discard the first reading and determine the
average of the four others (S1). Make two further determinations (S2 and S3). Calculate the average
of the values S1, S2 and S3. The average volume of 0.1M sodium hydroxide used should be about
0.12 ml per minute with limits of 0.08 ml to 0.16 ml.
Carry out three determinations in the same manner for the test suspension (T1, T2 and T3). If the
quantity of 0.1M sodium hydroxide used is outside the limits 0.08 ml to 0.16 ml per minute, the assay
is repeated with a quantity of test suspension which is more suitable but situated between 0.4 ml and
0.6 ml. Otherwise the quantity of the substance to be examined is adjusted to comply with the
conditions of the test. Calculate the average of the values T1, T2 and T3.
Calculate the activity in Ph. Eur. Units per milligram from the expression:
n m1
A
n1 m
n = average volume of 0.1M sodium hydroxide used per minute during the titration of the test
suspension,
n1 = average volume of 0.1M sodium hydroxide used per minute during the titration of the
reference suspension,
m = mass in milligrams of the substance to be examined,
m1 = mass in milligrams of the reference preparation,
A = activity of pancreas powder (amylase and lipase) BRP in Ph. Eur. Units per milligram.
Amylolytic activity The amylolytic activity is determined by comparing the rate at which a
suspension of pancreas powder hydrolyses a substrate of starch solution with the rate at which a
suspension of pancreas powder (amylase and lipase) BRP hydrolyses the same substrate under the same
conditions.
29-55
Starch solution. To a quantity of starch BRP equivalent to 2.0 g of the dried substance add 10 ml of
water R and mix. (Determine the water content of starch BRP prior to the test by heating at 120C for
4 h). Add this suspension, whilst stirring continuously, to 160 ml of boiling water R. Wash the
container several times with successive quantities, each of 10 ml, of water R and add the washings to
the hot starch solution. Heat to boiling, stirring continuously. Cool to room temperature and dilute
to 200 ml with water R. Use the solution on the day of preparation.
Test suspension. Triturate a quantity of the substance to be examined equivalent to about 1500 Ph.
Eur. U. of amylolytic activity with 60 ml of phosphate buffer solution pH 6.8 R1 for 15 min. Transfer
quantitatively to a volumetric flask and dilute to 100.0 ml with phosphate buffer solution pH 6.8 R1.
Reference suspension. Prepare a suspension of pancreas powder (amylase and lipase) BRP as described for
the test suspension, using a quantity equivalent to about 1500 Ph. Eur. U.
In a test tube 200 mm long and 22 mm in diameter, fitted with a ground-glass stopper, place 25.0 ml
of starch solution, 10.0 ml of phosphate buffer solution pH 6.8 R1 and 1.0 ml of a 11.7 g/l solution of
sodium chloride R. Close the tube, shake and place in a water-bath at 25.0 0.1C. When the
temperature equilibrium has been reached, add 1.0 ml of the test suspension and start the
chronometer. Mix and place the tube in the water-bath. After exactly 10 min, add 2 ml of 1M hydrochloric acid. Transfer the mixture quantitatively to a 300 ml conical flask fitted with a ground-glass
stopper. Whilst shaking continuously, add 10.0 ml of 0.05M iodine and immediately, 45 ml of 0.1M
sodium hydroxide. Allow to stand in the dark at a temperature between 15C and 25C for 15 min.
Add 4 ml of a mixture of 1 volume of sulphuric acid R and 4 volumes of water R. Titrate the excess of
iodine with 0.1M sodium thiosulphate using a microburette. Carry out a blank titration adding the 2 ml
of 1M hydrochloric acid before introducing the test suspension. Carry out the titration of the reference
suspension in the same manner.
Calculate the amylolytic activity in Ph. Eur. Units per milligram from the expression:
( n n) m 1
A
(n1 n1) m
n = number of millilitres of 0.1M sodium thiosulphate used in the titration of the test
suspension,
n1 = number of millilitres of 0.1M sodium thiosulphate used in the titration of the reference
suspension,
n = number of millilitres of 0.1M sodium thiosulphate used in the blank titration of the test
suspension,
n1 = number of millilitres of 0.1M sodium thiosulphate used in the blank titration of the reference suspension,
m = mass in milligrams of the substance to be examined,
m1 = mass in milligrams of the reference preparation,
A = activity of pancreas powder (amylase and lipase) BRP in Ph. Eur. Units per milligram.
STORAGE
Store in an airtight container, at a temperature below 15C.
__________________________________________________________________________________________________________ Ph Eur
29-56
Pancreatin
Definition Pancreatin is a preparation of mammalian pancreas containing enzymes having protease,
lipase and amylase activity. It may contain Sodium Chloride. Pancreatin contains in 1 mg not less
than 1.4 Units of free protease activity, not less than 20 Units of lipase activity and not less than 24
Units of amylase activity.
Production Pancreatin is prepared in conditions designed to minimise the degree of microbial
contamination.
Characteristics A white or buff amorphous powder; free from unpleasant odour.
Soluble or partly soluble in water forming a slightly turbid solution; practically insoluble in ethanol
(96%) and in ether.
Identification
A. Triturate 0.5 g with 10 ml of water and adjust to pH 8.0 by the addition of 1M sodium hydroxide
using cresol red solution as indicator. Divide the resulting solution into two equal portions. Boil one
portion [solution (1)] and leave the other untreated [solution (2)]. To each add a few shreds of congo
red fibrin, warm to 38 to 40 and maintain at this temperature for 1 hour. Solution (2) is stained red
and solution (1) is colourless or not more than slightly pink.
B. Triturate 0.25 g with 10 ml of water and adjust to pH 8.0 by the addition of 1M sodium hydroxide
using cresol red solution as indicator. Divide the resulting solution into two equal portions. Boil one
portion [solution (1)] and leave the other untreated [solution (2)]. Dissolve 0.1 g of soluble starch in
100 ml of boiling water, boil for 2 minutes, cool and dilute to 150 ml with water. Add solution (1) to
half the starch mucilage and solution (2) to the remainder and maintain the mixtures at 38 to 40 for
5 minutes. To 1 ml of each mixture add 10 ml of iodinated potassium iodide solution. The liquid
containing solution (2) retains the colour of the solution of iodine and the liquid containing solution
(1) acquires an intense blue colour.
Fat Extract 1 g with petroleum spirit (boiling range, 40 to 60) for 3 hours in an apparatus for the
continuous extraction of drugs, Appendix XI F, evaporate the extract and dry the residue at 105 for 2
hours. The residue weighs not more than 30 mg.
than 5.0% of its weight. Use 0.5 g.
Microbial contamination 1 g is free from Escherichia coli; 10 g is free from Salmonella, Appendix
XVI B1.
Assay Carry out the assay of pancreatin described below.
Storage Pancreatin should be kept in a well-closed container and stored at a temperature not
exceeding 15.
Labelling The label states (1) the minimum number of Units of activity of free protease, lipase and
amylase per mg; (2) the name of any added substance; (3) the date after which the material is not
intended to be used; (4) the conditions under which it should be stored.
Action and use Enzymes used in pancreatic deficiency.
Preparations
Pancreatin Granules
Pancreatin Tablets
ASSAY OF PANCREATIN
The free protease, lipase and amylase activities of pancreatin are determined by the following
methods.
Standard Preparation and Units
The Standard Preparation is the appropriate FIP Standard which has been adopted as an official
preparation by the European Pharmacopoeia Commission and is available as pancreas powder
(protease) EPBRP or pancreas powder (amylase and lipase) EPBRP as appropriate.
The Unit of protease activity is contained in that amount of the Standard Preparation that, under
the conditions of the assay, hydrolyses casein at an initial rate such that there is liberated per minute
an amount of peptides not precipitated by trichloroacetic acid that gives the same absorbance at
275 nm as one micromole of tyrosine. The Unit of lipase activity is contained in that amount of the
Standard Preparation that, under the conditions of the assay, liberates one micro-equivalent of acid
per minute at pH 9.0 and 37. The Unit of amylase activity is contained in that amount of the
standard preparation that, under the conditions of the assay, decomposes starch at an initial rate such
that one micro-equivalent of glycosidic linkage is hydrolysed per minute.
For free protease activity
Method SOLUTION OF THE STANDARD PREPARATION Triturate for 5 minutes a quantity of the
29-57
Standard Preparation containing approximately 100 Units of protease activity with 25 ml of calcium
chloride solution cooled to 5. Dilute to 100 ml with the cooled calcium chloride solution and then dilute
a sufficient quantity of the resulting suspension to 100 ml with borate buffer pH 7.5, cooled to 5, so
that 1 ml of the final solution contains 0.065 Units of protease activity.
SOLUTION OF THE SUBSTANCE BEING EXAMINED Triturate for 5 minutes a quantity of the substance
being examined containing approximately 100 Units of protease activity with 25 ml of calcium chloride
solution cooled to 5. Dilute to 100 ml with the cooled calcium chloride solution and then dilute a
sufficient quantity of the resulting suspension to 100 ml with borate buffer pH 7.5, cooled to 5, so
that the estimated free protease activity corresponds approximately to the activity of the solution of
the Standard Preparation.
Label 16 test tubes with the following identification in duplicate; S1, S2, S3, S1B, S2B, S3B, U and
UB. To tubes S1 and S1B add 2.0 ml and to tubes S2, S2B, U and UB, 1.0 ml of borate buffer pH 7.5.
Then to tubes S1 and S1B add 1.0 ml, to tubes S2 and S2B, 2.0 ml and to tubes S3 and S3B, 3.0 ml of
the solution of the Standard Preparation. Add 2.0 ml of the solution of the substance being examined
to tubes U and UB. To each of the control tubes (S1B, S2B, S3B and UB) add 5.0 ml of a 5% w/v
solution of trichloroacetic acid and mix. Place a stirring rod in each tube and warm to, and maintain at,
35 in a water bath. Add 5.0 ml of concentrated casein substrate to each of the control tubes and mix.
At accurately timed intervals add 5.0 ml of concentrated casein substrate, previously warmed to 35,
to tubes S1, S2, S3 and U and mix immediately. After exactly 30 minutes, in the same order, stop the
reaction in tubes S1, S2, S3 and U by adding 5.0 ml of a 5% w/v solution of trichloroacetic acid and
mix thoroughly. Remove all the test tubes from the water bath and allow to stand at room
temperature for 20 minutes. Filter the contents of the tubes through suitable filter paper1, collect the
filtrates and refilter through the same paper. The filtrates must be free from haze.
Measure the absorbances of the filtrates at the maximum at 275 nm, Appendix II B, using in the
reference cell a mixture of 6.0 ml of borate buffer pH 7.5 and 5.0 ml of the 5% w/v solution of
trichloroacetic acid that has been filtered in the same way. Correct the mean absorbances of the filtrates
from tubes S1, S2 and S3 by subtracting the mean absorbances of the filtrates from the corresponding
control tubes S1B, S2B and S3B.
Prepare a reference curve by plotting the mean corrected absorbances against the potency of the
dilution of the solution of the Standard Preparation used. Calculate the corrected mean absorbance
of the substance being examined by subtracting the mean absorbance of the filtrates from tubes UB
from that of the filtrates from tubes U. Using the corrected mean absorbance, determine the potency
of the solution of the substance being examined from the reference curve and calculate the free
protease activity per mg of the substance being examined by taking into account the dilution factors.
The test is not valid unless the corrected absorbances are between 0.15 and 0.60.
For lipase activity
Apparatus Use a reaction vessel of about 50-ml capacity fitted with a device that will maintain a
temperature of 36.5 to 37.5, a magnetic stirrer and a lid with holes for the insertion of electrodes,
the tip of a burette, a tube for the admission of nitrogen and the introduction of reagents. An
automatic or manual titration apparatus may be used. In the latter case, the burette is graduated in
5-l divisions and the pH meter is provided with a wide reading scale and glass and calomel
electrodes. After each test the reaction vessel is evacuated by suction and washed several times with
water, the washings being removed each time by suction.
Method Carry out the assay under nitrogen. In a small mortar cooled to 0 to 4 triturate carefully
an amount of the substance being examined containing approximately 2500 Units of lipase activity
with 1 ml of cooled lipase solvent until a very fine suspension is obtained (about 10 minutes). Dilute
with cooled lipase solvent, transfer quantitatively to a graduated flask and dilute to 100.0 ml with the
cooled solvent; use immediately.
Transfer 29.5 ml of olive oil substrate emulsion to the assembled reaction vessel equilibrated at 36.5
to 37.5 and adjust the pH to 9.2 with 0.1M sodium hydroxide. Add about 0.5 ml of the suspension of
the substance being examined and record the time at which the pH reaches 9.0. Add continuously
from a micrometer syringe sufficient 0.1M sodium hydroxide VS to maintain the pH at 9.0. Record the
volume of 0.1M sodium hydroxide VS consumed at 1-minute intervals for 5 minutes. Discounting the
first reading, calculate the mean rate of alkali consumption U. If necessary, dilute with sufficient lipase
solvent to produce an average alkali consumption of 0.08 to 0.16 ml of 0.1M sodium hydroxide VS per
minute. Repeat the procedure using the Standard Preparation in place of the substance being examined and calculate the mean rate of alkali consumption, S. Calculate the potency (PL ) of the
substance being examined in Units per mg from the expression:
U ws
R
S w
where U = the mean volume in ml of 0.1M sodium hydroxide VS used per minute in the
titration of the substance being examined,
S = the mean volume in ml of 0.1M sodium hydroxide VS used per minute in the
titration of the Standard Preparation,
PL =
29-58
w = weight in mg of the substance being examined,
ws = weight in mg of the Standard Preparation,
R = potency of the Standard Preparation inUnits per mg.
Calculate the potency of the substance being examined using the average of three separate titrations
for both the substance being examined and the Standard Preparation.
For amylase activity
Method Triturate an amount of the substance being examined containing approximately 1500 Units
of amylase activity with 60 ml of 0.2M mixed phosphate buffer pH 6.8 for 15 minutes and add sufficient
0.2M mixed phosphate buffer mixed phosphate buffer pH 6.8 to produce 100 ml. To a stoppered tube
(200 mm 22 mm) add 25.0 ml of starch substrate, 10.0 ml of 0.2M mixed phosphate buffer pH 6.8 and
1.0 ml of 0.2M sodium chloride. Stopper the tube, mix the contents and place in a water bath at 24.9
to 25.1. When the temperature of the mixture has reached 25 add 1.0 ml of the solution of the
substance being examined and record the time of addition. Mix thoroughly and replace in the water
bath. After exactly 10 minutes add 2 ml of 1M hydrochloric acid to stop the reaction. Transfer the
contents of the tube to a stoppered 300-ml flask. While shaking continuously add 10.0 ml of 0.05M
iodine VS followed immediately by 45 ml of 0.1M sodium hydroxide. Allow to stand in the dark at a
temperature of 15 to 25 for 15 minutes. Add 4 ml of a mixture of 1 volume of sulphuric acid and 4
volumes of water and titrate with 0.1M sodium thiosulphate VS. Repeat the procedure but add the 2 ml
of 1M hydrochloric acid before the addition of the solution of the substance being examined. Prepare a
solution of the Standard Preparation in the same manner as described for the solution of the
substance being examined and repeat the procedure beginning at the words To a stoppered tube
but using 1.0 ml of this solution in place of the solution of the substance being examined.
Calculate the potency (PA) of the substance being examined in Units per mg from the expression
( B A ) ws
Q
( B s As ) w
where A = volume in ml of 0.1M sodium thiosulphate VS used in the titration of the substance
being examined,
As = volume in ml of 0.1M sodium thiosulphate VS used in the titration of the Standard
Preparation
B = volume in ml of 0.1M sodium thiosulphate VS used in the titration of the substance
being examined inactivated by the addition of 1M hydrochloric acid,
Bs = volume in ml of 0.1M sodium thiosulphate VS used in the titration of the Standard
Preparation inactivated by the addition of 1M hydrochloric acid,
Q = potency of the Standard Preparation in Units per mg
w = total weight in mg of the substance being examined in the solution prepared for
assay,
ws = total weight in mg of the Standard Preparation in the solution prepared for assay.
PA =
1A suitable filter paper complies with the following test. Filter 5 ml of a 5% w/v solution of
trichloroacetic acid through a sample of the paper and measure the absorbance of the filtrate at
275 nm, Appendix II B, using the unfiltered 5% w/v solution of trichloroacetic acid in the reference
cell. The absorbance is not more than 0.04.
29-59
Pancuronium Bromide
Me
H
OAc
Me
Me
N
H
2Br
Me
AcO
H
C35H60Br2N2O4
733
15500-66-0
Pancuronium Bromide complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Neuromuscular blocking substance used to produce relaxation during anaesthesia.
Preparation
Pancuronium Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Pancuronium bromide contains not less than 98.0 per cent and not more than the equivalent of
102.0 per cent of 1,1-(3,17-diacetoxy-5-androstan-2,16-ylene)bis(1-methylpiperidinium)
dibromide, calculated with reference to the anhydrous substance.
CHARACTERS
A white or almost white, crystalline powder, hygroscopic, very soluble or freely soluble in water,
freely soluble in alcohol, practically insoluble in ether.
IDENTIFICATION
obtained with pancuronium bromide CRS.
C. It gives reaction (a) of bromides (2.3.1).
TESTS
Appearance of solution Dissolve 50 mg in water R and dilute to 25 ml with the same solvent. The
solvent. The specific optical rotation is +38 to +42, calculated with reference to the anhydrous
substance.
Prepare the solutions immediately before use.
the same solvent.
Reference solution (a). Dissolve 50 mg of pancuronium bromide CRS in chloroform R and dilute to 5 ml
Reference solution (b). Dilute 0.1 ml of the test solution to 10 ml with chloroform R.
Reference solution (c). Dissolve 5 mg of dacuronium bromide CRS in chloroform R and dilute to 25 ml
Reference solution (d). Dissolve 10 mg of pancuronium bromide CRS in 1 ml of reference solution (c).
Apply separately to the plate 2 l of each solution. Develop in an unlined and non-saturated tank
over a path of 12 cm using a mixture of 5 volumes of a 200 g/l solution of sodium iodide R, 10
volumes of acetonitrile R and 85 volumes of 2-propanol R. Dry the plate in a stream of cold air and
spray with a 10 g/l solution of sodium nitrite R in methanol R. Allow to stand for 2 min and spray with
potassium iodobismuthate solution R and dry in a stream of cold air. In the chromatogram obtained with
the test solution: any spot corresponding to dacuronium bromide is not more intense than the prin-
29-60
cipal spot in the chromatogram obtained with reference solution (c) (2.0 per cent); any spot apart
from the principal spot and any spot corresponding to dacuronium bromide is not more intense than
the spot obtained in the chromatogram with reference solution (b) (1.0 per cent). The test is not
valid unless: the chromatogram obtained with reference solution (d) shows two distinct spots; the Rf
value of dacuronium bromide CRS relative to that of pancuronium bromide CRS is not less than 1.2; a
spot is clearly visible in the chromatogram obtained with reference solution (b).
ASSAY
Dissolve 0.2000 g in 50 ml of acetic anhydride R, heating if necessary. Titrate with 0.1M perchloric
acid, determining the end-point potentiometrically (2.2.20).
1 ml of 0.1M perchloric acid is equivalent to 36.63 mg of C35H60Br2N2O4.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
29-61
Papaveretum
Definition Papaveretum is a mixture of 253 parts of Morphine Hydrochloride
(C17H19NO3,HCl,3H2O, 375.9), 23 parts of Papaverine Hydrochloride (C20H21NO4,HCl, 375.9)
and 20 parts of Codeine Hydrochloride (C18H21NO3,HCl,2H2O, 371.9). It contains not less than
80.0% and not more than 88.4% of C17H19NO3,HCl, not less than 8.3% and not more than 9.2% of
C20H21NO4,HCl and not less than 6.6% and not more than 7.4% of C18H21NO3,HCl, calculated
with reference to the dried material.
Soluble in water; sparingly soluble in ethanol (96%).
Identification
A. Carry out the method for thin-layer chromatography, Appendix III A, using a silica gel precoated
plate (Merck silica gel 60 plates are suitable) and a mixture of 2 volumes of 13.5M ammonia, 6
volumes of ethanol (96%), 40 volumes of acetone and 40 volumes of toluene as the mobile phase.
Apply separately to the plate 10 l of each of four solutions in water containing (1) 1.5% w/v of the
substance being examined, (2) 1.28% w/v of morphine hydrochloride BPCRS, (3) 0.115% w/v of
papaverine hydrochloride BPCRS and (4) 0.10% w/v of codeine hydrochloride BPCRS. After removal of
the plate, dry it at 100 to 105 for 15 minutes, allow it to cool and spray with potassium
iodobismuthate solution and then with a 0.4% v/v solution of sulphuric acid. The chromatogram
obtained with solution (1) shows three principal spots corresponding to the principal spots in the
chromatograms obtained with solutions (2), (3) and (4).
B. Yields the reactions characteristics of chlorides, Appendix VI.
Acidity pH of a 1.5% w/v solution, 3.7 to 4.7, Appendix V L.
Clarity and colour of solution A 1.5% w/v solution in water is clear, Appendix IV A, and not more
intensely coloured than reference solution BY5, Appendix IV B, Method II.
Loss on drying When dried to constant weight at 130, loses not less than 10.0% and not more than
14.0% of its weight. Use 0.5 g.
Assay Carry out the method for liquid chromatography, Appendix III D, using three solutions in
methanol (60%) containing (1) 0.128% w/v of morphine hydrochloride BPCRS, 0.0115% w/v of
papaverine hydrochloride BPCRS and 0.010% w/v of codeine hydrochloride BPCRS, (2) 0.128% w/v of
morphine hydrochloride BPCRS and (3) 0.15% w/v of the substance being examined.
The chromatographic procedure may be carried out using a stainless steel column (10 cm
4.6 mm) packed with stationary phase C (5 m) (Nucleosil C18 is suitable), (b) as the mobile phase
with a flow rate of 2 ml per minute a solution containing 0.01M sodium acetate and 0.005M dioctyl
sodium sulphosuccinate in methanol (60%) adjusted to pH 5.5 with glacial acetic acid and (c) a detection
wavelength of 285 nm. If necessary, adjust the proportion of methanol in the mobile phase in the
range 55% v/v to 65% v/v so that the retention time of morphine in solution (2) is 4 to 5 minutes.
Adjust the pH of the mobile phase with either glacial acetic acid or 2M sodium hydroxide in order to
obtain optimum separation of the three principal components in solution (1). The retention times of
codeine and papaverine relative to that of morphine are about 1.3 and 1.7 respectively.
For anhydrous morphine hydrochloride Calculate the content of C17H19NO3,HCl using the
declared content of C17H19NO3,HCl in morphine hydrochloride BPCRS.
For papaverine hydrochloride Calculate the content of C20H21NO4,HCl using the declared
content of C20H21NO4,HCl in papaverine hydrochloride BPCRS.
For anhydrous codeine hydrochloride Calculate the content of C18H21NO3,HCl using the
declared content of C18H21NO3,HCl in codeine hydrochloride BPCRS.
Storage Papaveretum should be kept in a well-closed container and protected from light.
Preparations
Papaveretum Injection
29-62
Papaverine Hydrochloride
revised 1/01
OMe
OMe
,HCl
MeO
MeO
C20H21NO4,HCl
375.9
61-25-6
Papaverine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Papaverine hydrochloride contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of 1-(3,4-dimethoxybenzyl)-6,7-dimethoxyisoquinoline hydrochloride, calculated with
CHARACTERS
A white or almost white, crystalline powder or white or almost white crystals, sparingly soluble in
water, slightly soluble in alcohol.
IDENTIFICATION
A. Dissolve 25 mg in 0.01M hydrochloric acid and dilute to 100.0 ml with the same acid. Dilute 5.0 ml
of the solution to 250.0 ml with 0.01M hydrochloric acid. Examined between 230 nm and 270 nm
(2.2.25), the solution shows an absorption maximum at 250 nm. The specific absorbance at the
maximum is 1590 to 1670. Dilute 10.0 ml of the first solution to 100.0 ml with 0.01M hydrochloric
acid. Examined between 270 nm and 350 nm, the solution shows two absorption maxima, at 280 nm
to 290 nm and 303 nm to 313 nm. The specific absorbances at the maxima are 140 to 200 and 200
to 250, respectively.
B. To 10 ml of solution S (see Tests) add ammonia R dropwise and allow to stand. The precipitate,
washed and dried, melts (2.2.14) at 146C to 149C.
C. To about 10 mg add 3 ml of acetic anhydride R and, cautiously, 0.15 ml of sulphuric acid R; heat
on a water-bath for 3 min to 4 min. A yellow colour with a green fluorescence develops.
TESTS
Solution S Dissolve 0.4 g in carbon dioxide-free water R, heating gently if necessary, and dilute to
Readily carbonisable substances To 50 mg add 5 ml of sulphuric acid R. Allow to stand for
15 min. The solution is not more intensely coloured than reference solution R4 or Y4 (Method I,
2.2.2).
Foreign alkaloids Examine by thin-layer chromatography (2.2.27), using a TLC silica gel GF254
plate R.
the same solvent.
Reference solution. Dissolve 50 mg of codeine R in chloroform R and dilute to 100 ml with the same
solvent.
volumes of diethylamine R, 20 volumes of ethyl acetate R and 70 volumes of toluene R. Warm the plate
29-63
until the odour of diethylamine is no longer perceptible. Examine in ultraviolet light at 254 nm. Any
spot in the chromatogram obtained with the test solution, apart from the principal spot, is not more
Disregard any spot remaining at the starting point.
100C to 105C.
Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on the residue from the test for
loss on drying.
ASSAY
__________________________________________________________________________________________________________ Ph Eur
29-64
Paracetamol
NHAc
HO
C8H9NO2
151.2
103-90-2
Paracetamol complies with the requirements of the 3rd edition of the European Pharmacopoeia [0049]. These
Action and use Analgesic and antipyretic.
Preparations
Co-codamol Tablets
Co-dydramol Tablets
Co-proxamol Tablets
Paediatric Paracetamol Oral Solution
Paracetamol Oral Suspension
Paracetamol Suppositories
Paracetamol Tablets
Dispersible Paracetamol Tablets
Soluble Paracetamol Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Paracetamol contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of N-(4-hydroxyphenyl)acetamide, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder, sparingly soluble in water, freely soluble in alcohol, very slightly soluble
in ether and in methylene chloride.
IDENTIFICATION
B. Dissolve 50 mg in methanol R and dilute to 100.0 ml with the same solvent. To 1.0 ml of the
solution add 0.5 ml of 0.1M hydrochloric acid and dilute to 100.0 ml with methanol R. Protect the
solution from bright light and immediately measure the absorbance (2.2.25) at the absorption
maximum at 249 nm. The specific absorbance at the maximum is 860 to 980.
obtained with paracetamol CRS. Examine the substances prepared as discs.
D. To 0.1 g add 1 ml of hydrochloric acid R, heat to boiling for 3 min, add 10 ml of water R and cool.
No precipitate is formed. Add 0.05 ml of 0.0167M potassium dichromate. A violet colour develops
which does not change to red.
E. It gives the reaction of acetyl (2.3.1). Heat over a naked flame.
TESTS
coating substance.
Test solution (a). Transfer 1.0 g of the finely powdered substance to be examined to a stoppered
15 ml glass or polytetrafluoroethylene centrifuge tube, add 5.0 ml of peroxide-free ether R, shake by
mechanical means for 30 min and centrifuge for 15 min or until a clear supernatant liquid is
obtained.
Test solution (b). Dissolve 0.10 g of the substance to be examined in methanol R and dilute to 4.0 ml
Reference solution (a). Dissolve 50 mg of chloroacetanilide R in methanol R and dilute to 10.0 ml with
the same solvent. Dilute 2.0 ml of this solution to 100 ml with methanol R.
Reference solution (b). Dilute 5.0 ml of reference solution (a) to 8.0 ml with methanol R.
Reference solution (c). Dissolve 0.25 g of chloroacetanilide R and 0.1 g of the substance to be examined
in methanol R and dilute to 100 ml with the same solvent.
29-65
Apply separately to the plate 200 l of test solution (a), 20 l of reference solution (a), 20 l of test
solution (b), 20 l of reference solution (b) and 20 l of reference solution (c). Develop immediately,
in an unsaturated tank, over a path of 10 cm using a mixture of 10 volumes of methanol R, 40
volumes of 1,1,1-trichloroethane R and 50 volumes of di-isopropyl ether R. Allow the plate to dry in air
and examine in ultraviolet light at 254 nm. Any spot corresponding to chloroacetanilide in the
chromatogram obtained with test solution (a) is not more intense than the spot in the chromatogram
obtained with reference solution (a) (0.005 per cent). Any spot in the chromatogram obtained with
test solution (b), apart from the principal spot and any spot corresponding to chloroacetanilide, is not
more intense than the spot in the chromatogram obtained with reference solution (b) (0.25 per cent).
The test is not valid unless the chromatogram obtained with reference solution (c) shows two clearly
separated spots.
4-Aminophenol Dissolve 0.50 g in a mixture of equal volumes of methanol R and water R and dilute
to 10.0 ml with the same mixture of solvents. Add 0.2 ml of a freshly prepared solution containing
10 g/l of sodium nitroprusside R and 10 g/l of anhydrous sodium carbonate R, mix and allow to stand for
30 min. Prepare a standard at the same time in the same manner using 10.0 ml of the mixture of
methanol R and water R containing 0.50 g of 4-aminophenol-free paracetamol R and 0.5 ml of a 0.05 g/l
solution of 4-aminophenol R in a mixture of equal volumes of methanol R and water R. Any blue colour
in the test solution is not more intense than that in the standard (50 ppm).
obtained by diluting lead standard solution (100 ppm Pb) R with the mixture of acetone R and water R.
100C to 105C.
ASSAY
Dissolve 0.300 g in a mixture of 10 ml of water R and 30 ml of dilute sulphuric acid R. Boil under a
reflux condenser for 1 h, cool and dilute to 100.0 ml with water R. To 20.0 ml of the solution add
40 ml of water R, 40 g of ice, 15 ml of dilute hydrochloric acid R and 0.1 ml of ferroin R. Titrate with
0.1M ammonium and cerium sulphate until a yellow colour is obtained. Carry out a blank titration.
1 ml of 0.1M ammonium and cerium sulphate is equivalent to 7.56 mg of C8H9NO2.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
29-66
Hard Paraffin
revised 1/01
Hard Paraffin complies with the requirements of the 3rd edition of the European Pharmacopoeia [1034].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Hard paraffin is a purified mixture of solid saturated hydrocarbons generally obtained from
petroleum. It may contain a suitable antioxidant.
CHARACTERS
A colourless or white mass, practically insoluble in water, freely soluble in methylene chloride,
practically insoluble in alcohol. The melted substance is free from fluorescence in daylight.
IDENTIFICATION
spectrum of hard paraffin.
B. It complies with the test for acidity or alkalinity (see Tests).
C. Melting point (2.2.14): 50C to 61C.
TESTS
Acidity or alkalinity To 15 g add 30 ml of boiling water R and shake vigorously for 1 min. Allow to
cool and to separate. To 10 ml of the aqueous layer add 0.1 ml of phenolphthalein solution R. The
solution is colourless. Not more than 1.0 ml of 0.01M sodium hydroxide is required to change the
colour of the indicator to red. To a further 10 ml of the aqueous layer add 0.1 ml of methyl red
solution R. The solution is yellow. Not more than 0.5 ml of 0.01M hydrochloric acid is required to
change the colour of the indicator to red.
Polycyclic aromatic hydrocarbons Use reagents for ultraviolet absorption spectrophotometry. Dissolve
0.50 g in 25 ml of heptane R and place in a 125 ml separating funnel with unlubricated ground-glass
parts (stopper, stopcock). Add 5.0 ml of dimethyl sulphoxide R. Shake vigorously for 1 min and allow
to stand until two clear layers are formed. Transfer the lower layer to a second separating funnel, add
2 ml of heptane R and shake the mixture vigorously. Allow to stand until two clear layers are formed.
Separate the lower layer and measure its absorbance (2.2.25) between 265 nm and 420 nm using as
the compensation liquid the clear lower layer obtained by vigorously shaking 5.0 ml of dimethyl
sulphoxide R with 25 ml of heptane R for 1 min. Prepare a 7.0 mg/l reference solution of naphthalene R
in dimethyl sulphoxide R and measure the absorbance of this solution at the maximum at 278 nm
using dimethyl sulphoxide R as the compensation liquid. At wavelengths from 265 nm to 420 nm, the
absorbance of the test solution is not greater than one-third that of the reference solution at 278 nm.
Sulphates (2.4.13). Introduce 2.0 g of the melted substance to be examined into a 50 ml groundglass-stoppered separating funnel. Add 30 ml of boiling distilled water R and shake vigorously for
1 min. Filter. 15 ml of the filtrate complies with the limit test for sulphates (150 ppm).
STORAGE
LABELLING
The label states, where applicable, the name and concentration of any added antioxidant.
__________________________________________________________________________________________________________ Ph Eur
29-67
Light Liquid Paraffin

revised 1/01
Light Liquid Paraffin complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Light Liquid Paraffin Eye Drops
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Light liquid paraffin is a purified mixture of liquid saturated hydrocarbons.
CHARACTERS
It has the characters described in the monograph on Liquid paraffin (0239).
IDENTIFICATION
B. In a test tube cautiously boil 1 ml with 1 ml of 0.1M sodium hydroxide, with continuous shaking, for
about 30 s. On cooling to room temperature two phases separate. To the aqueous phase add 0.1 ml
of phenolphthalein solution R. The colour turns to red.
C. It complies with the test for viscosity (see Tests).
TESTS
It complies with the tests prescribed in the monograph on Liquid paraffin, (0239) with the following
modifications:
Viscosity (2.2.9): 25 mPa.s to 80 mPa.s.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
29-68
Liquid Paraffin
revised 1/01
Liquid Paraffin complies with the requirements of the 3rd edition of the European Pharmacopoeia [0239].
Action and use Faecal softener.
Preparations
Liquid Paraffin Oral Emulsion
Liquid Paraffin and Magnesium Hydroxide Oral Emulsion
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Liquid paraffin, is a purified mixture of liquid saturated hydrocarbons obtained from petroleum.
CHARACTERS
A colourless, transparent, oily liquid, free from fluorescence in daylight. Practically insoluble in water,
slightly soluble in alcohol, miscible with hydrocarbons.
IDENTIFICATION
B. In a test tube cautiously boil 1 ml with 1 ml of 0.1M sodium hydroxide, with continuous shaking, for
about 30 s. On cooling to room temperature two phases separate. To the aqueous phase add 0.1 ml
of phenolphthalein solution R. The colour turns to red.
C. It complies with the test for viscosity (see Tests).
TESTS
Acidity or alkalinity To 10 ml add 20 ml of boiling water R and shake vigorously for 1 min.
Separate the aqueous layer and filter. To 10 ml of the filtrate, add 0.1 ml of phenolphthalein solution R.
The solution is colourless. Not more than 0.1 ml of 0.1M sodium hydroxide is required to change the
colour of the indicator to pink.
Viscosity (2.2.9): 110 mPa.s to 230 mPa.s.
Polycyclic aromatic hydrocarbons Use reagents for spectrophotometry. Introduce 25.0 ml into a
125 ml separating funnel with unlubricated ground-glass parts (stopper, stopcock). Add 25 ml of
hexane R which has been previously shaken twice with one-fifth its volume of dimethyl sulphoxide R.
Mix and add 5.0 ml of dimethyl sulphoxide R. Shake vigorously for 1 min and allow to stand until two
clear layers are formed. Transfer the lower layer to a second separating funnel, add 2 ml of hexane R
and shake the mixture vigorously. Allow to stand until two clear layers are formed. Separate the lower
layer and measure its absorbance (2.2.25) between 260 nm and 420 nm, using as the compensation
liquid the clear lower layer obtained by vigorously shaking 5.0 ml of dimethyl sulphoxide R with 25 ml
of hexane R for 1 min. Prepare a 7.0 mg/l reference solution of naphthalene R in trimethylpentane R
and measure the absorbance of the solution at the maximum at 275 nm, using trimethylpentane R as
the compensation liquid. At no wavelength between 260 nm and 420 nm does the absorbance of the
test solution exceed one-third that of the reference solution at 275 nm.
Readily carbonisable substances Use a ground-glass-stoppered tube about 125 mm long and
18 mm in internal diameter, graduated at 5 ml and 10 ml; wash with chromic acid cleansing mixture R,
rinse with water R and dry. Introduce 5 ml of the substance to be examined and add 5 ml of nitrogenfree sulphuric acid R (95.0 per cent m/m to 95.5 per cent m/m H2SO4). Insert the stopper and shake as
vigorously as possible, in the longitudinal direction of the tube, for 5 s. Loosen the stopper,
immediately place the tube in a water-bath, avoiding contact of the tube with the bottom or side of
the bath, and heat for 10 min. After 2 min, 4 min, 6 min and 8 min, remove the tube from the bath
and shake as vigorously as possible, in the longitudinal direction of the tube for 5 s. At the end of
10 min of heating, remove the tube from the water-bath and allow to stand for 10 min. Centrifuge at
2000 g for 5 min. Transfer 4 ml of the upper layer into a clean test tube. The solution is not more
intensely coloured (Method I, 2.2.2) than 4 ml of a mixture of 0.6 ml of standard solution B and
9.4 ml of a 10 g/l solution of hydrochloric acid R. The lower layer is not more intensely coloured
(Method I, 2.2.2) than a mixture of 0.5 ml of blue primary solution, 1.5 ml of red primary solution,
3.0 ml of yellow primary solution and 2 ml of a 10 g/l solution of hydrochloric acid R.
29-69
Solid paraffins Dry a suitable quantity of the substance to be examined by heating at 100C for 2 h
and cool in a desiccator over sulphuric acid R. Place in a glass tube with an internal diameter of about
25 mm, close the tube and immerse in a bath of iced water. After 4 h, the liquid is sufficiently clear
for a black line, 0.5 mm wide, to be easily seen against a white background held vertically behind the
tube.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
29-70
White Soft Paraffin

White Petroleum Jelly
Definition White Soft Paraffin is a semi-solid mixture of hydrocarbons obtained from petroleum and
bleached.
Characteristics A white, translucent, soft unctuous mass, retaining these characteristics on storage
and when melted and allowed to cool without stirring; not more than slightly fluorescent by daylight,
even when melted; odourless when rubbed on the skin.
Practically insoluble in water; soluble in chloroform, in ether and in petroleum spirit (boiling range, 40
to 60), the solutions sometimes showing a slight opalescence; practically insoluble in ethanol (96%).
Acidity or alkalinity To 10 g add 20 ml of boiling water, shake vigorously for 1 minute, cool, allow
to separate and filter the aqueous layer. To 10 ml of the filtrate add 0.1 ml of phenolphthalein solution.
The solution is colourless and not more than 0.1 ml of 0.1M sodium hydroxide VS is required to
change the colour of the solution to pink.
Light absorption Absorbance of a 0.05% w/v solution in 2,2,4-trimethylpentane at 290 nm, not more
than 0.5, Appendix II B.
Drop point 42 to 60, when determined by the following method and using an Ubbelohde
apparatus complying with British Standard 894:1956 (Specification for Ubbelohde apparatus for flow
and drop points). Heat the substance being examined, with stirring, to 118 to 122, to ensure
uniformity, and then cool to 103 to 107. Warm the metal cup to 103 to 107 in an oven, remove it
from the oven, place on a clean plate or ceramic tile and pour sufficient of the melted sample into the
cup to fill it completely. Allow the filled cup to cool for 30 minutes on the tile and then place it in a
water bath at 24 to 26 for a further 30 to 40 minutes. Level the surface of the sample with a single
stroke of a knife or razor blade, avoiding any working of the sample. Push the cup, without lateral
movement, into the metal case as far as the rim stop and wipe away the excess of the substance that is
squeezed out of the bottom of the tube, ensuring that the air vents are not blocked. Fit the
thermometer, with the cup attached, through the bored cork to the boiling tube such that the bottom
of the cup is 24 to 26 mm above the bottom of the boiling tube. Fix the boiling tube vertically within
the beaker so that at least two thirds of its length is immersed in the liquid contained in the beaker.
Adjust the temperature of the outer bath so that the temperature of the substance rises at the rate of
1 per minute. The temperature at which the first drop of melted liquid falls from the metal cup is
regarded as the drop point of the substance. Carry out not fewer than three determinations, each
time with a fresh sample of the substance being examined. The difference between the readings must
not exceed 3. The mean of three readings is taken as the drop point of the substance.
Polycyclic aromatic hydrocarbons To 1.0 g of the substance being examined in a separating
funnel add 50 ml of hexane and shake to dissolve, warming gently if necessary. Shake the solution
with 20 ml of dimethyl sulphoxide for 1 minute, allow to stand until two clear layers are produced and
transfer the lower layer to a second separating funnel. Repeat the extraction with a further 20 ml of
dimethyl sulphoxide. Add 20 ml of hexane to the combined extracts, shake for 1 minute, allow to stand
until two clear layers are produced, discard the upper layer and dilute the washed, lower layer to
50 ml with dimethyl sulphoxide (solution A). Measure the light absorption of a 4-cm layer of solution A
in the range
265 nm to 420 nm, Appendix II B, using in the reference cell the clear lower layer obtained by
shaking 10 ml of dimethyl sulphoxide with 25 ml of hexane for 1 minute. Measure the absorbance of a
4-cm layer of a solution containing 6.0 g per ml of naphthalene in dimethyl sulphoxide at 278 nm,
Appendix II B, using dimethyl sulphoxide in the reference cell.
The absorbance of solution A at all wavelengths in the range 265 to 420 nm is not more than that of
the naphthalene solution at 278 nm.
Foreign organic matter Heat 1 g until fumes appear. No acrid odour is evolved.
Storage White Soft Paraffin should be protected from light.
29-71
Yellow Soft Paraffin

Yellow Petroleum Jelly
1/01
Yellow Soft Paraffin complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Yellow soft paraffin is a purified mixture of semi-solid hydrocarbons, obtained from petroleum. It
may contain a suitable antioxidant.
CHARACTERS
A yellow, translucent, unctuous mass, slightly fluorescent in daylight when melted, practically
insoluble in water, soluble in methylene chloride, practically insoluble in alcohol and in glycerol.
IDENTIFICATION
A. The drop point (2.2.17) is 40C to 60C and does not differ by more than 5C from the value
stated on the label, with the following modification to fill the cup: heat the substance to be examined
at 118C to 122C, with stirring to ensure uniformity, then cool to 100C to 107C. Warm the metal
cup at 103C to 107C in an oven, remove it from the oven, place on a clean plate or ceramic tile and
pour a sufficient quantity of the melted sample into the cup to fill it completely. Allow the filled cup
to cool for 30 min on the ceramic tile and place it in a water-bath at 24C to 26C for a further
30 min to 40 min. Level the surface of the sample with a single stroke of a knife or razor blade,
avoiding compression of the sample.
B. Examine by infrared absorption spectrophotometry (2.2.24) comparing with the Ph. Eur. reference
spectrum of yellow soft paraffin. The spectrum obtained shows major peaks similar in position and
intensity to the Ph. Eur. reference spectrum.
C. Melt 2 g and when a homogenous phase is obtained, add 2 ml of water R and 0.2 ml of 0.05M
iodine. Shake. Allow to cool. The solid upper layer is violet-pink.
D. It complies with the test for appearance (see Tests).
TESTS
Appearance The substance is yellow. Melt 12 g on a water-bath. The melted mass is not more
intensely coloured than a mixture of 7.6 volumes of yellow primary solution and 2.4 volumes of red
primary solution (Method II, 2.2.2).
Acidity or alkalinity To 10 g add 20 ml of boiling water R and shake vigorously for 1 min. Allow to
cool and decant. To 10 ml of the aqueous layer add 0.1 ml of phenolphthalein solution R. The solution
is colourless. Not more than 0.5 ml of 0.01M sodium hydroxide is required to change the colour of the
indicator to red.
Consistency (2.9.9). The consistency is 100 to 300.
Polycyclic aromatic hydrocarbons Use reagents for ultraviolet absorption spectrophotometry. Dissolve
1.0 g in 50 ml of hexane R which has been previously shaken twice with one-fifth its volume of
dimethyl sulphoxide R. Transfer the solution to a 125 ml separating funnel with unlubricated groundglass parts (stopper, stopcock). Add 20 ml of dimethyl sulphoxide R. Shake vigorously for 1 min and
allow to stand until two clear layers are formed. Transfer the lower layer to a second separating
funnel. Repeat the extraction with a further 20 ml of dimethyl sulphoxide R. Shake vigorously the
combined lower layers with 20 ml of hexane R for 1 min. Allow to stand until two clear layers are
formed. Separate the lower layer and dilute to 50.0 ml with dimethyl sulphoxide R. Measure the
absorbance (2.2.25) between 260 nm and 420 nm using a path length of 4 cm and using as the
compensation liquid the clear lower layer obtained by vigorously shaking 10 ml of dimethyl
sulphoxide R with 25 ml of hexane R for 1 min. Prepare a 9.0 mg/l reference solution of naphthalene R
in dimethyl sulphoxide R and measure the absorbance of this solution at the maximum at 278 nm
using a path length of 4 cm and using dimethyl sulphoxide R as the compensation liquid. At no
wavelength in the range of 260 nm to 420 nm does the absorbance of the test solution exceed that of
the reference solution at 278 nm.
STORAGE
29-72
LABELLING
The label states:
the nominal drop point,
where applicable, the name and concentration of any added antioxidant.
__________________________________________________________________________________________________________ Ph Eur
29-73
Paraldehyde
H
Me
Me
H
H
Me
C6H12O3
132.2
123-63-7
Paraldehyde complies with the requirements of the 3rd edition of the European Pharmacopoeia [0351]. These
Preparation
Paraldehyde Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Paraldehyde is 2,4,6-trimethyl-1,3,5-trioxane, the cyclic trimer of acetaldehyde. It may contain a
suitable quantity of an antioxidant.
CHARACTERS
A colourless or slightly yellow, transparent liquid. It solidifies on cooling to form a crystalline mass.
Soluble in water, but less soluble in boiling water, miscible with alcohol, with ether and with essential
oils.
IDENTIFICATION
A. Solution S (see Tests) is clear (2.2.1) but becomes turbid on warming.
B. To 5 ml add 0.1 ml of dilute sulphuric acid R and heat. Acetaldehyde, recognisable by its odour, is
evolved.
C. To 5 ml of solution S in a test-tube add 5 ml of ammoniacal silver nitrate solution R and heat in a
water-bath. Silver is deposited as a mirror on the wall of the tube.
TESTS
Solution S Dissolve 20.0 ml in carbon dioxide-free water R and dilute to 200.0 ml with the same
solvent.
Acidity To 50.0 ml of solution S add 0.05 ml of phenolphthalein solution R. Not more than 1.5 ml of
Distillation range (2.2.11). Not more than 10 per cent distils below 123C and not less than 95 per
cent distils below 126C.
Freezing point (2.2.18): 10C to 13C.
Acetaldehyde To 5.0 ml add a mixture of 0.2 ml of methyl orange solution R, 5 ml of alcohol (60 per
cent V/V) R and 5 ml of alcoholic hydroxylamine solution R and shake. Not more than 0.8 ml of 0.5M
sodium hydroxide is required to change the colour of the indicator to pure yellow.
Peroxides Place 50.0 ml of solution S in a ground-glass-stoppered flask, add 5 ml of dilute sulphuric
acid R and 10 ml of potassium iodide solution R, close the flask and allow to stand protected from light
for 15 min. Titrate with 0.1M sodium thiosulphate using 1 ml of starch solution R as indicator. Allow to
stand for 5 min and, if necessary complete the titration. Not more than 2.0 ml of 0.1M sodium
thiosulphate is required.
Non-volatile residue Heat 5.0 ml in a tared evaporating dish on a water-bath and dry at 105C for
1 h. The residue weighs not more than 3 mg (0.6 g/l).
STORAGE
Store in a small, well-filled, airtight container, protected from light, in a cool place. If the substance
has solidified the whole contents of the container must be liquefied before use.
LABELLING
The label states the name and quantity of any added antioxidant.
__________________________________________________________________________________________________________ Ph Eur
30-1
Parnaparin Sodium
OSO3Na
O
OR
R2
O
O
O
NHR 1
OH
HO
O
R3
OH
OR
COONa
OH
OR
OH
NHSO3Na
OSO3Na
n = 1 to 21, R = H or SO3Na, R1 = SO3Na or COCH3

R2 = H and R3 = CO2Na or R2 = CO2Na and R3 = H
Parnaparin Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Parnaparin sodium is the sodium salt of a low-molecular-mass heparin that is obtained by radicalcatalysed depolymerisation, with hydrogen peroxide and with a cupric salt, of heparin from bovine or
porcine intestinal mucosa. The majority of the components have a 2-O-sulpho--L-idopyranosuronic
acid structure at the non-reducing end and a 2-N,6-O-disulpho-D-glucosamine structure at the
reducing end of their chain.
Parnaparin sodium complies with the monograph on Low-molecular-mass heparins (0828), with the
modifications and additional requirements below:
5000.
The degree of sulphatation is 2.0 to 2.6 per disaccharide unit.
milligram calculated with reference to the dried substance. The ratio of anti-factor Xa activity to antifactor IIa activity is between 1.5 and 3.0.
PRODUCTION
The animals from which parnaparin is derived must fulfil the requirements for the health of
animals suitable for human consumption to the satisfaction of the competent authority.
IDENTIFICATION
Carry out identification test C as described in the monograph on Low-molecular-mass heparins (0828).
The following requirements apply.
lower than 3000 is not more than 30 per cent. The mass percentage of chains between 3000 and
8000 ranges between 50 per cent and 60 per cent.
TESTS
Appearance of solution Dissolve 1.5 g in 10 ml of water R. The solution is clear (2.2.1) and not
Copper Not more than 10 ppm, determined by atomic absorption spectrophotometry (Method I,
2.2.23) and calculated with reference to the dried substance.
__________________________________________________________________________________________________________ Ph Eur
30-2
Passion Flower
Passiflora
Passion Flower complies with the requirements of the 3rd edition of the European Pharmacopoeia [1459].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Passion flower consists of the fragmented or cut, dried aerial parts of Passiflora incarnata L. It may
also contain flowers and/or fruits. It contains not less than 1.5 per cent of total flavonoids expressed
as vitexin (C21H20O10; Mr 432.4), calculated with reference to the dried drug.
CHARACTERS
IDENTIFICATION
A. The green to greenish-grey or brownish stem is ligneous, hollow, longitudinally striated, glabrous
or very slightly pubescent, with a diameter that is generally less than 8 mm. The green or greenishbrown leaves are alternate, finely dentate and pubescent, deeply divided into three acute lobes of
which the central lobe is the largest. The midrib is much more prominent on the lower surface. The
petiole is pubescent and bears two dark-coloured nectaries near the lamina. The tendrils are very
numerous and grow from the axils of the leaves; they are fine, smooth, round and terminated in
cylindrical spirals. The radiate flowers, if present, have three small bracts and a corolla consisting of
five white, elongated petals with several rows of filiform, petaloid appendices. If present, the greenish
to brownish fruit is flattened and oval; it contains several flattened, brownish-yellow, pitted seeds.
B. Reduce to a powder (355). The powder is light green. Examine under a microscope using chloral
hydrate solution R. The powder shows fragments of the leaf epidermis with sinuous walls and
anomocytic stomata (2.8.3); numerous cluster crystals of calcium oxalate isolated or aligned along
the veins; many isolated or grouped fibres from the stems associated with pitted vessels and tracheids;
uniseriate trichomes with one to three thin-walled cells, straight or slightly curved, ending in a point
or sometimes a hook. In addition the powder shows, if flowers are present, papillose epidermises of
the petals and appendages and pollen grains with a reticulate exine; and if mature fruits are present,
scattered brown tannin cells and brownish-yellow, pitted fragments of the testa.
C. Examine the chromatograms obtained in the test for other species of Passiflora.
The chromatogram obtained with the test solution shows below the zone due to rutin in the chromatogram obtained with the reference solution a zone of intense yellow fluorescence, above it a zone of
green fluorescence (diglycosylflavone), below the zone due to hyperoside in the chromatogram
obtained with the reference solution a zone of yellow fluorescence (iso-orientin) and above a zone of
green fluorescence (isovitexin), above the zone due to hyperoside in the chromatogram obtained with
the reference solution a zone of brownish-yellow fluorescence (orientin) and above it a zone of green
fluorescence (vitexin). These latter two zones may be absent. Further zones may be present.
TESTS
Other species of Passiflora Examine by thin-layer chromatography (2.2.27), using a TLC silica gel
plate R.
Test solution. To 1.0 g of the powdered drug (355) add 5 ml of methanol R. Heat to boiling under a
reflux condenser for 10 min. Cool and filter.
Reference solution. Dissolve with heating 2.0 mg of rutin R and 2.0 mg of hyperoside R in 10 ml of
methanol R.
mixture of 10 volumes of anhydrous formic acid R, 10 volumes of water R, 30 volumes of methyl ethyl
ketone R and 50 volumes of ethyl acetate R. Allow the plate to dry in air. Spray with a 10 g/l solution
of diphenylboric acid aminoethyl ester R in methanol R and then with a 50 g/l solution of macrogol 400 R
in methanol R. Allow the plate to dry in air for 30 min. Examine the plate in ultraviolet light at
365 nm. The chromatogram obtained with the reference solution shows in the lower third a zone of
yellowish-brown fluorescence due to rutin and in the middle third a zone of yellowish-brown fluorescence due to hyperoside. The chromatogram obtained with the test solution shows no intense zones
of greenish-yellow or orange-yellow fluorescence between the zone due to diglycosylflavones and that
due to iso-orientin (P. coerulea and P. edulis).
30-3
ASSAY
Stock solution. In a 100 ml round-bottomed flask, introduce 0.200 g of the powdered drug (250) and
add 40 ml of alcohol (60 per cent V/V) R. Heat in a water-bath at 60C under a reflux condenser for
30 min while shaking frequently. Allow to cool and filter the mixture through a plug of absorbent
cotton in a 100 ml flask. Transfer the absorbent cotton with the drug residue into the roundbottomed flask. Add 40 ml of alcohol (60 per cent V/V) R and heat again in a water-bath at 60C
under reflux for 10 min. Allow to cool and filter the mixture and the first filtrate from the 100 ml
flask through a paper filter in the 100 ml volumetric flask. Dilute to 100 ml with the same solvent,
while rinsing the flask, round-bottomed flask and filter.
Test solution. Introduce 5.0 ml of stock solution into a flask. Evaporate to dryness under reduced
pressure and take up the residue with 10 ml of a mixture of 10 volumes of methanol R and 100
volumes of glacial acetic acid R. Add 10 ml of a solution consisting of 25 g/l of boric acid R and 20 g/l
of oxalic acid in anhydrous formic acid R and dilute to 25.0 ml with anhydrous acetic acid R.
Compensation solution. Into a second flask introduce 5.0 ml of the stock solution. Evaporate to dryness
under reduced pressure and take up the residue with 10 ml of a mixture of 10 volumes of methanol R
and 100 volumes of glacial acetic acid R. Add 10 ml of formic acid R and dilute to 25.0 ml with
anhydrous acetic acid R.
Measure the absorbance (2.2.25) of the test solution after 30 min by comparison with the
compensation solution at 401 nm.
Calculate the percentage content of total flavonoids, expressed as vitexin, from the expression:
A 0.8
m
taking the value of the specific absorbance to be 628.

A = absorbance of the test solution at 401 nm,
m = mass of the substance to be examined, in grams.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
30-4
Pefloxacin Mesilate
corrected 1/01
eN
Et
N
N
,CH3SO3H
COOH
O
C17H20FN3O3,CH4SO3,2H2O
465.5 70458-95-6
Pefloxacin Mesilate complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Pefloxacin Mesilate Dihydrate [1383]. These requirements are reproduced after the heading Definition
below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Pefloxacin mesilate dihydrate contains not less than 98.5 per cent and not more than the equivalent
of 101.5 per cent of 1-ethyl-6-fluoro-7-(4-methylpiperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3carboxylic acid methanesulphonate, calculated with reference to the anhydrous substance.
CHARACTERS
A fine, white or almost white powder, freely soluble in water, slightly soluble in alcohol, very slightly
soluble in methylene chloride.
IDENTIFICATION
A. Dissolve separately 0.1 g of the substance to be examined and 0.1 g of pefloxacin mesilate dihydrate
CRS in 10 ml of water R. Add 5 ml of 1M sodium hydroxide. Adjust the pH of the solution to 7.4 0.1
with phosphoric acid R and shake with two quantities, each of 30 ml, of methylene chloride R. Combine
the organic layers and dry over anhydrous sodium sulphate R. Evaporate to dryness. Examine the
residues by infrared absorption spectrophotometry (2.2.24) comparing the spectra obtained. Examine
the residues as discs prepared using potassium bromide R.
Test solution. Dissolve 40 mg in water R and dilute to 1 ml with the same solvent.
Reference solution. Dissolve 60 mg of methanesulphonic acid R in water R and dilute to 10 ml with the
same solvent.
of water R, 10 volumes of ammonia R, 20 volumes of butanol R and 65 volumes of acetone R. Allow
the plate to dry in air. Spray with a 0.4 g/l solution of bromocresol purple R in alcohol (50 per cent
V/V) R, adjusted to pH 10 using 1M sodium hydroxide. The spot in the chromatogram obtained with
the test solution is similar in position, colour and size to the spot in the chromatogram obtained with
the reference solution.
TESTS
Appearance of solution (2.2.1). Examined within an hour after its preparation, solution S is not
more opalescent than reference suspension II (2.2.1) and not more intensely coloured than intensity
3 of the range of reference solutions of the most appropriate colour (Method II, 2.2.2).
pH (2.2.3). Dilute 1 ml of solution S and dilute to 10 ml with carbon dioxide-free water R. The pH of
the solution is 3.5 to 4.5.
Reference solution (a). Dissolve 5.0 mg of pefloxacin impurity B CRS and 25.0 mg of pefloxacin impurity
C CRS in the mobile phase and dilute to 50.0 ml with the mobile phase. Dilute 1.0 ml of this
Reference solution (b). Dissolve 10.0 mg of norfloxacin impurity A CRS (corresponding to pefloxacin
impurity F) in the mobile phase and dilute to 100.0 ml with the mobile phase. Dilute 1.0 ml of this
30-5
vinyl polymer for chromatography R (5 m),
as mobile phase at a flow rate of 1 ml/min a mixture prepared as follows: 30 volumes of acetonitrile R, 70 volumes of a solution containing 2.70 g/l of cetyltrimethylammonium bromide R and
6.18 g/l of boric acid R (exactly adjusted to pH 8.30 with 1M sodium hydroxide), and 0.2 volumes
of thiodiethylene glycol R,
as detector a spectrophotometer set at 258 nm and 273 nm.
Inject 20 l of reference solution (a). Record the chromatogram at 273 nm. The test is not valid
unless the resolution between the peaks corresponding to impurity B and impurity C is at least 1.5.
Inject 20 l of the test solution and 20 l of reference solution (b). Record the chromatograms of the
test solution at 258 nm and 273 nm for four times the retention time of pefloxacin (about 60
min). Record the chromatograms of reference solution (b) at 258 nm. When the chromatograms are
recorded in the prescribed conditions, the relative retention times are indicated in the table:
Table 14601
Approximate relative
retention time
Correction
factor
Impurity E
0.2
Impurity D
0.3
Impurity A
0.5
Impurity G
0.8
1.4
Pefloxacin
Impurity C
1.7
1.8
2.4
2.4
1.8
3.5
1.0
Impurity B
Impurity H
Impurity F
From the chromatogram obtained at 258 nm with the test solution, calculate the percentage content
of impurity C, F, G and H using the area of the principal peak in the chromatogram obtained at
258 nm with reference solution (b) (external standardisation) taking into account the correction
factors indicated in the table.
From the chromatogram obtained at 273 nm with the test solution, calculate the percentage
content of impurity A, B, D and E and of any unknown impurity from the areas of the peaks in the
chromatogram obtained with the test solution by the normalisation procedure, disregarding the peaks
with an area less than 0.0005 times that of the principal peak in the chromatogram obtained with the
test solution.
None of the impurities has a content of more than 0.5 per cent and not more than three impurities
have a content between 0.2 per cent and 0.5 per cent. The sum of the contents of the impurities is
not more than 1.0 per cent.
Heavy metals (2.4.8). 0.250 g complies with limit test E for heavy metals (10 ppm). Prepare the
Water (2.5.12). 7.0 per cent to 8.5 per cent, determined on 50.0 mg by the semi-micro determination of water using a mixture of 10 volumes of methanol R and 50 volumes of methylene chloride R.
ASSAY
Dissolve 0.200 g in 15.0 ml of anhydrous acetic acid R and add 75.0 ml of acetic anhydride R. Titrate
1 ml of 0.1M perchloric acid is equivalent to 21.48 mg of C18H24FN3O6S.
STORAGE
IMPURITIES
N
Et
N
COOH
O
A. 1-ethyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid
(demethylated pefloxacin or norfloxacin),
30-6
e
Et
N
Cl
COOH
O
B. 1-ethyl-6-chloro-7-(4-methylpiperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid
(chlorinated homologue of pefloxacin),
Et
N
COOH
N
N
Me
C. 1-ethyl-6-fluoro-5-(4-methylpiperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid
(isopefloxacin),
O
e
Et
N
COOH
O
D. 1-ethyl-6-fluoro-7-(4-methyl-4-oxide-piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3carboxylic acid (N-oxide of pefloxacin),

e
Et
N
F
O
E. 1-ethyl-6-fluoro-7-(4-methylpiperazin-1-yl)-quinoline-4(1H)-one (decarboxylated
pefloxacin),
Et
l
COOH
O
F. 7-chloro-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (N-ethyl acid)

(norfloxacin impurity A),
Et
l
COOEt
O
G. ethyl 7-chloro-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylate (N-ethyl ester),

Et
N
COOH
Cl
H. 5-chloro-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (iso-N-ethyl acid).

__________________________________________________________________________________________________________ Ph Eur
30-7
Penbutolol Sulphate
H OH
NHBu t
,H2SO4
C36H58N2O4,H2SO4
681
38363-32-5
Penbutolol Sulphate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Penbutolol sulphate contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of di[(2S)-1-(2-cyclopentylphenoxy)-3-[(1,1-dimethylethyl)amino]propan-2-ol]
sulphate, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, slightly soluble in water, soluble in methanol, practically
insoluble in cyclohexane.
IDENTIFICATION
First identification: A, C, D.
obtained with penbutolol sulphate CRS.
Reference solution. Dissolve 40 mg of penbutolol sulphate CRS in 1 ml of methanol R.
10 volumes of glacial acetic acid R, 20 volumes of water R, 35 volumes of butanol R and 35 volumes of
ethyl acetate R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal
spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution.
C. Dissolve 50 mg in a mixture of 5 ml of water R and 1 ml of 0.1M hydrochloric acid. The solution
gives reaction (a) of sulphates (2.3.1).
D. It complies with the test for specific optical rotation (see Tests).
TESTS
Solution S Dissolve 1.00 g in methanol R and dilute to 20.0 ml with the same solvent.
Acidity or alkalinity To 4 ml of solution S add 4 ml of carbon dioxide-free water R. Add 0.1 ml of
methyl red solution R and 0.2 ml of 0.01M sodium hydroxide. The solution is yellow. Add 0.4 ml of
0.01M hydrochloric acid. The solution is red.
Specific optical rotation (2.2.7). 23 to 25 determined on solution S, calculated with reference
Reference solution (a). Dissolve 4.0 mg of the substance to be examined and 1.0 mg of penbutolol
impurity A CRS in 5.0 ml of a mixture of 40 volumes of mobile phase B and 60 volumes of mobile
phase A.
Reference solution (b). Dilute 1.0 ml of the test solution to 200.0 ml with a mixture of 40 volumes of
mobile phase B and 60 volumes of mobile phase A.
30-8
Reference solution (c). Dilute 1.0 ml of reference solution (b) to 10.0 ml with a mixture of 40 volumes
of mobile phase B and 60 volumes of mobile phase A.
Reference solution (d). Dissolve 5.0 mg of penbutolol impurity A CRS in a mixture of 40 volumes of
solvents. Dilute 2.0 ml to 10.0 ml with the same mixture of solvents.
Mobile phase A. Mix 39 volumes of acetonitrile for chromatography R and 61 volumes of
methanol R,
Mobile phase B. Dissolve 11 g of sodium heptanesulphonate R in 1000 ml of water R, add 5.0 ml of
triethylamine R and adjust to pH 2.7 with phosphoric acid R,
Time
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
(min)
0 15
15 35
35 36
60
60 80
80 60
40
40 20
20 40
isocratic
linear gradient
linear gradient

Inject 10 l of reference solution (b) and adjust the sensitivity of the system so that the height of the
second peak (penbutolol) is at least 20 per cent of the full scale of the recorder. The resolution
between the two principal peaks must be at least 6.0.
Inject 10 l of each of the other solutions. In the chromatogram obtained with the test solution, the
area of any peak corresponding to impurity A is not greater than the area of the principal peak in the
chromatogram obtained with reference solution (d) (0.5 per cent), the area of any peak, apart from
the principal peak and that of impurity A, is not greater than the area of the principal peak in the
chromatogram obtained with reference solution (b) (0.5 per cent) and the sum of the areas of all the
peaks, apart from the principal peak and the peak corresponding to impurity A, is not greater than
twice the area of the principal peak in the chromatogram obtained with reference solution (b) (1 per
cent). Disregard any peak with an area less than that of the principal peak in the chromatogram
Heavy metals (2.4.8). 1.0 g complies with limit test F for heavy metals (10 ppm). Prepare the
100C to 105C.
ASSAY
STORAGE
IMPURITIES
H OH
O
H
N
CH3
Me Me
A. (2S)-1-[2-(cyclopent-1-enyl)phenoxy]-3-[(1,1-dimethylethyl)amino]propan-2-ol.
__________________________________________________________________________________________________________ Ph Eur
30-9
Penicillamine
H
NH2
HS
COOH
Me
Me
C5H11NO2S
149.2
52-67-5
Penicillamine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0566]. These
Action and use Used in treatment of rheumatoid arthritis and in treatment of lead poisoning.
Preparation
Penicillamine Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Penicillamine contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent
of (2S)-2-amino-3-methyl-3-sulphanylbutanoic acid, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, freely soluble in water, slightly soluble in alcohol,
IDENTIFICATION
A. Dissolve 0.5 g in a mixture of 0.5 ml of hydrochloric acid R and 4 ml of warm acetone R, cool in
iced water and initiate crystallisation by scratching the wall of the tube with a glass rod. A white
precipitate is formed. Filter with the aid of vacuum, wash with acetone R and dry with suction. A
10 g/l solution of the precipitate is dextrorotatory.
B. Examine the chromatograms obtained in the test for penicillamine disulphide. The principal peak
in the chromatogram obtained with the test solution is similar in position and approximate size to the
Test solution. Dissolve 10 mg of the substance to be examined in 4 ml of water R.
Reference solution. Dissolve 10 mg of penicillamine CRS in 4 ml of water R.
18 volumes of glacial acetic acid R, 18 volumes of water R and 72 volumes of butanol R. Dry the plate
at 100C to 105C for 5 min to 10 min and expose to iodine vapour for 5 min to 10 min. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to
the principal spot in the chromatogram obtained with the reference solution.
D. Dissolve 40 mg in 4 ml of water R and add 2 ml of phosphotungstic acid solution R. Allow to stand
for 5 min. A blue colour develops.
TESTS
Appearance of solution Solution S is clear (2.2.1) and not more intensely coloured than intensity 6
of the range of reference solutions of the most appropriate colour (Method II, 2.2.2).
pH (2.2.3). Dilute 1 ml of solution S to 10 ml with carbon dioxide-free water R. The pH of this solution is 4.5 to 5.5.
Specific optical rotation (2.2.7). Dissolve 0.500 g in 1M sodium hydroxide and dilute to 10.0 ml
with the same solvent. The specific optical rotation is 61.0 to 65.0, calculated with reference to
Penicillamine disulphide Examine by liquid chromatography (2.2.29).
Test solution. Dissolve 40 mg of the substance to be examined in the mobile phase and dilute to
Reference solution (a). Dissolve 40 mg of penicillamine CRS in the mobile phase and dilute to 10.0 ml
30-10
Reference solution (b). Dissolve 20 mg of penicillamine disulphide CRS in the mobile phase and dilute to
50.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase.
as mobile phase at a flow rate of 2.0 ml per minute a solution containing 0.1 g/l of sodium
edetate R and 2 g/l of methanesulphonic acid R,
In the chromatogram obtained with the test solution the area of any peak due to penicillamine
disulphide is not greater than the area of the corresponding peak in the chromatogram obtained with
reference solution (b) (1 per cent).
Ultraviolet-absorbing substances. Dissolve 0.100 g in water R and dilute to 50.0 ml with the
same solvent. The absorbance (2.2.25) of the solution at 268 nm is not greater than 0.07 (about
0.5 per cent of penilloic acid).
Mercury. Not more than 10 ppm of Hg, determined by atomic absorption spectrometry (Method I,
2.2.23).
Test solution. To 1.00 g of the substance to be examined add 10 ml of water R and 0.15 ml of
perchloric acid R and swirl until dissolution is complete. Add 1.0 ml of a 10 g/l solution of ammonium
pyrrolidinedithiocarbamate R which has been washed immediately before use three times, each time
with an equal volume of methyl isobutyl ketone R. Mix and add 2.0 ml of methyl isobutyl ketone R and
shake for 1 min. Dilute to 25.0 ml with water R and allow the layers to separate; use the methyl
isobutyl ketone layer.
Reference solutions. Dissolve a quantity of mercuric oxide R equivalent to 0.108 g of HgO in the smallest
necessary volume of dilute hydrochloric acid R and dilute to 1000.0 ml with water R (100 ppm Hg).
Prepare the reference solutions in the same manner as the test solution but using instead of the
substance to be examined suitable volumes of the solution containing 100 ppm of Hg.
Measure the absorbance at 254 nm using a mercury hollow-cathode lamp as source of radiation and
an air-acetylene flame. Set the zero of the instrument using a methyl isobutyl ketone layer obtained as
described for the test solution but omitting the substance to be examined.
Penicillin Carry out all the operations in a penicillin-free atmosphere and with equipment reserved for this
test. Sterilise the equipment at 180C for 3 h and the buffer solutions at 121C for 20 min before use.
Test solution (a). Dissolve 1.000 g of the substance to be examined in 8 ml of buffer solution pH 2.5 R
and add 8 ml of ether R. Shake vigorously for 1 min. Repeat the extraction and combine the ether
layers. Add 8 ml of buffer solution pH 2.5 R. Shake for 1 min, allow to settle and quantitatively
separate the upper layer, taking care to eliminate the aqueous phase completely (penicillin is unstable at
pH 2.5; carry out operations at this pH within 6 min to 7 min). Add 8 ml of phosphate buffer solution pH
6.0 R2; shake for 5 min, allow to settle, separate the aqueous layer and check that the pH is 6.0.
Test solution (b). To 2 ml of test solution (a) add 20 l of penicillinase solution R and incubate at 37C
for 1 h.
Reference solution (a). Dissolve 5 mg of benzylpenicillin sodium R in 500 ml of phosphate buffer solution
pH 6.0 R2. Dilute 0.25 ml of this solution to 200.0 ml with buffer solution pH 2.5 R. Carry out the
extraction using 8 ml of this solution as described for test solution (a).
Reference solution (b). To 2 ml of reference solution (a), add 20 l of penicillinase solution R and
incubate at 37C for 1 h.
Reference solution (c). Prepare the solution as described for test solution (a) but omitting the substance
to be examined (blank).
Liquefy a suitable nutrient medium such as that described below and inoculate it at a suitable
temperature with a culture of Micrococcus flavus (ATCC 9341) to give 5 104 micro-organisms per
millilitre or a different quantity if necessary to obtain the required sensitivity and formation of clearly
defined inhibition zones of suitable diameter. Immediately pour the inoculated medium into five Petri
dishes 10 cm in diameter to give uniform layers 2 mm to 5 mm deep. The medium may alternatively
consist of two layers, only the upper layer being inoculated. Store the dishes so that no appreciable
growth or death of the micro-organisms occurs before use and so that the surface of the medium is
dry at the time of use. In each dish, place five stainless steel hollow cylinders 6 mm in diameter on
the surface of the agar evenly spaced on a circle with a radius of about 25 mm and concentric with
the dish. For each dish, place in separate cylinders 0.15 ml of test solutions (a) and (b) and reference
solutions (a), (b) and (c). Maintain at 30C for at least 24 h. Measure the diameters of the inhibition
zones to at least 0.1 mm. The test is valid if reference solution (a) gives a clear inhibition zone and if
reference solutions (b) and (c) give no inhibition zone. If test solution (a) gives an inhibition zone,
30-11
this is caused by penicillin if test solution (b) gives no inhibition zone. If this is so, the average diameter of the inhibition zones given by test solution (a) for the five Petri dishes is less than the average
diameter of the inhibition zones given by reference solution (a) (0.1 ppm).
Nutrient medium (pH 6.0)
Peptone
5g
Yeast extract
1.5 g
Meat extract
1.5 g
Sodium chloride
3.5 g
Agar
15 g
Distilled water R
1000 ml
diphosphorus pentoxide R at 60C at a pressure not exceeding 670 Pa.
ASSAY
STORAGE
IMPURITIES
Me S
HOOC
S Me
Me Me
H2N H
COOH
H NH2
A. 3,3-(disulphanediyl)bis[(2S)-2-amino-3-methylbutanoic] acid (penicillamine disulphide),

H
O
R
COOH
Me
H
N
Me
H H
B. penicillin.
_______________________________________________________________________________________________________________________ Ph Eur
30-12
Diluted Pentaerythrityl Tetranitrate

O2NO
ONO2
O2NO
ONO2
C5H8N4O12
316.1
78-11-5
Diluted Pentaerythrityl Tetranitrate complies with the requirements of the 3rd edition of the European
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Diluted pentaerythrityl tetranitrate is a dry mixture of pentaerythrityl tetranitrate and Lactose monohydrate (187) or Mannitol (559). It contains not less than 95.0 per cent m/m and not more than
105.0 per cent m/m of the declared content of 2,2-bis(hydroxymethyl)propane-1,3-diyl tetranitrate.
CHARACTERS
Undiluted pentaerythrityl tetranitrate is a white or slightly yellowish powder, practically insoluble in
water, soluble in acetone, slightly soluble in alcohol.
The solubility of the diluted product depends on the diluent and its concentration.
IDENTIFICATION
A. The melting point (2.2.14) of the residue obtained with the substance to be examined in
identification test B is 138C to 142C.
B. Shake a quantity of the substance to be examined and a quantity of diluted pentaerythrityl
tetranitrate CRS, each corresponding to 25 mg of pentaerythrityl tetranitrate with 10 ml of acetone R
for 5 min. Filter, evaporate to dryness at a temperature below 40C and dry the residue over
diphosphorus pentoxide R at a pressure of 0.7 kPa for 16 h. Examine the residue obtained with the
substance to be examined by infrared absorption spectrophotometry (2.2.24), comparing with the
spectrum of the residue obtained with diluted pentaerythrityl tetranitrate CRS. Examine the residues
prepared as discs.
Test solution. Shake a quantity of the substance to be examined corresponding to 10 mg of
pentaerythrityl tetranitrate with 10 ml of alcohol R for 5 min and filter.
Reference solution. Shake a quantity of diluted pentaerythrityl tetranitrate CRS corresponding to 10 mg of
pentaerythrityl tetranitrate with 10 ml of alcohol R for 5 min and filter.
20 volumes of ethyl acetate R and 80 volumes of toluene R. Dry the plate in a current of air. Spray with
freshly prepared potassium iodide and starch solution R. Expose to ultraviolet light at 254 nm for
15 min. Examine in daylight. The principal spot in the chromatogram obtained with the test solution
is similar in position, colour and size to the principal spot in the chromatogram obtained with the
reference solution.
Test solution. Shake a quantity of the substance to be examined corresponding to 0.10 g of lactose or
mannitol with 10 ml of water R. Filter if necessary.
Reference solution (a). Dissolve 0.10 g of lactose R in water R and dilute to 10 ml with the same
solvent.
Reference solution (b). Dissolve 0.10 g of mannitol R in water R and dilute to 10 ml with the same
solvent.
Reference solution (c). Mix equal volumes of reference solutions (a) and (b).
Apply separately to the plate 1 l of each solution and thoroughly dry the starting points. Develop
over a path of 15 cm using a mixture of 10 volumes of water R, 15 volumes of methanol R, 25
volumes of anhydrous acetic acid R and 50 volumes of ethylene chloride R, measured accurately since a
slight excess of water produces cloudiness. Dry the plate in a current of warm air. Repeat immediately the development after renewing the mobile phase. Dry the plate in a current of warm air. Spray
with 4-aminobenzoic acid solution R. Dry the plate in a current of cold air until the acetone is removed.
Heat the plate at 100C for 15 min. Allow to cool and spray with a 2 g/l solution of sodium
30-13
principal spot in the chromatogram obtained with reference solution (a) for lactose or to the principal spot in the chromatogram obtained with reference solution (b) for mannitol. The test is not valid
unless the chromatogram obtained with reference solution (c) shows two clearly separated spots.
TESTS
Inorganic nitrates Examine by thin-layer chromatography (2.2.27), using a TLC silica gel plate R.
Test solution. Shake a quantity of the substance to be examined corresponding to 0.10 g of
pentaerythrityl tetranitrate with 5 ml of alcohol R and filter.
Reference solution. Dissolve 10 mg of potassium nitrate R in 1 ml of water R and dilute to 100 ml with
alcohol R.
15 volumes of glacial acetic acid R, 30 volumes of acetone R and 60 volumes of toluene R. Dry the
plate thoroughly in a current of air until the acetic acid is completely removed. Spray copiously with
freshly prepared potassium iodide and starch solution R. Expose the plate to ultraviolet light at 254 nm
for 15 min. Examine in daylight. Any spot corresponding to nitrate ion in the chromatogram
obtained with the test solution is not more intense than the spot in the chromatogram obtained with
the reference solution (0.5 per cent, calculated as potassium nitrate).
obtained with reference solution (c) is at least 20 per cent of the full scale of the recorder.
The test is not valid unless in the chromatogram obtained with reference solution (e), the resolution between the peaks corresponding to glyceryl trinitrate and to pentaerythrityl tetranitrate is at
least 2.0.
Inject 20 l of test solution (a) and 20 l of reference solution (c) and record the chromatogram of
test solution (a) for at least five times the retention time of pentaerythrityl tetranitrate. In the
chromatogram obtained with test solution (a); the area of any peak, apart from the principal peak is
(c) (0.3 per cent); the sum of the areas of all the peaks, apart from the principal peak, is not greater
than twice the area of the principal peak in the chromatogram obtained with reference solution (c)
(0.6 per cent). Disregard any peak with an area less than 0.2 times that of the principal peak in the
chromatogram obtained with reference solution (c).
ASSAY
Test solution (a). Sonicate for 15 min a quantity of the substance to be examined corresponding to
25.0 mg of pentaerythrityl tetranitrate in 20 ml of methanol R and dilute to 25.0 ml with the mobile
phase. Filter through a suitable membrane filter.
Reference solution (a). Sonicate for 15 min a quantity of diluted pentaerythrityl tetranitrate CRS corresponding to 25.0 mg of pentaerythrityl tetranitrate in 20 ml of methanol R and dilute to 25.0 ml with
the mobile phase. Filter through a suitable membrane filter.
Reference solution (c). Dilute 0.3 ml of reference solution (b) to 10.0 ml with the mobile phase.
Reference solution (d). Sonicate for 15 min a quantity of glyceryl trinitrate solution CRS corresponding
to 20.0 mg of glyceryl trinitrate in 20 ml of methanol R and dilute to 25.0 ml with the mobile phase.
Filter through a suitable membrane filter. Dilute 1.0 ml of the filtrate to 10.0 ml with the mobile
phase.
Reference solution (e). To 1 ml of reference solution (b), add 1 ml of reference solution (d) and dilute
to 10 ml with the mobile phase.
as mobile phase at a flow rate of 2 ml/min a mixture of 40 volumes of water R and 60 volumes
of methanol R,
When the chromatograms are recorded in the prescribed conditions the retention time of pentaerythrityl tetranitrate is about 8 min. Inject 20 l of reference solution (b). Adjust the sensitivity of
the system so that the height of the principal peak in the chromatogram obtained is at least 50 per
cent of the full scale of the recorder. Inject reference solution (b) six times. The assay is not valid
unless the relative standard deviation for the area of the principal peak is at most 2.0 per cent. Inject
test solution (b) and reference solution (b) alternately.
30-14
STORAGE
Store protected from light and heat.
LABELLING
The label states:
the content of pentaerythrityl tetranitrate as a percentage,
the diluent used.
IMPURITIES
A. inorganic nitrates,
OH
O2N
O
O2N
NO2
B. pentaerythrityl trinitrate,
NO2
O NO2
O
O2N
O2N
O2N
O
NO2
NO2
O
O2N
C. tripentaerythrityl octanitrate,
O2N
NO2
O
O2N
O
O2N
O
O
O
O
NO2
NO2
D. dipentaerythrityl hexanitrate.
__________________________________________________________________________________________________________ Ph Eur
30-15
Pentagastrin
Boc-Ala-Trp-Met-Asp-Phe
Boc=tert-butyloxycarbonyl
C37H49N7O9S
767.9
5534-95-2
Definition Pentagastrin is a pentapeptide that stimulates gastric secretion of acid. It contains not less
than 97.0% and not more than 103.0% of C37H49N7O9S, calculated with reference to the dried
substance.
Practically insoluble in water; soluble in 5M ammonia and in dimethylformamide; slightly soluble in
ethanol (96%).
Identification
ammonia exhibits maxima at 280 nm and 288 nm and an inflection at 275 nm.
B. Carry out the method for thin-layer chromatography, Appendix III A, using silica gel G as the coating substance on each of three plates and the following solvent systems as the mobile phases: (A) a
mixture of 75 volumes of butan-2-ol and 25 volumes of a 3% v/v solution of 13.5M ammonia, (B) the
upper layer produced by shaking together 50 volumes of water, 40 volumes of butan-1-ol and 10
volumes of glacial acetic acid and allowing to separate and (C) a mixture of 50 volumes of glacial acetic
acid, 25 volumes of ether and 25 volumes of water. Apply separately to each plate 2 l of each of two
solutions in 0.1M ammonia containing (1) 0.5% w/v of the substance being examined and (2)
pentagastrin BPCRS containing 0.5% w/v of pentagastrin. After removal of the plates, allow them to
dry in air, heat at 100 for 2 minutes, spray with a 1.0% w/v solution of 4-dimethylaminobenzaldehyde
in a mixture of 3 volumes of methanol and 1 volume of hydrochloric acid and heat at 100 until purple
spots are produced (about 2 minutes). Examine by transmitted light. On each plate, the principal
spot in the chromatogram obtained with solution (1) corresponds to that in the chromatogram
Light absorption Ratio of the absorbance of a 0.010% w/v solution in 0.01M ammonia at the maximum at 280 nm to that at the maximum at 288 nm, 1.12 to 1.22, Appendix II B.
Specific optical rotation In a 1% w/v solution in dimethylformamide, 25.0 to 29.0, calculated
with reference to the dried substance, Appendix V F.
Amino acids -Alanine 11.0 to 12.2%, aspartic acid 16.4 to 18.2%, methionine 18.4 to 20.4%,
phenylalanine, 20.4 to 22.6%, when determined by the following method. Examine using an amino
acid analyser according to the manufacturers instructions. Standardise the apparatus with 1 mg of an
equimolar mixture of -alanine, aspartic acid, methionine and phenylalanine. Place 1 mg of the
substance being examined in a rigorously cleaned hard-glass tube (10 cm 6 mm), add not less than
0.5 ml of 6M hydrochloric acid, immerse the tube in a freezing mixture at 5, evacuate to a pressure
not exceeding 0.133 kPa and seal. Heat for 16 hours at 110 to 115, cool, open the tube, transfer
the contents to a 10-ml flask with the aid of five 0.2-ml quantities of water and evaporate to dryness
over potassium hydroxide at a pressure of 2 kPa. Take up the residue in water and repeat the evaporation; repeat these operations. Dissolve the residue in a suitable buffer solution (pH 2.2) and dilute to
a suitable volume with the same buffer solution. Apply an aliquot of the solution to the amino acid
analyser. Choose conditions such that the peak given by the amino acid present in the largest amount
produces almost maximum deflection on the chart paper.
gel G as the coating substance and a mixture of 100 volumes of ether, 20 volumes of glacial acetic acid
and 10 volumes of water as the mobile phase. Apply separately to the plate 10 l of each of two
solutions of the substance being examined in a mixture of 24 volumes of methanol and 1 volume of
13.5M ammonia containing (1) 0.50% w/v and (2) 0.010% w/v. After removal of the plate, allow it to
dry in air, heat at 100 for 2 minutes, spray with a 1.0% w/v solution of 4-dimethylaminobenzaldehyde
in a mixture of 3 volumes of methanol and 1 volume of hydrochloric acid and heat at 100 until purple
spots are produced (about 2 minutes). Examine by transmitted light. Any secondary spot in the
Assay Dissolve 5 mg in sufficient 0.01M ammonia to produce 100 ml and measure the absorbance at
the maximum at 280 nm, Appendix II B. Calculate the content of C37H49N7O9S taking 70.0 as the
value of A(1%, 1 cm) at the maximum at 280 nm.
Storage Pentagastrin should be protected from light.
30-16
Action and use Peptide stimulating gastric secretion.
Preparation
Pentagastrin Injection
30-17
Pentamidine Isetionate
O
H2N
NH 2
NH
NH
,
C H 2SO 3H
C H 2OH
C19H24N4O2,2C2H6O4S
592.7
140-64-7
Pentamidine Isetionate complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Pentamidine Diisetionate [1137]. These requirements are reproduced after the heading Definition below.
Action and use Antiprotozoan.
Preparation
Pentamidine Injection
When pentamidine isethionate is prescribed or demanded, Pentamidine Isetionate shall be dispensed
or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Pentamidine diisetionate contains not less than 98.5 per cent and not more than the equivalent of
101.5 per cent of 4,4-[pentane-1,5-diylbis(oxy)]dibenzamidine di(2-hydroxyethanesulphonate),
CHARACTERS
A white or almost white powder or colourless crystals, hygroscopic, freely soluble in water, sparingly
soluble in alcohol, practically insoluble in methylene chloride.
IDENTIFICATION
First identification: B, C, F.
Second identification: A, C, D, E, F.
A. Dissolve 20.0 mg in alcohol R and dilute to 100.0 ml with the same solvent. Dilute 5.0 ml of this
solution to 100.0 ml with alcohol R. Examined between 230 nm and 340 nm (2.2.25), the solution
shows an absorption maximum at 265 nm. The specific absorbance at the maximum, calculated with
reference to the dried substance, is 520 to 560.
obtained with pentamidine diisetionate CRS. Examine the substances prepared as discs.
C. Dissolve 40 mg in 5 ml of water R and add dropwise with shaking 1 ml of a 10 g/l solution of
sodium chloride R. Allow to stand for 5 min. The mixture remains clear.
D. Dissolve 0.5 g in 5 ml of water R with heating to about 80C. Add 10 ml of 1M sodium hydroxide.
Cool in iced water and filter. To 2 ml of the solution add 0.2 ml of nitric acid R and then 0.2 ml of a
400 g/l solution of ammonium and cerium nitrate R in dilute nitric acid R. An orange-red colour is
produced. A blank test prepared at the same time in the same manner is yellow.
E. Dissolve about 30 mg and 30 mg of ninhydrin R in 5 ml of water R. Add 1 ml of a 20 g/l solution
of disodium tetraborate R. An abundant white precipitate forms slowly.
F. Treat 0.150 g by the oxygen-flask method (2.5.10). Use 10 ml of dilute hydrogen peroxide solution R
to absorb the combustion products. The solution gives reaction (a) of sulphates (2.3.1).
TESTS
solution is not more opalescent than reference suspension II (2.2.1) and not more intensely coloured
than intensity 6 of the range of reference solutions of the most appropriate colour (Method II, 2.2.2).
30-18
Reference solution (b). To 0.1 g in a conical flask, add 40 ml of water R and glass beads. Adjust to pH
10.5 with dilute sodium hydroxide solution R and boil under reflux for 20 min. Cool and dilute to 50 ml
with water R. Dilute 1 ml of the solution to 50 ml with the mobile phase.
as the mobile phase at a flow rate of 1 ml/min a mixture of 65 volumes of methanol R, and 35
volumes of a 30 g/l solution of ammonium acetate R previously adjusted to pH 7.5 using
triethylamine R,
Inject 10 l of reference solution (b). The test is not valid unless the chromatogram obtained shows
two principal peaks and the resolution between these peaks is greater than 2.0. Inject separately 10 l
of the test solution and 10 l of reference solution (a). Continue the chromatography for 3.5 times
the retention time of the principal peak. In the chromatogram obtained with the test solution, the
area of any peak apart from the principal peak is not greater than the area of the peak in the chromatogram obtained with reference solution (a) (0.2 per cent), and the total area of all the peaks apart
from the principal peak is not greater than twice the area of the peak in the chromatogram obtained
with reference solution (a) (0.4 per cent). Disregard any peak with an area less than 0.1 times the
area of the peak in the chromatogram obtained with reference solution (a).
100C to 105C.
ASSAY
Dissolve 0.250 g in 50 ml of dimethylformamide R. Add 0.25 ml of thymol blue solution R. Titrate with
0.1M tetrabutylammonium hydroxide, under a current of nitrogen R, until the colour of the indicator
changes to blue. Carry out a blank titration.
1 ml of 0.1M tetrabutylammonium hydroxide is equivalent to 29.63 mg of C23H36N4O10S2.
STORAGE
IMPURITIES
O
O
5
H2N
NH2
NH
A. 4-[[5-(4-amidinophenoxy)pentyl]oxy]benzenecarboxamide.
__________________________________________________________________________________________________________ Ph Eur
30-19
Pentazocine
HO
H
N
Me
Me
H
H
CM e2
and enantiom er
C19H27NO
285.4
359-83-1
Pentazocine complies with the requirements of the 3rd edition of the European Pharmacopoeia [1462]. These
Action and use Analgesic.
Preparation
Pentazocine Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Pentazocine contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of (2RS,6RS,11RS)-6,11-dimethyl-3-(3-methylbut-2-enyl)-1,2,3,4,5,6-hexahydro-2,6-methano-3benzazocin-8-ol, calculated with reference to the dried substance.
CHARACTERS
A white or almost white powder, practically insoluble in water, freely soluble in methylene chloride
and soluble in alcohol.
IDENTIFICATION
spectrum for pentazocine (form A).
TESTS
Absorbance (2.2.25). Dissolve 0.100 g in a mixture of 20 ml of water R and 1 ml of 1M hydrochloric
acid, and dilute to 100.0 ml with water R. To 10.0 ml add 1 ml of 1M hydrochloric acid and dilute to
100.0 ml with water R. The specific absorbance at the maximum at 278 nm is 0.67 to 0.71, calculated with reference to the dried substance.
plate R.
Reference solution (a). Dilute 1 ml of the test solution to 100 ml with methylene chloride R.
Reference solution (c). Dilute 5 ml of reference solution (a) to 20 ml with methylene chloride R.
Apply separately to the plate 10 l of each solution. Develop over a path corresponding to two thirds
of the plate height using a mixture of 3 volumes of isopropylamine R, 3 volumes of methanol R and 94
254 nm. Heat the plate at 100C to 105C for 15 min, allow to cool, expose to iodine vapour and reexamine under ultraviolet light at 254 nm. By each method of visualisation: any spot in the chromatogram obtained with the test solution, apart from the principal spot, is not more intense than the
spot obtained with reference solution (a) (1 per cent); not more than one such spot is more intense
than the spot in the chromatogram obtained with reference solution (b) (0.5 per cent) and not more
than four such spots are more intense than the spot in the chromatogram obtained with reference
pressure not exceeding 0.7 kPa for 4 h.
30-20
ASSAY
1 ml of 0.1M perchloric acid is equivalent to 28.54 mg of C19H27NO.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
30-21
Pentazocine Hydrochloride
O
,HCl
Me
Me
CMe2
H
H
and enantiomer
C19H27NO,HCl
321.9
64024-15-3
Pentazocine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Pentazocine Capsules
Pentazocine Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Pentazocine hydrochloride contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of (2RS,6RS,11RS)-6,11-dimethyl-3-(3-methylbut-2-enyl)-1,2,3,4,5,6-hexahydro2,6-methano-3-benzazocin-8-ol hydrochloride, calculated with reference to the dried substance.
CHARACTERS
A white or almost white powder, sparingly soluble in water, soluble in alcohol and sparingly soluble
IDENTIFICATION
spectrum of pentazocine hydrochloride.
TESTS
6.0.
Absorbance (2.2.25). Dissolve 0.100 g in a mixture of 20 ml of water R and 1 ml of 1M hydrochloric
acid, and dilute to 100.0 ml with water R. To 10.0 ml add 1 ml of 1M hydrochloric acid and dilute to
100.0 ml with water R. The specific absorbance at the maximum at 278 nm is 0.59 to 0.63, calculated with reference to the dried substance.
plate R.
Test solution. Dissolve 0.20 g in 3 ml of methanol R and dilute to 10 ml with methylene chloride R.
Reference solution (a). Dilute 1 ml of the test solution to 100 ml with methylene chloride R.
Reference solution (c). Dilute 5 ml of reference solution (a) to 20 ml with methylene chloride R.
Apply separately to the plate 10 l of each solution. Develop over a path corresponding to two-thirds
of the plate height using a mixture of 3 volumes of isopropylamine R, 3 volumes of methanol R and 94
254 nm. Heat the plate at 100C to 105C for 15 min, allow to cool, expose to iodine vapour and reexamine under ultraviolet light at 254 nm. By each method of visualisation: any spot in the chromatogram obtained with the test solution, apart from the principal spot, is not more intense than the
spot obtained with reference solution (a) (1 per cent); not more than one such spot is more intense
than the spot in the chromatogram obtained with reference solution (b) (0.5 per cent); and not more
than four such spots are more intense than the spot in the chromatogram obtained with reference
30-22
ASSAY
Dissolve 0.250 g in 50 ml of alcohol R. Add 5 ml of 0.01M hydrochloric acid. Carry out a potentiometric titration (2.2.20), using 0.1M sodium hydroxide. Read the volume added between the two
points of inflection.
1 ml of 0.1M sodium hydroxide is equivalent to 32.19 mg of C19H28CINO.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
30-23
Pentobarbital
H
N
O
Prn
Et
H
O
NH
Me
and enantiomer
C11H18N2O3
226.3
76-74-4
Pentobarbital complies with the requirements of the 3rd edition of the European Pharmacopoeia [0200]. These
When pentobarbitone is prescribed or demanded, Pentobarbital shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Pentobarbital contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of 5-ethyl-5-(1-methylbutyl)-1H,3H,5H-pyrimidine-2,4,6-trione, calculated with reference to the
dried substance.
CHARACTERS
ethanol and in ether. It forms water-soluble compounds with alkali hydroxides and carbonates and
with ammonia.
IDENTIFICATION
substance to be examined and pentobarbital CRS and determine the melting point of the mixture. The
difference between the melting points (which are about 133C) is not greater than 2C.
the same solvent.
Reference solution. Dissolve 0.1 g of pentobarbital CRS in alcohol R and dilute to 100 ml with the same
solvent.
chloroform R. Examine immediately in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the
C. To about 10 mg add about 10 mg of vanillin R and 2 ml of sulphuric acid R. Mix and heat on a
water-bath for 2 min. A reddish-brown colour develops. Cool and add cautiously 5 ml of ethanol R.
The colour becomes violet and then blue.
TESTS
Appearance of solution Dissolve 1.0 g in a mixture of 4 ml of dilute sodium hydroxide solution R and
6 ml of water R. The solution is clear (2.2.1) and not more intensely coloured than reference solution
Y6 (Method II, 2.2.2).
coating substance.
the same solvent.
30-24
Isomer Dissolve 0.3 g in 5 ml of a 50 g/l solution of anhydrous sodium carbonate R, heating slightly if
necessary. Add a solution of 0.3 g of nitrobenzyl chloride R in 10 ml of alcohol R and heat under a
reflux condenser for 30 min. Cool to 25C, filter and wash the precipitate with five quantities, each of
5 ml, of water R. In a small flask, heat the precipitate with 25 ml of alcohol R under a reflux condenser
until dissolved (about 10 min). Cool to 25C, if necessary scratching the wall of the flask with a glass
rod to induce crystallisation, and filter. The precipitate, washed with two quantities, each of 5 ml, of
water R and dried at 100C to 105C for 30 min, melts (2.2.14) at 136C to 148C.
100C to 105C.
ASSAY
_______________________________________________________________________________________________________________________ Ph Eur
30-25
Pentobarbital Sodium
O
Prn
H
N
Et
H
ONa
N
Me O
and enantiomer
C11H17N2NaO3
248.3
57-33-0
Pentobarbital Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Pentobarbital Tablets
When pentobarbitone sodium is prescribed or demanded, Pentobarbital Sodium shall be dispensed
or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Pentobarbital sodium contains not less than 99.0 per cent and not more than the equivalent of
101.5 per cent of the sodium derivative of 5-ethyl-5-(1-methylbutyl)-1H,3H, 5H-pyrimidine-2,4,6trione, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder, hygroscopic, very soluble in water, practically insoluble in ether.
IDENTIFICATION
A. Dissolve 1 g in 10 ml of water R and add 5 ml of dilute acetic acid R. A white, crystalline precipitate
is formed. Filter, wash the precipitate with water R and dry at 100C to 105C. Determine the melting point (2.2.14) of the precipitate. Mix equal parts of the precipitate and pentobarbital CRS and
determine the melting point of the mixture. The difference between the melting points (which are
about 131C) is not greater than 2C.
Test solution. Dissolve 25 mg of the precipitate obtained in identification test A in alcohol R and dilute
Reference solution. Dissolve 25 mg of pentobarbital CRS in alcohol R and dilute to 25 ml with the same
solvent.
layer from a mixture of 5 volumes of concentrated ammonia R, 15 volumes of alcohol R and 80 volumes
of chloroform R. Examine immediately in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the
C. To about 10 mg add about 10 mg of vanillin R and 2 ml of sulphuric acid R. Mix and heat on a
water-bath for 2 min. A reddish-brown colour develops. Cool and add cautiously 5 ml of ethanol R.
The colour becomes violet and then blue.
D. Ignite 1 g. The residue gives reaction (a) of sodium (2.3.1).
TESTS
pH (2.2.3). Dissolve 1.0 g in carbon dioxide-free water R and dilute to 10.0 ml with the same solvent.
The pH measured immediately after preparation of the solution is 9.6 to 11.0.
coating substance.
same solvent.
30-26
of chloroform R. Examine immediately in ultraviolet light at 254 nm. Any spot in the chromatogram
chromatogram obtained with the reference solution (0.5 per cent). Spray with diphenylcarbazone
examine immediately in daylight. Any spot in the chromatogram obtained with the test solution,
apart from the principal spot, is not more intense than the spot in the chromatogram obtained with
the reference solution (0.5 per cent).
Free pentobarbital Not more than 3.5 per cent. Dissolve 2.00 g in 75 ml of dimethylformamide R,
heating gently if necessary. Add 0.25 ml of a 10 g/l solution of thymol blue R in dimethylformamide R.
Titrate with 0.1M sodium methoxide until the colour changes from olive-green to blue. Carry out a
blank titration.
1 ml of 0.1M sodium methoxide is equivalent to 22.63 mg of pentobarbital.
Isomer Dissolve 0.3 g in 5 ml of a 50 g/l solution of anhydrous sodium carbonate R. Add a solution of
0.3 g of nitrobenzyl chloride R in 10 ml of alcohol R and heat under a reflux condenser for 30 min.
Cool to 25C, if necessary scratching the wall of the container with a glass rod to induce crystallisation. Filter and wash the precipitate with five quantities, each of 5 ml, of water R. In a small flask,
heat the precipitate with 25 ml of alcohol R under a reflux condenser until dissolved (about 10 min).
Cool to 25C, if necessary scratching the wall of the flask with a glass rod to induce crystallisation,
and filter. The precipitate, washed with two quantities, each of 5 ml, of water R and dried at 100C
to 105C for 30 min, melts (2.2.14) at 136C to 148C.
Heavy metals (2.4.8). Dissolve 1.0 g in water R and dilute to 10.0 ml with the same solvent. To
9 ml of the solution, add 3 ml of dilute acetic acid R and 3 ml of buffer solution pH 3.5 R and filter.
Dilute the filtrate to 18 ml with water R. 12 ml of the solution complies with limit test A for heavy
metals (20 ppm). In preparing the test solution, replace the buffer solution with water R. Prepare the
100C to 105C.
ASSAY
Dissolve 0.200 g in 15 ml of a 127.5 g/l solution of silver nitrate R in pyridine R. Titrate with 0.1M
ethanolic sodium hydroxide until a pure blue colour is obtained, using 0.5 ml of thymolphthalein solution R as indicator. Carry out a blank titration.
1 ml of 0.1M ethanolic sodium hydroxide is equivalent to 24.83 mg of C11H17N2NaO3.
STORAGE
_______________________________________________________________________________________________________________________ Ph Eur
30-27
Pentoxifylline / Oxpentifylline
H3C
N
O
Me
Me
C13H18N4O3
278.3
6493-05-6
Pentoxifylline complies with the requirements of the 3rd edition of the European Pharmacopoeia [0851].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Pentoxifylline contains not less than 99.0 per cent and not more than the equivalent of 101.0 per
cent of 3,7-dimethyl-1-(5-oxohexyl)-3,7-dihydro-1H-purine-2,6-dione, calculated with reference to
CHARACTERS
A white or almost white, crystalline powder, soluble in water, freely soluble in methylene chloride,
sparingly soluble in alcohol, very slightly soluble in ether.
IDENTIFICATION
obtained with pentoxifylline CRS. Examine the substances prepared as discs.
D. It gives the reaction of xanthines (2.3.1).
TESTS
(2.2.1) and not more intensely coloured than reference solution Y7 (Method II, 2.2.2).
Acidity To 8 ml of solution S add 12 ml of water R and 0.05 ml of bromothymol blue solution R1. The
solution is green or yellow. Not more than 0.2 ml of 0.01M sodium hydroxide is required to change the
coating substance.
Test solution (b). Dilute l ml of test solution (a) to 10 ml with methanol R.
Reference solution (a). Dissolve 20 mg of pentoxifylline CRS in methanol R and dilute to 10 ml with the
same solvent.
Reference solution (c). Dissolve 20 mg of pentoxifylline CRS and 20 mg of theophylline CRS in
30-28
15 volumes of methanol R and 85 volumes of ethyl acetate R. Allow the plate to dry in air and examine
in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with test solution (a), apart
from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (b) (0.2 per cent). The test is not valid unless the chromatogram obtained with reference solution (c) shows two clearly separated principal spots.
Chlorides (2.4.4). Place 20 ml of solution S in a separating funnel and shake with two quantities,
each of 20 ml, of 2-methylpropan-1-ol R. Dilute 10 ml of the aqueous layer to 15 ml with water R. The
solution complies with the limit test for chlorides (100 ppm).
diphosphorus pentoxide R at 60C at a pressure not exceeding 700 Pa.
ASSAY
Dissolve 0.200 g in 5 ml of anhydrous acetic acid R. Add 20 ml of acetic anhydride R. Titrate with 0.1M
perchloric acid determining the end-point potentiometrically (2.2.20).
STORAGE
IMPURITIES
A. theobromine.
_______________________________________________________________________________________________________________________ Ph Eur
30-29
Peppermint Leaf
Peppermint Leaf complies with the requirements of the 3rd edition of the European Pharmacopoeia [0406].
Action and use Carminative.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Peppermint leaf consists of the whole or cut dried leaves of Mentha piperita L. The whole drug
contains not less than 12 ml/kg of essential oil. The cut drug contains not less than 9 ml/kg of
essential oil.
CHARACTERS
Peppermint leaf has a characteristic and penetrating odour and a characteristic aromatic taste; it is
green to brownish-green, with brownish-violet veins in some varieties. The petioles are green to
brownish-violet.
IDENTIFICATION
A. The leaf is entire, broken or cut, thin, fragile; the entire leaf is 3 cm to 9 cm long and 1 cm to
3 cm wide and often crumpled. The lamina is oval or lanceolate, the apex acuminate, the margin
sharply dentate and the base asymmetrical. Venation is pinnate, prominent on the lower surface, with
lateral veins leaving the midrib at about 45. The lower surface is slightly pubescent and secretory
trichomes are visible under a lens (6) as bright yellowish points. The petiole is grooved, usually up
to 1 mm in diameter and 0.5 cm to 1 cm long.
B. Reduce to a powder (355). The powder is brownish-green. Examine under a microscope using
chloral hydrate solution R. The powder shows the following diagnostic characteristics: leaf-tissue
fragments with cells of the epidermis having sinuous-wavy walls and the cuticle striated over the veins
and diacytic stomata predominantly present on the lower epidermis; epidermis fragments from near
the leaf margin with isodiametric cells straighter-walled showing distinct beading and pitting in
anticlinal walls; covering trichomes short, conical, unicellular or bicellular, or elongated, uniseriate
with three to eight cells with striated cuticle; glandular trichomes of two types: (a) unicellular base
with small, rounded unicellular head 15 m to 25 m in diameter; (b) unicellular base with enlarged
oval head 55 m to 70 m in diameter composed of eight radiating cells; dorsiventral mesophyll
fragment with a single palisade layer and four to six layers of spongy parenchyma; yellowish crystals
of menthol under the cuticle of secretory cells. Calcium oxalate crystals are absent.
Test solution. To 0.2 g of the recently powdered drug add 2 ml of methylene chloride R, shake for a few
minutes and filter. Evaporate the filtrate to dryness at about 40C and dissolve the residue in 0.1 ml
of toluene R.
Reference solution. Dissolve 50 mg of menthol R, 20 l of cineole R, 10 mg of thymol R and 10 l of
menthyl acetate R in toluene R and dilute to 10 ml with the same solvent.
Apply separately to the plate as bands 10 l of the reference solution and 20 l of the test solution.
Develop over a path of 15 cm using a mixture of 5 volumes of ethyl acetate R and 95 volumes of
toluene R. Allow the plate to dry in air until the odour of the solvent is no longer perceptible and
examine in ultraviolet light at 254 nm. The chromatogram obtained with the test solution may show
a light quenching zone situated just below the level of the zone (thymol) in the chromatogram
obtained with the reference solution (carvone, pulegone). Spray with anisaldehyde solution R and
examine in daylight while heating for 5 min to 10 min at 100C to 105C. The chromatogram
obtained with the reference solution shows, in order of increasing Rf value: in the lower third a deepblue to violet zone (menthol); a violet-blue to brown zone (cineole); a pink zone (thymol); and a
bluish-violet zone (menthyl acetate). The chromatogram obtained with the test solution shows: a
zone due to menthol (the most intense); a faint zone due to cineole; at Rf values between those of the
cineole and thymol zones in the chromatogram obtained with the reference solution, it may show
light pink, or bluish-grey or greyish-green zones (carvone, pulegone, isomenthone); in the middle of
the chromatogram, a bluish-violet zone (menthyl acetate) and just below it a greenish-blue zone
(menthone); an intense reddish-violet zone (hydrocarbons) appears near the solvent front; other less
intensely coloured zones also appear.
TESTS
Foreign matter (2.8.2). Carry out the determinations using 10 g of the drug.
Foreign organs. Not more than 5 per cent of stems; the diameter of the stems is not greater than
1.5 mm.
30-30
Foreign elements. Not more than 2 per cent. Not more than 8 per cent of the leaves show brown stains
due to Puccinia menthae.
ASSAY
Carry out the determination of essential oils in vegetable drugs (2.8.12). Use 20.0 g of crushed drug,
a 500 ml flask, 200 ml of water R as the distillation liquid and 0.50 ml of xylene R in the graduated
tube. Distil at a rate of 3 ml to 4 ml per minute for 2 h.
STORAGE
_______________________________________________________________________________________________________________________ Ph Eur
30-31
Peppermint Oil
Peppermint Oil complies with the requirements of the 3rd edition of the European Pharmacopoeia [0405].
Action and use Flavour; used in treatment of irritable bowel syndrome.
Preparations
Gastro-resistant Peppermint Oil Capsules
Concentrated Peppermint Emulsion
Peppermint Spirit
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Peppermint oil is obtained by steam distillation from the fresh overground parts of the flowering plant
of Mentha piperita L.
CHARACTERS
A colourless, pale yellow or pale greenish-yellow liquid with a characteristic odour and taste followed
by a sensation of cold, miscible with alcohol, with ether and with methylene chloride.
IDENTIFICATION
A. Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable silica gel
Test solution. Dissolve 0.1 g of the substance to be examined in toluene R and dilute to 10 ml with the
same solvent.
Reference solution. Dissolve 10 mg of thymol R, 10 l of menthyl acetate R, 20 l of cineole R and 50 mg
of menthol R in toluene R and dilute to 10 ml with the same solvent.
Apply separately to the plate as bands 10 l of the reference solution and 20 l of the test solution.
Develop over a path of 15 cm using a mixture of 5 volumes of ethyl acetate R and 95 volumes of
toluene R. Allow the plate to dry in air until the odour of the solvent is no longer perceptible and
examine in ultraviolet light at 254 nm. The chromatogram obtained with the test solution may show
quenching zones (carvone, pulegone) situated just below the level of the zone (thymol) in the chromatogram obtained with the reference solution. Spray with anisaldehyde solution R and examine in
daylight for 5 min to 10 min while heating at 100C to 105C. The chromatogram obtained with the
reference solution shows, in order of increasing Rf value: an intense blue to violet zone (menthol) in
the lower third; a violet-blue to brown zone (cineole); a pink zone (thymol); and a violet-blue zone
(menthyl acetate). In the chromatogram obtained with the test solution: there is a zone due to
menthol (the most intense) and a faint zone due to cineole; at Rf values between those of the cineole
and thymol zones in the chromatogram obtained with the reference solution, there may be light pink
or greyish-blue or greenish-grey zones (carvone, pulegone, isomenthone); in the middle of the chromatogram, there is a violet-blue zone (menthyl acetate) and just below it a greenish-blue zone
(menthone); an intense violet-red zone (hydrocarbons) appears near the solvent front and below it a
brownish-yellow zone (menthofuran); other less intensely coloured zones also appear.
B. Examine the chromatograms obtained in the test for chromatographic profile. The retention time
of the principal peaks in the chromatogram obtained with the test solution is similar to that of the
principal peaks in the chromatogram obtained with the reference solution. Carvone and pulegone
may be absent from the chromatogram obtained with the test solution.
TESTS
mixture of the solvents.
Optical rotation (2.2.7). The angle of optical rotation is 10 to 30.
Fatty oils and resinified essential oils (2.8.7). It complies with the test for fatty oils and resinified
essential oils.
Reference solution. Dissolve 0.1 g of limonene R, 0.2 g of cineole R, 0.4 g of menthone R, 0.1 g of
30-32
menthofuran R, 0.1 g of isomenthone R, 0.4 g of menthyl acetate R, 0.6 g of menthol R, 0.2 g of
pulegone R and 0.1 g of carvone R in 1 ml of hexane R.
a fused-silica capillary column 60 m long and about 0.25 mm in internal diameter coated with
macrogol 20,000 R as the bonded phase,
of 2C per minute to 180C and maintaining at 180C for 5 min and maintaining the temperature of
the injection port and of the detector at 220C.
prescribed conditions, the components elute in the order indicated in the composition of the reference solution. Record the retention times of these substances.
The test is not valid unless: the number of theoretical plates calculated from the limonene peak at
110C is at least 30,000; the resolution between the peaks corresponding to limonene and cineole is
at least 1.5.
Inject about 0.2 l of the test solution. Using the retention times determined from the chromatogram obtained with the reference solution, locate the components of the reference solution on the
chromatogram obtained with the test solution (disregard the peak due to hexane).
Determine the percentage content of the components by the normalisation procedure.
Limonene
1.0 to 5.0 per cent
Cineole
Menthone
Menthofuran
1.0 to 9.0 per cent
Isomenthone
Menthyl acetate
Menthol
Pulegone
Carvone
The ratio of cineole content to limonene content is greater than two.
STORAGE
The type chromatogram is given for information and guidance in application of the analytical method. It is
not part of the requirements of the monograph.
Fig. 405-1 Type chromatogram for peppermint oil

1. Limonene
4. Menthofuran
7. Menthol
2. Cineole
5. Isomenthone
8. Pulegone
3. Menthone 6. Menthyl acetate
9.Carvone
_______________________________________________________________________________________________________________________ Ph Eur
30-33
Pepsin
1/01
Pepsin complies with the requirements of the 3rd edition of the European Pharmacopoeia for Pepsin Powder
Action and use Proteolytic enzyme.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Pepsin powder is prepared from the gastric mucosa of pigs, cattle or sheep. It contains gastric
proteinases, active in acid medium (pH 1 to 5). It has an activity not less than 0.5 Ph. Eur. U. per
milligram, calculated with reference to the dried substance.
PRODUCTION
The animals from which pepsin powder is derived must fulfil the requirements for the health of
animals suitable for human consumption to the satisfaction of the competent authority.
It must have been shown to what extent the method of production allows inactivation or removal
of any contamination by viruses or other infectious agents.
CHARACTERS
A white or slightly yellow, crystalline or amorphous powder, hygroscopic, soluble in water, practically
insoluble in alcohol. The solution in water may be slightly opalescent with a weak acidic reaction.
IDENTIFICATION
In a mortar, pound 30 mg of fibrin blue R. Suspend in 20 ml of dilute hydrochloric acid R2. Filter the
suspension on a filter paper and wash with dilute hydrochloric acid R2 until a colourless filtrate is
obtained. Perforate the filter paper and wash the fibrin blue R through it into a conical flask using
20 ml of dilute hydrochloric acid R2. Shake before use. Dissolve a quantity of the substance to be
examined, equivalent to not less than 20 Ph. Eur. U., in 2 ml of dilute hydrochloric acid R2 and adjust
to pH 1.60.1. Add 1 ml of this solution to a test-tube containing 4 ml of the fibrin blue suspension,
mix and place in a water-bath at 25C with gentle shaking. Prepare a blank solution at the same time
and in the same manner using 1 ml of water R. After 15 min of incubation the blank solution is
colourless and the test solution is blue.
TESTS
diphosphorus pentoxide R at a pressure not exceeding 670 Pa for 4 h.
Microbial contamination. Total viable aerobic count (2.6.12) not more than 104 micro-organisms
per gram, determined by plate-count. It complies with the tests for Escherichia coli and Salmonella
(2.6.13).
ASSAY
The activity of pepsin powder is determined by comparing the quantity of peptides, non-precipitable
by trichloroacetic acid solution R and assayed using the phosphomolybdotungstic reagent R, which are
released per minute from a substrate of haemoglobin solution R, with the quantity of such peptides
released by pepsin powder BRP from the same substrate in the same conditions.
Avoid shaking and foaming during preparation of the test and reference solutions.
Test solution. Immediately before use, prepare a solution of the substance to be examined expected to
contain 0.5 Ph. Eur. U. per millilitre in dilute hydrochloric acid R2; before dilution to volume, adjust to
pH 1.6 0.1, if necessary, using 1M hydrochloric acid.
Reference solution. less than 15 min before use, prepare a solution of pepsin powder BRP containing 0.5
Ph. Eur. U. per millilitre in dilute hydrochloric acid R2; before dilution to volume, adjust to pH
1.6 0.1, if necessary, using 1M hydrochloric acid.
Designate tubes in duplicate T, Tb, S1, S1b, S2, S2b, S3, S3b; designate a tube B.
Add dilute hydrochloric acid R2 to the tubes as follows:
B: 1.0 ml
S1 and S1b: 0.5 ml
S2, S2b and T and Tb: 0.25 ml
Add the reference solution to the tubes as follows:
S1 and S1b: 0.5 ml
30-34
S2 and S2b: 0.75 ml
S3 and S3b: 1.0 ml
Add 0.75 ml of the test solution to tubes T and Tb.
Add 10.0 ml of trichloroacetic acid solution R to tubes S1b, S2b, S3b, Tb and B. Mix by shaking.
Place the tubes and haemoglobin solution R in a water bath at 25 0.1C. When temperature
equilibrium is reached, add 5.0 ml of haemoglobin solution R to tubes B, S1b, S2b, S3b and Tb. Mix.
At time zero add 5.0 ml of haemoglobin solution R successively and at intervals of 30 s to tubes S1,
S2, S3 and T.
Mix immediately after each addition.
Exactly 10 min after adding the haemoglobin solution R, stop the reaction by adding, at intervals of
30 s, 10.0 ml of trichloroacetic acid solution R to tubes S1, S2, S3 and T (the use of a fast-flowing or
blow-out pipette is recommended) and mix.
Filter the contents of each tube (samples and blanks) twice through the same suitable filter paper
previously washed with a 50 g/l solution of trichloroacetic acid R, then with water R and dried. Discard
the first 5 ml of filtrate. Place 3.0 ml of each filtrate separately in a tube containing 20 ml of water R.
Mix.
A suitable filter paper complies with the following test: filter 5 ml of a 50 g/l solution of
trichloroacetic acid R through a 7 cm disc of white filter paper: the absorbance (2.2.25) of the
filtrate, measured at 275 nm using unfiltered trichloroacetic acid R solution as the compensation
liquid, is less than 0.04.
Add to each tube 1.0 ml of sodium hydroxide solution R and 1.0 ml of phosphomolybdotungstic reagent R,
beginning with the blanks and then the samples of each set, in a known order.
Table 06821
Tubes
S1
S1b
S2
S2b
Dilute hydrochloric acid R2 (ml)
0.5
0.5
0.25
0.25
Reference solution (ml)
0.5
0.5
0.75
0.75
S3
1.0
S3b
Trichloroacetic acid solution R (ml)

Mix
Water bath at 25C
+
+
Haemoglobin solution R (ml)

Mix
Haemoglobin solution R (ml)
Mix
Water bath at 25C
Trichloroacetic acid solution R (ml)
10.0
0.25
0.25
1.0
0.75
0.75
10.0
+
+
Tb
1.0
Test solution (ml)

10.0
+
+
5.0
5.0
5.0
5.0
5.0
10.0
+
5.0
5.0
Filter
Mix
10.0
5.0
5.0
10.0
10.0
+
+
10.0
+
+
+
10.0
+
A schematic presentation of the above operations is shown in the Table 0682.-1.

After 15 min measure the absorbance (2.2.25) of solutions S1, S2, S3, S1b, S2b, S3b and T at
540 nm using the filtrate obtained from tube B as the compensation liquid. Correct the average
absorbance values for the filtrates obtained from tubes S1, S2 and S3 by subtracting the average values
obtained for the filtrates from tubes S1b, S2b, S3b respectively.
Draw a calibration curve of the corrected values against volume of reference solution used. Determine the activity of the substance to be examined using the corrected absorbance for the test solution
(T Tb) together with the calibration curve and taking into account the dilution factors.
STORAGE
LABELLING
The label states the activity in European Pharmacopoeia Units per milligram.
_______________________________________________________________________________________________________________________ Ph Eur
30-35
Pergolide Mesilate
1/01
H
S
N
Me
,H3C
SO3H
N
H
C20H30N2O3S2
Pr n
410.6
66104-23-2
Pergolide Mesilate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1555].
Action and use Dopamine receptor agonist.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Pergolide mesilate contains not less than 97.5 per cent and not more than 102.0 per cent of
(6aR,9R,10aR)-9-[(methylsulphanyl)methyl]-7-propyl-4,6,6a,7,8,9,10,10a-octahydroindolo[4,3fg]quinoline monomethanesulphonate, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, slightly soluble in water, sparingly soluble in methanol,
slightly soluble in alcohol and in methylene chloride, very slightly soluble in acetone.
IDENTIFICATION
A. The specific optical rotation (2.2.7) is 17 to 23, calculated with reference to the dried
substance and determined on a solution prepared as follows: dissolve 0.25 g in dimethylformamide R
obtained with pergolide mesilate CRS. Examine the substances prepared as discs.
TESTS
Reference solution (a). Dilute 1.0 ml of the test solution to 100.0 ml with methanol R. Dilute 1.0 ml of
Reference solution (b). Dissolve 10 mg of 4,4-dimethoxybenzophenone R in methanol R and dilute to
10 ml with the same solvent. To 1 ml of this solution add 2 ml of the test solution and dilute to
100 ml with methanol R. Dilute 1 ml of this solution to 10 ml with methanol R.
Mobile phase A. Mix 5.0 ml of morpholine for chromatography R with 995 ml of water R and adjust the
pH to 7.0 with phosphoric acid R. Use within 24 h.
Mobile phase B. Mix equal volumes of acetonitrile R, methanol R and tetrahydrofuran R,
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
0 35
70 0
30 100
35 40
0 70
100 30
Linear gradient
Return to initial
conditions
40 50
70
30
Re-equilibration
30-36
obtained with 20 l of reference solution (a) is at least 90 per cent of the full scale of the recorder.
Inject 20 l of reference solution (b). The test is not valid unless, in the chromatogram obtained,
the resolution between the peaks corresponding to 4,4-dimethoxybenzophenone (first peak) and
pergolide (second peak) is at least 2.0.
Inject 20 l of the test solution and 20 l of reference solution (a).
solution (a) (0.1 per cent); the sum of the areas of all the peaks, apart from the principal peak, is not
greater than 5 times the area of the principal peak in the chromatogram obtained with reference
solution (a) (0.5 per cent). Disregard any peak with an area less than 0.2 times that of the principal
peak in the chromatogram obtained with reference solution (a) (0.02 per cent).
100-105C for 1 h.
ASSAY
Solution A. Dissolve 5.0 mg of DL-methionine R in 500 ml of 0.01M hydrochloric acid. Add 500 ml of
methanol R and mix.
Test solution. Dissolve 65.0 mg of the substance to be examined in solution A and dilute to 100.0 ml
with the same solution. Dilute 10.0 ml to 100.0 ml with solution A.
Reference solution. Dissolve 65.0 mg of pergolide mesilate CRS in solution A and dilute to 100.0 ml with
the same solution. Dilute 10.0 ml to 100.0 ml with solution A.
a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with basedeactivated octylsilyl silica gel for chromatography R (5 m),
as mobile phase at a flow rate of 1 ml/min a mixture of 1 volume of acetonitrile R, 1 volume of
methanol R and 2 volumes of a mixture prepared as follows: dissolve 2.0 g of sodium
octanesulphonate R in water R, add 1.0 ml of anhydrous acetic acid R and dilute to 1000 ml with
water R,
Inject 20 l of the reference solution. When the chromatogram is recorded in the prescribed
conditions, the retention time of pergolide is about 9 min. Adjust the sensitivity of the system so that
the height of the principal peak in the chromatogram obtained with the reference solution is at least
50 per cent of the full scale of the recorder.
The assay is not valid unless the symmetry factor of the peak due to pergolide is at most 1.5.
Inject 20 l of the test solution.
Calculate the percentage content of C20H30N2O3S2 from the areas of the peaks and the declared
content of pergolide mesilate CRS.
STORAGE
IMPURITIES
H
Prn
N
R
H
Qualified impurities: A.
Other detectable impurities: B.
A. R = SO-CH3: (6aR,9R,10aR)-9-[(methylsulphinyl)methyl]-7-propyl-4,6,6a,7,8,9,10,10aoctahydroindolo[4,3-fg]quinoline (pergolide sulphoxide),
B. R = SO2-CH3: (6aR,9R,10aR)-9-[(methylsulphonyl)methyl]-7-propyl-4,6,6a,7,8,9,10,10aoctahydroindolo[4,3-fg]quinoline (pergolide sulphone).
__________________________________________________________________________________________________________ Ph Eur
30-37
Perphenazine
N
OH
Cl
C21H26ClN3OS
404.0
58-39-9
Perphenazine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0629].
Action and use Antipsychotic; antiemetic.
Preparation
Perphenazine Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Perphenazine contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of 2-[4-[3-(2-chlorophenothiazin-10-yl)propyl]piperazin-1-yl]ethanol, calculated with reference to
CHARACTERS
A white or yellowish-white, crystalline powder, practically insoluble in water, freely soluble in
methylene chloride, soluble in alcohol, sparingly soluble in ether. It dissolves in dilute solutions of
hydrochloric acid.
IDENTIFICATION
First identification: A, C
B. Dissolve 10 mg in methanol R and dilute to 100 ml with the same solvent. Dilute 10 ml of the
solution to 100 ml with methanol R. Examined between 230 nm and 350 nm (2.2.25), the solution
shows two absorption maxima, at 257 nm and 313 nm. The ratio of the absorbance measured at the
obtained with perphenazine CRS. Examine the substances prepared as discs.
D. Examine by thin-layer chromatography (2.2.27), using kieselguhr G R as the coating substance.
Impregnate the plate by placing it in a closed tank containing the necessary quantity of the impregnation mixture containing 2.5 per cent V/V of phenoxyethanol R and 7.5 per cent V/V of formamide R in
acetone R so that the plate dips about 5 mm beneath the surface of the liquid. When the impregnation
mixture has risen at least 17 cm from the lower edge of the plate, remove the plate and use immediately for chromatography. Carry out the chromatography in the same direction as the impregnation.
the same solvent.
Reference solution. Dissolve 20 mg of perphenazine CRS in chloroform R and dilute to 10 ml with the
same solvent.
Apply separately to the plate 2 l of each solution. Develop in the dark over a path of 15 cm using a
mixture of 2 volumes of diethylamine R and 100 volumes of light petroleum R, saturated with
phenoxyethanol (add phenoxyethanol R 6 volumes to 8 volumes to the above mixture of solvents
until there is a persistent cloudiness after shaking, decant, and use the supernatant liquid, even if it is
cloudy). Expose the plate to ultraviolet light at 365 nm and examine after a few minutes. The principal spot in the chromatogram obtained with the test solution is similar in position, fluorescence and
size to the principal spot in the chromatogram obtained with the reference solution. Dry the plate at
120C for 20 min, allow to cool and spray with a 10 per cent V/V solution of sulphuric acid R in
alcohol R. The principal spot in the chromatogram obtained with the test solution is of the same
colour as the principal spot in the chromatogram obtained with the reference solution.
30-38
TESTS
Appearance of solution Dissolve 0.20 g in 10 ml of methanol R. The solution is clear (2.2.1).
coating substance. Prepare the solutions immediately before use.
the same solvent.
1 volume of concentrated ammonia R, 14 volumes of water R and 85 volumes of butanol R. Allow the
plate to dry in air. Examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained
with the test solution, apart from the principal spot, is not more intense than the spot in the chromatogram obtained with the reference solution (0.5 per cent).
65C for 4 h.
ASSAY
Dissolve 0.1500 g in 25 ml of glacial acetic acid R. Titrate with 0.1M perchloric acid determining the
1 ml of 0.1M perchloric acid is equivalent to 20.20 mg of C21H26ClN3OS.
STORAGE
_______________________________________________________________________________________________________________________ Ph Eur
30-39
Peru Balsam
Peru Balsam complies with the requirements of the 3rd edition of the European Pharmacopoeia [0754]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Peru balsam is the balsam obtained from the scorched and wounded trunk of Myroxylon balsamum (L.)
Harms var. pereirae (Royle) Harms. It contains not less than 45.0 per cent m/m and not more than
70.0 per cent m/m of esters, mainly benzyl benzoate and benzyl cinnamate.
CHARACTERS
A dark brown, viscous liquid which is transparent and yellowish-brown when viewed in a thin layer;
the liquid is not sticky, it is non-drying and does not form threads; practically insoluble in water, freely
soluble in ethanol, not miscible with fatty oils, except for castor oil.
IDENTIFICATION
A. Dissolve 0.20 g in 10 ml of alcohol R. Add 0.2 ml of ferric chloride solution R1. A green to olive-green
colour develops.
Test solution. Dissolve 0.5 g of the substance to be examined in 10 ml of ethyl acetate R.
Reference solution. Dissolve 4 mg of thymol R, 30 mg of benzyl cinnamate R and 80 l of benzyl
benzoate R in 5 ml of ethyl acetate R.
Apply separately to the plate as bands 20 mm by 3 mm 10 l of each solution. Develop twice over a
path of 10 cm using a mixture of 0.5 volumes of glacial acetic acid R, 10 volumes of ethyl acetate R and
90 volumes of hexane R. Allow the plate to dry in air, examine in ultraviolet light at 254 nm and mark
the quenching zones. The chromatogram obtained with the reference solution shows in the upper third
two quenching zones, the higher one corresponding to benzyl benzoate and the lower one to benzyl
cinnamate. The chromatogram obtained with the test solution shows two quenching zones at the same
levels and of approximately the same size. Spray the plate with a freshly prepared 200 g/l solution of
phosphomolybdic acid R in alcohol R, using 10 ml for a plate 200 mm square and examine the plate in
daylight while heating at 100C to 105C for 5 min to 10 min. The zones due to benzyl benzoate and
benzyl cinnamate are coloured blue against a yellow background. The chromatogram obtained with
the reference solution shows at about the middle a violet-grey zone (thymol). In the chromatogram
obtained with the test solution, a blue zone (nerolidol) is seen just below the level of the zone due to
thymol in the chromatogram obtained with the reference solution. Just below the zone due to
nerolidol, no blue zone is seen corresponding to a quenching zone seen when examined in ultraviolet
light at 254 nm (colophony). In the upper and lower part of the chromatogram obtained with the test
solution, other faint blue zones may be seen.
TESTS
Saponification value (2.5.6). 230 to 255, determined on the residue obtained in the assay.
Artificial balsams Shake 0.20 g with 6 ml of light petroleum R1. The light petroleum solution is clear
and colourless and the whole of the insoluble parts of the balsam stick to the wall of the test-tube.
Fatty oils Shake 1 g with 3 ml of a 1000 g/l solution of chloral hydrate R. The resulting solution is as
clear as the 1000 g/l solution of chloral hydrate R.
Turpentine Evaporate to dryness 4 ml of the solution obtained in the test for artificial balsams. The
residue has no odour of turpentine.
ASSAY
To 2.50 g in a separating funnel add 7.5 ml of dilute sodium hydroxide solution R and 40 ml of peroxidefree ether R and shake vigorously for 10 min. Separate the lower layer and shake it with three quantities,
each of 15 ml, of peroxide-free ether R. Combine the ether layers, dry over 10 g of anhydrous sodium
sulphate R and filter. Wash the sodium sulphate with two quantities, each of 10 ml, of peroxide-free
ether R. Combine the ether layers and evaporate to dryness. Dry the residue (esters) at 100C to 105C
for 30 min and weigh.
STORAGE
_______________________________________________________________________________________________________________________ Ph Eur
30-40
Pethidine Hydrochloride
1/01
NMe
COOEt
C15H22ClNO2
283.8
50-13-5
Pethidine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Opoid analgesic
Preparations
Pethidine Injection
Pethidine Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ethyl 1-methyl-4-phenylpiperidine-4-carboxylate hydrochloride.
CHARACTERS
Appearance: white, crystalline powder.
Solubility: very soluble in water, freely soluble in alcohol.
PRODUCTION
If intended for use in the manufacture of parenteral dosage forms, the manufacturing process is
validated to show that the content of impurity B is not more than 0.1 ppm.
IDENTIFICATION
B. Infrared absorption spectrophotometry (2.2.24).
Comparison: Ph. Eur. reference spectrum of pethidine hydrochloride.
C. Dissolve 0.1 g in 10 ml of ethanol R and add 10 ml of picric acid solution R. A crystalline precipitate
is formed which, when washed with water R and dried at 100-105C, melts (2.2.14) at 186C to
193C. Mix equal quantities of the precipitate and the substance to be examined and determine the
melting point of the mixture. The melting point is at least 20C lower than that of the precipitate.
D. To 5 ml of Solution S (see Tests) add 5 ml of water R. The solution gives reaction (a) of chlorides
(2.3.1).
TESTS
Appearance of solution Solution S is clear (2.2.1) and colourless (2.2.2, Method II).
Acidity or alkalinity To 10 ml of solution S add 0.2 ml of methyl red solution R and 0.2 ml of 0.01M
sodium hydroxide. The solution is yellow. Add 0.3 ml of 0.01M hydrochloric acid. The solution is red.
Impurity B: if intended for non-parenteral use, maximum 10 ppm.
Liquid chromatography (2.2.29).
Test solution (a). Dissolve 0.100 g of the substance to be examined in a mixture of 20 volumes of
Test solution (b). Dissolve 0.125 g of the substance to be examined in a mixture of 20 volumes of
Reference solution (a). Dilute 0.5 ml of test solution (a) to 100.0 ml with a mixture of 20 volumes of
acetonitrile R and 80 volumes of water R.
Reference solution (b). Dissolve 10.0 mg of pethidine impurity A CRS in a mixture of 20 volumes of
Reference solution (c). Dissolve 12.5 mg of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine R in a mixture
30-41
of 20 volumes of acetonitrile R and 80 volumes of water R and dilute to 10.0 ml with the same mixture
of solvents. Dilute 1.0 ml of the solution to 100.0 ml with a mixture of 20 volumes of acetonitrile R
and 80 volumes of water R.
Reference solution (d). Dilute 5.0 ml of reference solution (b) and 1.0 ml of reference solution (c) to
100.0 ml with a mixture of 20 volumes of acetonitrile R and 80 volumes of water R.
Column:
size: l = 0.25 m, = 4.0 mm,
stationary phase: spherical end-capped octadecylsilyl silica gel for chromatography R (5 m) with a
specific surface area of 340 m2/g, a pore size of 10 nm and a carbon loading of 19 per cent.
Mobile phase:
mobile phase A: mix equal volumes of a 42.0 g/l solution of sodium perchlorate R and of a 11.6 g/l
solution of phosphoric acid R. Adjust to pH 2.0 with triethylamine R;
mobile phase B: acetonitrile R.
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
0 15
80 75
75 55
55
20 25
25 45
45
55 80
80
45 20
20
15 31
31 40
40 41
41 50
Flow rate: 1.0 ml/min

Injection: 50 l; inject test solution (b) and reference solution (d).
Relative retention with reference to pethidine (retention time = about 24 min): impurity B = about
0.66; impurity A = about 0.68.
System suitability: reference solution (d):
signal-to-noise ratio: minimum10 for the first peak,
peak-to-valley ratio: minimum 4, where hp = height above the baseline of the peak due to impurity B, and hv = height above the baseline of the lowest point of the curve separating this peak
from the peak due to impurity A,
Limit:
impurity B: not more than the area of the corresponding peak in the chromatogram obtained
with reference solution (d).
Related substances. Liquid chromatography (2.2.29), as described for the test for impurity B.
Injection: 20 l; inject test solution (a) and reference solution (a).
Limit:
any impurity: not more than the area of the principal peak in the chromatogram obtained with
reference solution (a) (0.5 per cent),
total: not more than twice the area of the principal peak in the chromatogram obtained with
reference solution (a) (1.0 per cent).
disregard limit: 0.1 times the area of the principal peak inthe chromatogram obtained with
Loss on drying (2.2.32): maximum 0.5 per cent, determined on 1.000 g by drying in an oven at
100-105C.
ASSAY
Dissolve 0.200 g in 30 ml of anhydrous acetic acid R. Add 5 ml of mercuric acetate solution R. Titrate
with 0.1M perchloric acid, using 0.1 ml of crystal violet solution R as indicator, until the colour changes
from violet-blue to green.
STORAGE
In an airtight container, protected from light.
LABELLING
The label states, where applicable, that the substance is suitable for use in the manufacture of
parenteral dosage forms.
30-42
IMPURITIES
NR1
R2
A. R1 = CH3, R2 = H: 1-methyl-4-phenylpiperidine (MPP),

C. R1 = CH3, R2 = CO2H: 1-methyl-4-phenylpiperidine-4-carboxylic acid,
D. R1 = CH3, R2 = CO2-CH3: methyl 1-methyl-4-phenylpiperidine-4-carboxylate,
E. R1 = H, R2 = CO2-CH2-CH3: ethyl 4-phenylpiperidine-4-carboxylate,
F. R1 = CH2-C6H5, R2 = CO2H: 1-benzyl-4-phenylpiperidine-4-carboxylic acid,
G. R1 = CH3, R2 = CO2-CH(CH3)2: 1-methylethyl 1-methyl-4-phenylpiperidine-4-carboxylate,
H. R1 = CH2-C6H5, R2 = CO2-CH2-CH3: ethyl 1-benzyl-4-phenylpiperidine-4-carboxylate,
J. R1 = CH2-CH3, R2 = CO2-CH2-CH3: ethyl 1-ethyl-4-phenylpiperidine-4-carboxylate,
NMe
B. 1-methyl-4-phenyl-1,2,3,6-tetrahydropiperidine (MPTP),
NMe
and enantiomer
COOEt
I. ethyl (4RS)-1-methyl-4-phenyl-1,2,3,4-tetrahydropiperidine-4-carboxylate.
__________________________________________________________________________________________________________ Ph Eur
30-43
Phenazone
Me
Me
N
N
Ph
O
C11H12N2O
188.2
60-80-0
Phenazone complies with the requirements of the 3rd edition of the European Pharmacopoeia [0421]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Phenazone contains not less than 99.0 per cent and not more than the equivalent of 100.5 per cent of
2,3-dimethyl-1-phenyl-3-pyrazolin-5-one, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder or colourless crystals, very soluble in water, in alcohol
and in methylene chloride, sparingly soluble in ether.
IDENTIFICATION
obtained with phenazone CRS. Examine the substances prepared as discs using potassium bromide R.
C. To 1 ml of solution S (see Tests) add 4 ml of water R and 0.25 ml of dilute sulphuric acid R. Add
1 ml of sodium nitrite solution R. A green colour develops.
D. To 1 ml of solution S add 4 ml of water R and 0.5 ml of ferric chloride solution R2. A red colour
develops which is discharged on the addition of dilute sulphuric acid R.
TESTS
Acidity or alkalinity To 10 ml of solution S add 0.1 ml of phenolphthalein solution R. The solution is
colourless. Add 0.2 ml of 0.01M sodium hydroxide. The solution is red. Add 0.25 ml of methyl red
solution R and 0.4 ml of 0.01M hydrochloric acid. The solution is red or yellowish-red.
Sulphates (2.4.13). Dissolve 1.5 g in distilled water R and dilute to 15 ml with the same solvent. The
solution complies with the limit test for sulphates (100 ppm).
60C for 6 h.
ASSAY
Dissolve 0.150 g in 20 ml of water R. Add 2 g of sodium acetate R and 25.0 ml of 0.05M iodine. Allow
to stand protected from light for 30 min. Add 25 ml of methylene chloride R and shake until the
precipitate dissolves. Titrate with 0.1M sodium thiosulphate, using 1 ml of starch solution R, added
towards the end of the titration, as indicator. Carry out a blank titration.
1 ml of 0.05M iodine is equivalent to 9.41 mg of C11H12N2O.
STORAGE
_______________________________________________________________________________________________________________________ Ph Eur
30-44
Phenelzine Sulphate
H
N
NH2 ,H2SO4
C8H12N2,H2SO4
234.3
156-51-4
Definition Phenelzine Sulphate is phenethylhydrazine hydrogen sulphate. It contains not less than
98.0% and not more than 100.5% of C8H12N2,H2SO4, calculated with reference to the dried
substance.
Characteristics A white powder or pearly platelets; odour, pungent.
Freely soluble in water; practically insoluble in chloroform, in ethanol (96%) and in ether.
Identification
sulphuric acid exhibits three well-defined maxima, at 252, 258 and 263 nm. The absorbances at the
maxima are about 0.62, about 0.77 and about 0.58, respectively.
B. Dissolve 0.1 g in 5 ml of water, make alkaline with 5M sodium hydroxide and add 1 ml of cupritartaric solution R1. A red precipitate is produced.
Assay Dissolve 0.25 g in 50 ml of water, add 1.5 g of sodium hydrogen carbonate and 50 ml of 0.05M
iodine VS, close the flask and allow to stand for 90 minutes. Add 20 ml of 2M hydrochloric acid and
titrate with 0.1M sodium thiosulphate VS using starch mucilage, added towards the end of the titration,
as indicator. Repeat the operation without the substance being examined. The difference between the
titrations represents the amount of iodine required. Each ml of 0.05M iodine VS is equivalent to
5.857 mg of C8H12N2,H2SO4.
Storage Phenelzine Sulphate should be kept in a well-closed container and protected from light.
Action and use Monoamine oxidase inhibitor.
Preparation
Phenelzine Tablets
30-45
Phenindamine Tartrate
H
H
OH
COOH
, HOOC
NMe
OH
and enantiomer
C19H19N,C4H6O6
411.5
569-59-5
Definition Phenindamine Tartrate is (RS)-2,3,4,9-tetrahydro-2-methyl-9-phenyl-1H-indeno[2,1c]pyridine hydrogen (2R,3R)-tartrate. It contains not less than 98.5% and not more than 101.0% of
C19H19N,C4H6O6, calculated with reference to the dried substance.
Characteristics A white or almost white, voluminous powder; odourless or almost odourless.
Sparingly soluble in water; slightly soluble in ethanol (96%); practically insoluble in chloroform and
in ether.
Identification
A. The light absorption, Appendix II B, in the range 230 to 300 nm of a 0.004% w/v solution exhibits
a maximum only at 259 nm. The absorbance at the maximum is about 0.88.
B. Dissolve 25 mg in 5 ml of sulphuric acid. An orange-brown colour is produced which is discharged
when the solution is carefully diluted with 20 ml of water.
C. Dissolve 0.5 g in 15 ml of hot water, add a slight excess of 5M sodium hydroxide, filter and
neutralise the filtrate to litmus paper with 2M hydrochloric acid. The solution yields reaction B
characteristic of tartrates, Appendix VI.
D. Melting point, 160 to 162, Appendix V A. When heated to 163 it resolidifies and it melts again
at about 168, with decomposition.
1 g.
Assay Dissolve 0.7 g in 40 ml of warm water, cool, add 10 ml of dilute sodium carbonate solution and
extract with successive quantities of 25, 10 and 10 ml of chloroform, washing each extract with the
same 15 ml of water and filtering into a dry flask. Titrate the combined extracts, which should be
clear and free from droplets of water, with 0.05M perchloric acid VS using oracet blue B solution as
indicator. Each ml of 0.05M perchloric acid VS is equivalent to 20.57 mg of C19H19N,C4H6O6.
Storage Phenindamine Tartrate should be kept in a well-closed container and protected from light.
30-46
Phenindione
O
O
C15H10O2
222.2
83-12-5
Definition Phenindione is 2-phenylindane-1,3-dione. It contains not less than 98.0% and not more
than 100.5% of C15H10O2, calculated with reference to the dried substance.
Characteristics Soft, white or creamy white crystals; odourless or almost odourless.
Very slightly soluble in water; slightly soluble in ethanol (96%) and in ether; freely soluble in chloroform. Solutions are yellow to red.
Identification
phenindione (RS 268).
B. Dissolve 0.1 g in 30 ml of ethanol (96%) with the aid of heat, cool and add sufficient ethanol
(96%) to produce 50 ml. Dilute 10 ml to 250 ml with 0.1M sodium hydroxide and further dilute 5 ml
to 100 ml with 0.1M sodium hydroxide. The absorbance of the resulting solution at the maximum at
278 nm is about 0.54 and at the maximum at 330 nm is about 0.16, Appendix II B.
C. To 1 g add 50 ml of ethanol (96%) and 0.5 ml of aniline, heat gently under a reflux condenser for
3 hours, cool in ice and filter. The melting point of the residue, after washing with 2 ml of ethanol
(96%) and recrystallising from chloroform, is about 225, Appendix V A.
III A, using a silica gel F254 precoated plate (Merck silica gel 60 F254 plates are suitable) and as the
mobile phase a solution containing 0.02% w/v of butylated hydroxytoluene in a mixture of 80 volumes
of toluene, 20 volumes of ethyl acetate and 4 volumes of glacial acetic acid. Allow the solvent front to
ascend 4 cm, remove the plate and dry it in a current of cold air for 1 minute. Without delay apply
separately to the plate 10 l of each of three solutions of the substance being examined in dichloromethane containing (1) 1.0% w/v, (2) 0.020% w/v and (3) 0.0050% w/v and develop immediately.
After removal of the plate, allow it to dry in a current of warm air and examine under ultraviolet light
(254 nm). Any secondary spot in the chromatogram obtained with solution (1) is not more intense
than the spot in the chromatogram obtained with solution (2) (2%) and not more than one such spot
is more intense than the spot in the chromatogram obtained with solution (3) (0.5%).
Assay To 0.3 g add 50 ml of ethanol (96%) and warm until solution is effected. Cool to room
temperature, add 10 ml of a 10% v/v solution of bromine in ethanol (96%) and allow to stand for 10
minutes, shaking occasionally. Add 1 g of 2-naphthol and shake until the colour of the bromine is
discharged. Remove any vapour of bromine in the flask with a current of air, add 50 ml of water and
10 ml of dilute potassium iodide solution and titrate the liberated iodine with 0.1M sodium thiosulphate
VS using starch mucilage as indicator. Each ml of 0.1M sodium thiosulphate VS is equivalent to
11.11 mg of C15H10O2.
Storage Phenindione should be kept in a well-closed container.
Action and use Anticoagulant.
Preparation
Phenindione Tablets
30-47
Pheniramine Maleate
H
CH2CH2NMe2
COOH
COOH
and enantiomer
C16H20N2,C4H4O4
356.5
132-20-7
Pheniramine Maleate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Pheniramine maleate contains not less than 98.0 per cent and not more than the equivalent of
102.0 per cent of (3RS)-N,N-dimethyl-3-phenyl-3-(pyridin-2-yl)propan-1-amine (Z)-butenedioate,
CHARACTERS
A white, crystalline powder, very soluble in water, freely soluble in alcohol, in methanol and in
methylene chloride.
IDENTIFICATION
320 nm (2.2.25), the solution shows a shoulder at 261 nm and an absorption maximum at 265 nm.
obtained with pheniramine maleate CRS. Examine the substances prepared as discs.
D. Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable silica gel
Test solution. Dissolve 0.10 g of the substance to be examined in methanol R and dilute to 5.0 ml with
the same solvent.
Reference solution (a). Dissolve 65 mg of maleic acid R in methanol R and dilute to 10 ml with the same
solvent.
Reference solution (b). Dissolve 0.10 g of pheniramine maleate CRS in methanol R and dilute to 5.0 ml
of water R, 7 volumes of anhydrous formic acid R, 20 volumes of methanol R and 70 volumes of diisopropyl ether R. Examine in ultraviolet light at 254 nm. The chromatogram obtained with the test
solution shows two clearly separated spots. The upper spot is similar in position and size to the spot
in the chromatogram obtained with reference solution (a). The lower spot is similar in position and
size to the spot in the chromatogram obtained with reference solution (b).
TESTS
pH (2.2.3). Dissolve 0.20 g in 20.0 ml of carbon dioxide-free water R. The pH of the solution is 4.5 to
5.5.
+0.10.
Test solution. Dissolve 20.0 mg of the substance to be examined in a mixture of 1 volume of acetonitrile R and 9 volumes of mobile phase A and dilute to 20.0 ml with the same mixture of solvents.
30-48
Reference solution (a). Dissolve 10.0 mg of 2-benzylpyridine R in 10.0 ml of the test solution. Dilute to
100.0 ml with a mixture of 1 volume of acetonitrile R and 9 volumes of mobile phase A.
Reference solution (b). Dilute 2.0 ml of the test solution to 100.0 ml with a mixture of 1 volume of
acetonitrile R and 9 volumes of mobile phase A. Dilute 1.0 ml of this solution to 10.0 ml with a
mixture of 1 volume of acetonitrile R and 9 volumes of mobile phase A.
a stainless steel column 0.30 m long and 3.9 mm in internal diameter packed with dimethyloctadecylsilyl silica gel for chromatography R (10 m),
Mobile phase A. A 5.056 g/l solution of sodium heptanesulphonate R adjusted to pH 2.5 with
phosphoric acid R,
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
0
035
3537
90
9062
6290
10
1038
3810
equilibration
linear gradient
linear gradient

Inject 20 l of each solution. Adjust the sensitivity of the system so that the height of the principal
peak in the chromatogram obtained with reference solution (b) is at least 50 per cent of the full scale
of the recorder. The test is not valid unless: the chromatogram obtained with reference solution (a)
shows three principal peaks (maleic acid, 2-benzylpyridine and pheniramine in order of elution); the
resolution between the peaks corresponding to 2-benzylpyridine and pheniramine is at least 8. In the
chromatogram obtained with the test solution: the area of any peak, apart from the principal peak
and the peak due to maleic acid, is not greater than the principal peak in the chromatogram obtained
with reference solution (b) (0.2 per cent); the sum of the areas of all the peaks, apart from the
principal peak and the peak due to maleic acid, is not greater than five times the area of the principal
peak in the chromatogram obtained with reference solution (b) (1 per cent). Disregard any peak with
an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (b).
60C in vacuo for 3 h.
ASSAY
STORAGE
IMPURITIES
N
A. 2-benzylpyridine,
B. 4-benzylpyridine,
NMe2
and enantiomer
C. (3RS)-N,N-dimethyl-3-phenyl-3-(pyridin-4-yl)propan-1-amine,
30-49
Me2N
NMe2
N
D. N,N,N,N-tetramethyl-3-phenyl-3-(pyridin-2-yl)pentane-1,5-diamine.
__________________________________________________________________________________________________________ Ph Eur
30-50
Phenobarbital
O
H
N
Et
O
NH
O
C12H12N2O3
232.2
50-06-6
Phenobarbital complies with the requirements of the 3rd edition of the European Pharmacopoeia [0201].
Action and use Sedative; anticonvulsant.
Preparations
Phenobarbital Elixir
Phenobarbital Tablets
When phenobarbitone is prescribed or demanded, Phenobarbital shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Phenobarbital contains not less than 99.0 per cent and not more than the equivalent of 101.0 per
cent of 5-ethyl-5-phenyl-1H,3H,5H-pyrimidine-2,4,6-trione, calculated with reference to the dried
substance.
CHARACTERS
alcohol, soluble in ether. It forms water-soluble compounds with alkali hydroxides and carbonates
and with ammonia.
IDENTIFICATION
substance to be examined and phenobarbital CRS and determine the melting point of the mixture.
The difference between the melting points (which are about 176C) is not greater than 2C.
obtained with phenobarbital CRS.
the same solvent.
Reference solution. Dissolve 0.1 g of phenobarbital CRS in alcohol R and dilute to 100 ml with the same
solvent.
chloroform R. Examine immediately in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the
D. It gives the reaction of non-nitrogen substituted barbiturates (2.3.1).
TESTS
6 ml of water R. The solution is clear (2.2.1) and not more intensely coloured than reference solution
Y6 (Method II, 2.2.2).
coating substance.
the same solvent.
30-51
100C to 105C.
ASSAY
_______________________________________________________________________________________________________________________ Ph Eur
30-52
Phenobarbital Sodium
H
N
Et
ONa
N
O
C12H11N2NaO3
254.2
57-30-7
Phenobarbital Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparations
Phenobarbital Injection
Phenobarbital Sodium Tablets
When phenobarbitone sodium is prescribed or demanded, Phenobarbital Sodium shall be dispensed
or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Phenobarbital sodium contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of the sodium derivative of 5-ethyl-5-phenyl-1H,3H,5H-pyrimidine-2,4,6-trione,
CHARACTERS
A white, crystalline powder, hygroscopic, freely soluble in carbon dioxide-free water (a small fraction
may be insoluble), soluble in alcohol, practically insoluble in methylene chloride and in ether.
IDENTIFICATION
A. Acidify 10 ml of solution S (see Tests) with dilute hydrochloric acid R and shake with 20 ml of
ether R. Separate the ether layer, wash with 10 ml of water R, dry over anhydrous sodium sulphate R
and filter. Evaporate the filtrate to dryness and dry the residue at 100C to 105C. Determine the
melting point (2.2.14) of the test residue. Mix equal parts of the residue and of phenobarbital CRS
and determine the melting point of the mixture. The difference between the two melting points
(which are about 176C) is not greater than 2C.
B. Examine by infrared absorption spectrophotometry (2.2.24), comparing the residue obtained
during identification test A with the spectrum obtained with phenobarbital CRS. If the spectra
obtained in the solid state show differences, dissolve the test residue and the reference substance
separately in ethanol R, evaporate to dryness and record the spectra again.
Test solution. Dissolve 0.10 g of the substance to be examined in alcohol (50 per cent V/V) R and dilute
Reference solution. Dissolve 90 mg of phenobarbital CRS in alcohol (50 per cent V/V) R and dilute to
of chloroform R. Examine immediately in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the
E. It gives reaction (a) of sodium (2.3.1).
TESTS
Solution S Dissolve 5.0 g in alcohol (50 per cent V/V) R and dilute to 50 ml with the same solvent.
30-53
pH (2.2.3). Dissolve 5.0 g as completely as possible in carbon dioxide-free water R and dilute to 50 ml
with the same solvent. The pH of the solution is not greater than 10.2.
coating substance.
Test solution. Dissolve 1.0 g of the substance to be examined in alcohol (50 per cent V/V) R and dilute
Reference solution. Dilute 0.5 ml of the test solution to 100 ml with alcohol (50 per cent V/V) R.
of chloroform R. Examine immediately in ultraviolet light at 254 nm. Spray with diphenylcarbazone
spot in the chromatogram obtained with the reference solution (0.5 per cent). Disregard any spot at
the starting-point.
150C for 4 h.
ASSAY
Dissolve 0.150 g in 2 ml of water R and add 8 ml of 0.05M sulphuric acid. Heat to boiling and cool.
Add 30 ml of methanol R and shake until dissolution is complete. Carry out a potentiometric titration
(2.2.20), using 0.1M sodium hydroxide. After the first point of inflexion, interrupt the addition of
sodium hydroxide, add 10 ml of pyridine R, mix and continue the titration. Read the volume added
between the two points of inflexion.
1 ml of 0.1M sodium hydroxide is equivalent to 25.42 mg of C12H11N2NaO3.
STORAGE
_______________________________________________________________________________________________________________________ Ph Eur
30-54
Phenol
OH
C6H6O
94.1
108-95-2
Phenol complies with the requirements of the 3rd edition of the European Pharmacopoeia [0631]. These
Action and use Antiseptic; antimicrobial preservative; antipruritic.
Preparations
Aqueous Phenol Injection
Oily Phenol Injection
Phenol and Glycerol Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Phenol contains not less than 99.0 per cent and not more than the equivalent of 100.5 per cent of
C6H6O.
CHARACTERS
Colourless or faintly pink or faintly yellowish crystals or crystalline masses, deliquescent, soluble in
water, very soluble in alcohol, in glycerol and in methylene chloride.
IDENTIFICATION
A. Dissolve 0.5 g in 2 ml of concentrated ammonia R. The substance dissolves completely. Dilute to
about 100 ml with water R. To 2 ml of the dilute solution add 0.05 ml of strong sodium hypochlorite
solution R. A blue colour develops and becomes progressively more intense.
B. To 1 ml of solution S (see Tests) add 10 ml of water R and 0.1 ml of ferric chloride solution R1. A
violet colour is produced which disappears on addition of 5 ml of 2-propanol R.
C. To 1 ml of solution S add 10 ml of water R and 1 ml of bromine water R. A pale-yellow precipitate
is formed.
TESTS
Acidity To 2 ml of solution S add 0.05 ml of methyl orange solution R. The solution is yellow.
Freezing point (2.2.18). Not less than 39.5C.
Residue on evaporation Not more than 0.05 per cent, determined by evaporating 5.000 g to
dryness on a water-bath and drying the residue at 100C to 105C for 1 h.
ASSAY
Dissolve 2.000 g in water R and dilute to 1000.0 ml with the same solvent. Transfer 25.0 ml of the
solution to a ground-glass-stoppered flask and add 50.0 ml of 0.0167M bromide-bromate and 5 ml of
hydrochloric acid R, close the flask, allow to stand with occasional swirling for 30 min and then allow
to stand for a further 15 min. Add 5 ml of a 200 g/l solution of potassium iodide R, shake and titrate
with 0.1M sodium thiosulphate until a faint yellow colour remains. Add 0.5 ml of starch solution R and
10 ml of chloroform R and continue the titration with vigorous shaking. Carry out a blank titration.
1 ml of 0.0167M bromide-bromate is equivalent to 1.569 mg of C6H6O.
STORAGE
_______________________________________________________________________________________________________________________ Ph Eur
30-55
Liquefied Phenol
Definition
Phenol
Purified Water
800 g
sufficient to produce 1000 g
Extemporaneous preparation The following directions apply.

Warm the Phenol on a water bath until it is melted, add the Purified Water and mix thoroughly.
Content of phenol, C6H6O 77.0 to 81.5% w/w.
Characteristics A colourless or faintly coloured liquid; odour, characteristic and not tarry; caustic.
Soluble in water; miscible with ethanol (96%), with ether and with glycerol.
Identification
A. Dissolve 0.6 g in 2 ml of 13.5M ammonia and dilute to 100 ml with water. To 2 ml of the resulting
solution add 0.05 ml of sodium hypochlorite solution. A blue colour is produced which becomes
progressively more intense.
B. Dilute 1 ml of a 15% w/v solution to 10 ml and add 0.1 ml of iron(III) chloride solution R1. A violet
colour is produced which is discharged on the addition of propan-2-ol.
C. To 1 ml of a 15% w/v solution add 10 ml of water and 1 ml of bromine water. A white or yellowish
white precipitate is produced.
Acidity To 2 ml of a 15% w/v solution add 0.05 ml of methyl orange solution. The solution is yellow.
Clarity and colour of solution A solution of 1.0 ml in 14 ml of water, at 20, is clear, Appendix
IV A, and not more intensely coloured than reference solution R7 or B7, Appendix IV B, Method II.
Non-volatile matter When heated on a water bath and dried at 105, leaves not more than 0.05%
w/v of residue.
Assay Dissolve 2.5 g in sufficient water to produce 1000 ml, transfer 25 ml to a 500-ml glassstoppered flask and add 50 ml of 0.05M bromine VS and 5 ml of hydrochloric acid, stopper, swirl
occasionally during 30 minutes and allow to stand for 15 minutes. Add 5 ml of a 20% w/v solution of
potassium iodide taking care to avoid loss of bromine, shake thoroughly and titrate with 0.1M sodium
thiosulphate VS until only a faint yellow colour remains. Add 0.1 ml of starch mucilage and 10 ml of
chloroform and complete the titration with vigorous shaking. Repeat the operation without the
material being examined. The difference between the titrations represents the amount of bromine
required. Each ml of 0.05M bromine VS is equivalent to 1.569 mg of C6H6O.
Storage Liquefied Phenol should be kept in a well-closed container and protected from light.
Liquefied Phenol may congeal or deposit crystals if stored at a temperature below 4. It should be
completely melted before use.
Action and use Antiseptic; antimicrobial preservative; antipruritic.
When Phenol is to be mixed with collodion, fixed oils or paraffins, melted Phenol should be used and
not Liquefied Phenol.
30-56
Phenolphthalein
1/01
OH
OH
O
O
C20H14O4
318.3
77-09-8
Phenolphthalein complies with the requirements of the 3rd edition of the European Pharmacopoeia [1584].
Action and use Laxative.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Phenolphthalein contains not less than 98.0 per cent and not more than the equivalent of 101.0 per
cent of 3,3-bis(4-hydroxyphenyl)-3H-isobenzofuran-1-one, calculated with reference to the dried
substance.
CHARACTERS
A white or almost white powder, practically insoluble in water, soluble in alcohol.
IDENTIFICATION
A. Dissolve 25.0 mg in alcohol R and dilute to 100.0 ml with the same solvent (solution A). To
2.0 ml of solution A add 5.0 ml of 1M hydrochloric acid and dilute to 50.0 ml with alcohol R (solution
A1). To 10.0 ml of solution A add 5.0 ml of 1M hydrochloric acid and dilute to 50.0 ml with alcohol R
(solution A2). To 2.0 ml of solution A add 5.0 ml of 1M sodium hydroxide and dilute to 50.0 ml with
alcohol R (solution B). Examined between 220 nm and 250 nm (2.2.25), solution A1 shows an
absorption maximum at 229 nm. The specific absorbance at the maximum at 229 nm is 922 to 1018.
Examined between 250 nm and 300 nm, solution A2 shows an absorption maximum at 276 nm. The
specific absorbance at the maximum at 276 nm is 142 to 158. Examined between 230 nm and
270 nm, solution B shows an absorption maximum at 249 nm. The specific absorbance at the
maximum at 249 nm is 744 to 822.
B. Dissolve about 10 mg in alcohol R. Add 1 ml of dilute sodium hydroxide solution R. The solution is
red. Add 5 ml of dilute sulphuric acid R. The colour disappears.
TESTS
Solution S To 2.0 g add 40 ml of distilled water R and heat to boiling. Cool and filter.
Appearance of solution Dissolve 0.20 g in 5 ml of alcohol R. The solution is clear (2.2.1) and not
Acidity or alkalinity To 10 ml of solution S add 0.15 ml of bromothymol blue solution R1. Add
0.05 ml of 0.01M hydrochloric acid, the solution is yellow. Add 0.10 ml of 0.01M sodium hydroxide, the
solution is blue.
plate R.
same solvent.
Reference solution (a). Dilute 1 ml of the test solution to 10 ml with alcohol R. Dilute 5 ml of this
solution to 100 ml with alcohol R.
Reference solution (b). Dissolve 25 mg of fluorene R in alcohol R, add 0.5 ml of the test solution and
dilute to 10 ml with alcohol R.
Apply to the plate 5 l of the test solution and 5 l of each of the reference solutions. Develop over a
path corresponding to two-thirds of the plate height using a mixture of 50 volumes of acetone R and
30-57
50 volumes of methylene chloride R. Allow the plate to dry in air. Examine in ultraviolet light at
254 nm and re-examine after exposure to ammonia vapour. Any spot in the chromatogram obtained
with the test solution, apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (a) (0.5 per cent). The test is not valid unless the chromatogram obtained with reference solution (b) shows 2 clearly separated spots.
Sulphates (2.4.13).15 ml of solution S complies with the limit test for sulphates (200 ppm).
Heavy metals (2.4.8). Heat 3 g with 50 ml of dilute hydrochloric acid R on a water-bath for 5 min and
filter. Evaporate the filtrate almost to dryness and dissolve the residue in 30 ml of water R.12 ml of
this solution complies with limit test A for heavy metals (10 ppm). Prepare the standard using 10 ml
of lead standard solution (1 ppm Pb) R.
100C to 105C.
ASSAY
Dissolve 0.100 g in 5 ml of dimethylformamide R. Add 5 ml of sodium carbonate solution R, 10 ml of
sodium hydrogen carbonate solution R, 35 ml of water R and 50.0 ml of 0.05M iodine. Add 10 ml of
methylene chloride R and 20 ml of dilute sulphuric acid R. Titrate the excess of iodine with 0.1M sodium
thiosulphate, using 0.3 ml of starch solution R added towards the end of the titration, as indicator.
1 ml of 0.05M iodine is equivalent to 3.979 mg of C20H14O4.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
30-58
Phenolsulphonphthalein
OH
OH
O
S
O
C19H14O5S
O
354.4
143-74-8
Phenolsulphonphthalein complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Phenolsulfonphthalein [0242]. These requirements are reproduced after the heading Definition below.
Action and use Diagnostic aid.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Phenolsulfonphthalein (phenol red) contains not less than 98.0 per cent and not more than the
equivalent of 102.0 per cent of 4,4-(3H-2,1-benzoxathiol-3-ylidene)diphenol S,S-dioxide, calculated
CHARACTERS
A bright-red to dark-red, crystalline powder, very slightly soluble in water, slightly soluble in alcohol.
IDENTIFICATION
A. Dissolve 10 mg in a 10 g/l solution of sodium carbonate R and dilute to 200.0 ml with the sodium
carbonate solution. Dilute 5.0 ml of the solution to 100.0 ml with a 10 g/l solution of sodium carbonate R. Examined between 400 nm and 630 nm (2.2.25), the solution shows an absorption maximum
at 558 nm. The specific absorbance at the maximum is 1900 to 2100.
B. Dissolve about 10 mg in 1 ml of dilute sodium hydroxide solution R and add 9 ml of water R. The
solution is deep red. To 5 ml of the solution add a slight excess of dilute sulphuric acid R. The colour
becomes orange.
C. To 5 ml of the solution prepared for identification test B add 1 ml of 0.0167M bromide-bromate and
1 ml of dilute hydrochloric acid R, shake and allow to stand for 15 min. Make alkaline with dilute
sodium hydroxide solution R. An intense violet-blue colour is produced.
TESTS
coating substance.
Test solution. Dissolve 0.1 g of the substance to be examined in 0.1M sodium hydroxide and dilute to
Reference solution. Dilute 0.5 ml of the test solution to 100 ml with 0.1M sodium hydroxide.
25 volumes of glacial acetic acid R, 25 volumes of water R and 100 volumes of tert-pentyl alcohol R.
Allow the plate to dry in air until the odour of solvent is no longer detectable and expose the plate to
the vapour from concentrated ammonia R. Examine in ultraviolet light at 254 nm. Not more than one
spot, apart from the principal spot, appears in the chromatogram obtained with the test solution and
this spot is not more intense than the spot in the chromatogram obtained with the reference solution
(0.5 per cent).
Insoluble matter To 1.0 g of the finely powdered substance to be examined add 12 ml of sodium
hydrogen carbonate solution R. Allow to stand for 1 h, shaking frequently. Dilute to 100 ml with
water R and allow to stand for 15 h. Centrifuge at 2000 g to 3000 g, for 30 min, decant the supernatant liquid and wash the residue with 25 ml of a 10 g/l solution of sodium hydrogen carbonate R and
then 25 ml of water R. Dry at 100C to 105C. The residue weighs not more than 5 mg (0.5 per
cent).
Loss on drying (2.2.32). Not more than 1.0 per cent, determined on 1.00 g of the powdered
substance to be examined by drying in an oven at 100C to 105C.
30-59
ASSAY
Dissolve 0.900 g in 15 ml of 1M sodium hydroxide and dilute to 250.0 ml with water R. To 10.0 ml of
the solution in a glass-stoppered flask add 25 ml of glacial acetic acid R, 20.0 ml of 0.0167M potassium
bromate, 5 ml of a 100 g/l solution of potassium bromide R and 5 ml of hydrochloric acid R. Allow to
stand protected from light for 15 min, add 10 ml of a 100 g/l solution of potassium iodide R and titrate
immediately with 0.1M sodium thiosulphate, using 0.1 ml of starch solution R as indicator.
1 ml of 0.0167M potassium bromate is equivalent to 4.43 mg of C19H14O5S.
_______________________________________________________________________________________________________________________ Ph Eur
30-60
Phenoxybenzamine Hydrochloride
H
Me
O
,HCl
Cl
and enantiomer
C18H22ClNO,HCl
340.3
63-92-3
Definition Phenoxybenzamine Hydrochloride is (RS)-benzyl(2-chloroethyl)1-methyl-2phenoxyethylamine hydrochloride. It contains not less than 98.5% and not more than 101.0% of
C18H22ClNO,HCl, calculated with reference to the dried substance.
Sparingly soluble in water; freely soluble in chloroform and in ethanol (96%).
Identification
phenoxybenzamine hydrochloride (RS 271).
B. Dissolve 0.5 g in 50 ml of ethanol-free chloroform and extract with three 20-ml quantities of 0.01M
hydrochloric acid. Filter the chloroform layer through absorbent cotton and dilute 5 ml of the filtrate
to 250 ml with ethanol-free chloroform. The light absorption of the resulting solution, Appendix II B, in
the range 250 to 350 nm exhibits two maxima, at 272 nm and 279 nm. The absorbances at the
maxima are about 1.1 and about 0.90, respectively.
C. Yields the reactions characteristic of chlorides, Appendix VI.
Melting point 137.5 to 140, Appendix V A.
gel G as the coating substance and a mixture of 80 volumes of acetone and 20 volumes of chloroform as
the mobile phase. Apply separately to the plate 10 l of each of two freshly prepared solutions of the
substance being examined in methanol containing (1) 2.0% w/v and (2) 0.010% w/v. After removal of
the plate, allow it to dry in air and spray with dilute potassium iodobismuthate solution. Any secondary
spot in the chromatogram obtained with solution (1) is not more intense than the spot in the chromatogram obtained with solution (2).
Assay Carry out Method I for non-aqueous titration, Appendix VIII A, using 0.6 g and oracet blue B
solution as indicator. Each ml of 0.1M perchloric acid VS is equivalent to 34.03 mg of
C18H22ClNO,HCl.
Action and use Alpha-adrenoceptor antagonist.
Preparation
Phenoxybenzamine Capsules
30-61
Phenoxyethanol
O
OH
C8H10O2
138.2
122-99-6
Phenoxyethanol complies with the requirements of the 3rd edition of the European Pharmacopoeia [0781].
Action and use Antimicrobial preservative; also used topically in treatment of bacterial infections.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Phenoxyethanol contains not less than 99.0 per cent and not more than the equivalent of 100.5 per
cent m/m of 2-phenoxyethanol.
CHARACTERS
A colourless, slightly viscous liquid, slightly soluble in water, miscible with acetone, with alcohol and
with glycerol, slightly soluble in arachis oil and in olive oil.
IDENTIFICATION
A. Refractive index (2.2.6): 1.537 to 1.539.
B. Dissolve 80.0 mg in water R and dilute to 100.0 ml with the same solvent. Dilute 10.0 ml of the
shows two absorption maxima, at 269 nm and at 275 nm. The specific absorbances at the maxima
are 95 to 105 and 75 to 85 respectively.
obtained with phenoxyethanol CRS.
D. Shake 2 ml with a mixture of 4 g of potassium permanganate R, 5.4 g of sodium carbonate R and
75 ml of water R for 30 min. Add 25 g of sodium chloride R and stir continuously for 60 min, filter and
acidify with hydrochloric acid R to about pH 1.7. The melting point of the precipitate, after
recrystallisation from water R, is 96C to 99C (2.2.14).
TESTS
Related substances Examine by gas chromatography (2.2.28), using methyl laurate R as internal
standard.
Internal standard solution. Dissolve 1.25 g of methyl laurate R in methylene chloride R and dilute to
Test solution (b). Dissolve 5.0 g of the substance to be examined in methylene chloride R, add 1.0 ml of
the internal standard solution and dilute to 10.0 ml with methylene chloride R.
Reference solution. To 1.0 ml of test solution (a), add 10.0 ml of the internal standard solution and
dilute to 100.0 ml with methylene chloride R.
earth for gas chromatography R (150 m to 180 m) impregnated with 3 per cent m/m of polymethylphenylsiloxane R,
detector at 200C.
Inject 1 l of the reference solution and adjust the sensitivity of the detector so that the heights of
the two peaks, apart from the solvent peak, are not less than 70 per cent of the full scale of the
recorder. The substances elute in the following order: phenoxyethanol and methyl laurate. The test is
not valid unless, in the chromatogram obtained with the reference solution, the resolution between
the peaks corresponding to phenoxyethanol and methyl laurate is not less than twelve.
30-62
Inject 1 l of test solution (a). In the chromatogram obtained, verify that there is no peak with the
same retention time as the internal standard.
Inject separately 1 l of test solution (b) and the reference solution. Continue the chromatography
for five times the retention time of phenoxyethanol (which is about 5 min). From the chromatogram
obtained with the reference solution, calculate the ratio (R) of the area of the peak due to
phenoxyethanol to the area of the peak due to the internal standard. From the chromatogram
obtained with test solution (b), calculate the ratio of the sum of the areas of any peaks, apart from the
principal peak, the peak due to the internal standard and the peak due to the solvent, to the area of
the peak due to the internal standard: this ratio is not greater than R (1.0 per cent).
Phenol Dissolve 1.00 g in 50 ml of methylene chloride R, add 1 ml of dilute sodium hydroxide solution R
and 10 ml of water R. Shake. Wash the upper layer with two quantities, each of 20 ml, of methylene
chloride R and dilute to 100.0 ml with water R. The absorbance (2.2.25) of the resulting solution at
the maximum at 287 nm is not more than 0.27 (0.1 per cent).
ASSAY
To 2.000 g in an acetylation flask fitted with an air condenser, add 10.0 ml of freshly prepared acetic
anhydride solution R1 and heat with frequent shaking in a water-bath for 45 min. Cool and carefully
add 10 ml of water R. Heat for a further 2 min. Cool, add 10 ml of butanol R, shake vigorously and
titrate the excess of acetic acid with 1M sodium hydroxide using 0.2 ml of phenolphthalein solution R as
indicator. Repeat the procedure without the substance to be examined. The difference between the
volumes used in the titrations represents the amount of acetic anhydride required for the acetylation
of the substance to be examined.
1 ml of 1M sodium hydroxide is equivalent to 0.1382 g of C8H10O2.
STORAGE
_______________________________________________________________________________________________________________________ Ph Eur
30-63
Phenoxymethylpenicillin
H
O
O
O
N
H H
C16H18N2O5S
COOH
350.4
Me
S
Me
87-08-1
Phenoxymethylpenicillin complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Phenoxymethylpenicillin is (2S,5R,6R)-3,3-dimethyl-7-oxo-6-[(phenoxyacetyl)amino]-4-thia-1azabicyclo[3.2.0]heptane-2-carboxylic acid, a substance produced by the growth of certain strains of
Penicillium notatum or related organisms on a culture medium containing an appropriate precursor, or
obtained by any other means. The sum of the percentage contents of phenoxymethylpenicillin and
4-hydroxyphenoxymethylpenicillin is not less than 95.0 per cent and not more than the equivalent of
100.5 per cent, calculated with reference to the anhydrous substance.
CHARACTERS
A white, crystalline powder, slightly hygroscopic, very slightly soluble in water, soluble in alcohol.
IDENTIFICATION
A. It complies with the test for pH (see Tests).
obtained with phenoxymethylpenicillin CRS.
C. Examine by thin-layer chromatography (2.2.27), using silanised silica gel H R as the coating
substance.
Test solution. Dissolve 25 mg of the substance to be examined in 5 ml of acetone R.
Reference solution (a). Dissolve 25 mg of phenoxymethylpenicillin CRS in 5 ml of acetone R.
Reference solution (b). Dissolve 25 mg of benzylpenicillin potassium CRS and 25 mg of
phenoxymethylpenicillin potassium CRS in 5 ml of water R.
of acetone R and 70 volumes of a 154 g/l solution of ammonium acetate R, adjusted to pH 5.0 with
glacial acetic acid R. Allow the plate to dry in air and expose it to iodine vapour until the spots appear.
D. Place about 2 mg in a test-tube about 150 mm long and 15 mm in diameter. Moisten with
0.05 ml of water R and add 2 ml of sulphuric acid-formaldehyde reagent R. Mix the contents of the tube
by swirling; the solution is reddish-brown. Place the test-tube on a water-bath for 1 min; a dark
reddish-brown colour develops.
TESTS
pH (2.2.3). Suspend 50 mg in 10 ml of carbon dioxide-free water R. The pH of the suspension is 2.4 to
4.0.
Specific optical rotation (2.2.7). Dissolve 0.250 g in butanol R and dilute to 25.0 ml with the same
substance.
20 l of reference solution (d) and elute isocratically with the chosen mobile phase until elution of the
phenoxymethylpenicillin peak. Adjust the sensitivity of the system to obtain a peak with a signal-to-
30-64
noise ratio of at least 3. Inject 20 l of reference solution (e). Inject 20 l of test solution (b) and start
the elution isocratically. Immediately after elution of the phenoxymethylpenicillin peak start the
following linear gradient.
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
020
600
0
060
40100
100
10040
2035
3550
Comment
linear gradient
isocratic
re-equilibration
Inject the dissolution mixture and use the same elution pattern to obtain a blank. In the chromatogram obtained with test solution (b), the area of any peak, apart from the principal peak and any peak
corresponding to 4-hydroxyphenoxymethylpenicillin, is not greater than the area of the principal peak
in the chromatogram obtained with reference solution (e) (1 per cent).
4-Hydroxyphenoxymethylpenicillin Not more than 4.0 per cent, calculated with reference to the
anhydrous substance and determined by liquid chromatography (2.2.29), as described under Assay.
ASSAY
Dissolution mixture. To 250 ml of 0.2M potassium dihydrogen phosphate R add 500 ml of water R and
adjust to pH 6.5 with an 8.4 g/l solution of sodium hydroxide R. Dilute to 1000 ml with water R.
Test solution (a). Dissolve 50.0 mg of the substance to be examined in the dissolution mixture and
dilute to 50.0 ml with the same mixture.
Test solution (b). Prepare immediately before use. Dissolve 80.0 mg of the substance to be examined in
the dissolution mixture and dilute to 20.0 ml with the same mixture.
Reference solution (a). Dissolve 55.0 mg of phenoxymethylpenicillin potassium CRS in the dissolution
mixture and dilute to 50.0 ml with the same mixture.
Reference solution (b). Dissolve 20.0 mg of 4-hydroxyphenoxymethylpenicillin CRS in the dissolution
mixture and dilute to 50.0 ml with the same mixture. Dilute 5.0 ml of the solution to 100.0 ml with
the dissolution mixture.
Reference solution (c). Dissolve 10 mg of phenoxymethylpenicillin potassium CRS and 10 mg of
benzylpenicillin sodium CRS in the dissolution mixture and dilute to 50 ml with the same mixture.
Reference solution (d). Dilute 1.0 ml of reference solution (a) to 20 ml with the dissolution mixture.
Dilute 1.0 ml of the solution to 50 ml with the dissolution mixture.
Reference solution (e). Dilute 1.0 ml of reference solution (a) to 25.0 ml with the dissolution mixture.
a column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for
Mobile phase A. Mix 10 volumes of phosphate buffer solution pH 3.5 R, 30 volumes of methanol R
and 60 volumes of water R,
Mobile phase B. Mix 10 volumes of phosphate buffer solution pH 3.5 R, 35 volumes of water R and
55 volumes of methanol R,
Equilibrate the column with a mobile phase ratio A:B of 60:40. Inject 20 l of reference solution (c).
The test is not valid unless, in the chromatogram obtained, the resolution between the two principal
peaks is at least 6.0 (if necessary, adjust the ratio A:B of the mobile phase) and the mass distribution
ratio for the second peak (phenoxymethylpenicillin) is 5.0 to 7.0. Inject reference solution (a) six
times. The test is not valid unless the relative standard deviation for the area of the principal peak is
at most 1.0 per cent. Inject alternately test solution (a) and reference solutions (a) and (b).
Calculate the percentage content of phenoxymethylpenicillin by multiplying the percentage content
of phenoxymethylpenicillin potassium by 0.902. Calculate the percentage content of 4-hydroxyphenoxymethylpenicillin.
STORAGE
Store protected from moisture.
IMPURITIES
A. benzylpenicillin,
30-65
COOH
B. phenoxyacetic acid,
COOH
O
N
Me
H2N
Me
S
H H
C. (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo-[3.2.0]heptane-2-carboxylic
acid (6-aminopenicillanic acid),
COOH
O
HO
H
N
Me
S
Me
H H
D. (2S,5R,6R)-3,3-dimethyl-7-oxo-6-[[2-(4-hydroxyphenoxy)acetyl]amino]-4-thia-1azabicyclo[3.2.0]heptane-2-carboxylic acid (4-hydroxyphenoxymethylpenicillin),

COOH
H
N *
O
O
HN
*
Me
S
Me
COOH
E. (4S)-2-[carboxy[(phenoxyacetyl)amino]methyl]-5,5-dimethylthiazolidine-4-carboxylic acid
(penicilloic acids of phenoxymethylpenicillin),
COOH
H
N
O
O
HN
*
Me
S
Me
and epimer at C*
F. (2RS,4S)-5,5-dimethyl-2-[[(phenoxyacetyl)amino]methyl]-thiazolidine-4-carboxylic acid
(penilloic acids of phenoxymethylpenicillin).
__________________________________________________________________________________________________________ Ph Eur
30-66
Phenoxymethylpenicillin Potassium
H
O
O
O
N
H H
C16H17KN2O5S
COOK
388.5
Me
S
Me
54-35-3
Phenoxymethylpenicillin Potassium complies with the requirements of the 3rd edition of the European
Preparations
Phenoxymethylpenicillin Oral Solution
Phenoxymethylpenicillin Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Phenoxymethylpenicillin potassium is the potassium salt of (2S,5R,6R)-3,3-dimethyl-7-oxo-6[(phenoxyacetyl)amino]4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid, a substance produced
by the growth of certain strains of Penicillium notatum or related organisms on a culture medium
containing an appropriate precursor, or obtained by any other means. The sum of the percentage
contents of phenoxymethylpenicillin potassium and 4-hydroxyphenoxymethylpenicillin potassium is
not less than 95.0 per cent and not more than the equivalent of 100.5 per cent, calculated with
CHARACTERS
A white, crystalline powder, freely soluble in water, practically insoluble in alcohol.
IDENTIFICATION
obtained with phenoxymethylpenicillin potassium CRS.
B. Examine by thin-layer chromatography (2.2.27), using silanised silica gel H R as the coating
substance.
Test solution. Dissolve 25 mg of the substance to be examined in 5 ml of water R.
Reference solution (a). Dissolve 25 mg of phenoxymethylpenicillin potassium CRS in 5 ml of water R.
Reference solution (b). Dissolve 25 mg of benzylpenicillin potassium CRS and 25 mg of
phenoxymethylpenicillin potassium CRS in 5 ml of water R.
glacial acetic acid R. Allow the plate to dry in air and expose it to iodine vapour until the spots appear.
C. Place about 2 mg in a test-tube about 150 mm long and 15 mm in diameter. Moisten with
by swirling; the solution is reddish-brown. Place the test-tube in a water-bath for 1 min; a dark
D. It gives reaction (a) of potassium (2.3.1).
TESTS
pH (2.2.3). Dissolve 50 mg in carbon dioxide-free water R and dilute to 10 ml with the same solvent.
Specific optical rotation (2.2.7). Dissolve 0.250 g in carbon dioxide-free water R and dilute to
25.0 ml with the same solvent. The specific optical rotation is +215 to +230, calculated with
30-67
20 l of reference solution (d) and elute isocratically with the chosen mobile phase until elution of the
phenoxymethylpenicillin peak. Adjust the sensitivity of the system to obtain a peak with a signal-tonoise ratio of at least 3. Inject 20 l of reference solution (e). Inject 20 l of test solution (b) and start
the elution isocratically. Immediately after elution of the phenoxymethylpenicillin peak start the
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
020
600
0
060
40100
100
10040
2035
3550
Comment
linear gradient
isocratic
re-equilibration
Inject the dissolution mixture and use the same elution pattern to obtain a blank. In the chromatogram obtained with test solution (b), the area of any peak, apart from the principal peak and any peak
corresponding to 4-hydroxyphenoxymethylpenicillin, is not greater than the area of the principal peak
in the chromatogram obtained with reference solution (e) (1 per cent).
4-Hydroxyphenoxymethylpenicillin potassium Not more than 4.0 per cent, calculated with
reference to the anhydrous substance and determined by liquid chromatography (2.2.29), as
described under Assay.
ASSAY
Dissolution mixture. To 250 ml of 0.2M potassium dihydrogen phosphate R add 500 ml of water R and
adjust to pH 6.5 with a 8.4 g/l solution of sodium hydroxide R. Dilute to 1000 ml with water R.
Test solution (a). Dissolve 50.0 mg of the substance to be examined in the dissolution mixture and
dilute to 50.0 ml with the same mixture.
Test solution (b). Prepare immediately before use. Dissolve 80.0 mg of the substance to be examined in
the dissolution mixture and dilute to 20.0 ml with the same mixture.
Reference solution (a). Dissolve 50.0 mg of phenoxymethylpenicillin potassium CRS in the dissolution
mixture and dilute to 50.0 ml with the same mixture.
Reference solution (b). Dissolve 20.0 mg of 4-hydroxyphenoxymethylpenicillin CRS in the dissolution
mixture and dilute to 50.0 ml with the same mixture. Dilute 5.0 ml of the solution to 100.0 ml with
the dissolution mixture.
Reference solution (c). Dissolve 10 mg of phenoxymethylpenicillin potassium CRS and 10 mg of
benzylpenicillin sodium CRS in the dissolution mixture and dilute to 50 ml with the same mixture.
Reference solution (d). Dilute 1.0 ml of reference solution (a) to 20 ml with the dissolution mixture.
Dilute 1.0 ml of the solution to 50 ml with the dissolution mixture.
Reference solution (e). Dilute 1.0 ml of reference solution (a) to 25.0 ml with the dissolution mixture.
Mobile phase A. Mix 10 volumes of phosphate buffer solution pH 3.5 R, 30 volumes of methanol R
and 60 volumes of water R,
Mobile phase B. Mix 10 volumes of phosphate buffer solution pH 3.5 R, 35 volumes of water R and
55 volumes of methanol R,
Equilibrate the column with a mobile phase ratio A:B of 60:40. Inject 20 l of reference solution (c).
The test is not valid unless, in the chromatogram obtained, the resolution between the two principal
peaks is at least 6.0 (if necessary, adjust the ratio A:B of the mobile phase) and the mass distribution
ratio for the second peak (phenoxymethylpenicillin) is 5.0 to 7.0. Inject reference solution (a) six
times. The test is not valid unless the relative standard deviation for the area of the principal peak is
at most 1.0 per cent. Inject alternately test solution (a) and reference solutions (a) and (b).
Calculate the percentage content of phenoxymethylpenicillin potassium. Calculate the percentage
content of 4-hydroxyphenoxymethylpenicillin potassium by multiplying the percentage content of
4-hydroxyphenoxymethylpenicillin by 1.104.
IMPURITIES
A. benzylpenicillin,
30-68
COOH
B. phenoxyacetic acid,
COOH
O
N
Me
H2N
Me
S
H H
C. (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo-[3.2.0]heptane-2-carboxylic
acid (6-aminopenicillanic acid),
COOH
O
HO
H
N
Me
S
Me
H H
D. (2S,5R,6R)-3,3-dimethyl-7-oxo-6-[[2-(4-hydroxyphenoxy)acetyl]amino]-4-thia-1azabicyclo[3.2.0]heptane-2-carboxylic acid (4-hydroxyphenoxymethylpenicillin),

COOH
H
N *
O
O
HN
*
Me
S
Me
COOH
E. (4S)-2-[carboxy[(phenoxyacetyl)amino]methyl]-5,5-dimethylthiazolidine-4-carboxylic acid
(penicilloic acids of phenoxymethylpenicillin),
COOH
H
N
O
O
HN
*
Me
S
Me
and epimer at C*
F. (2RS,4S)-5,5-dimethyl-2-[[(phenoxyacetyl)amino]methyl]thiazolidine-4-carboxylic acid
(penilloic acids of phenoxymethylpenicillin).
__________________________________________________________________________________________________________ Ph Eur
30-69
Phentolamine Mesilate
OH
Me
,CH3SO3H
N
N
HN
C17H19N3O,CH4O3S
377.5
65-28-1
Phentolamine Mesilate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Alpha-adrenoceptor antagonist.
Preparation
Phentolamine Injection
When phentolamine mesylate is prescribed or demanded, Phentolamine Mesilate shall be dispensed
or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Phentolamine mesilate contains not less than 98.0 per cent and not more than the equivalent of
100.5 per cent of 3-[[(4,5-dihydro-1H-imidazol-2-yl)methyl](4-methylphenyl)amino]phenol
methanesulphonate, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder, slightly hygroscopic, freely soluble in water and in alcohol, practically
insoluble in methylene chloride.
IDENTIFICATION
First identification: C, E
B. Dissolve 60.0 mg in water R and dilute to 100.0 ml with the same solvent. Dilute 5.0 ml of the
spectrum of phentolamine mesilate.
D. Dissolve 0.5 g in a mixture of 5 ml of alcohol R and 5 ml of a 10 g/l solution of hydrochloric acid R
and add 0.5 ml of a 5 g/l solution of ammonium vanadate R. A light green precipitate is produced.
E. Mix 50 mg with 0.2 g of sodium hydroxide R, heat to fusion and continue the heating for a few
seconds. Allow to cool and add 0.5 ml of warm water R. Acidify with dilute hydrochloric acid R and
heat. Sulphur dioxide is evolved, which turns moistened starch iodate paper R blue.
TESTS
Acidity Dissolve 0.1 g in carbon dioxide-free water R and dilute to 10 ml with the same solvent. Add
0.1 ml of methyl red solution R. If the solution is red, not more than 0.05 ml of 0.1M sodium hydroxide
is required to change the colour of the indicator to yellow.
coating substance.
same solvent.
Reference solution (a). Dilute 0.5 ml of the test solution to 100 ml with alcohol R.
Reference solution (b). Dilute 5 ml of reference solution (a) to 10 ml with alcohol R.
5 volumes of concentrated ammonia R, 15 volumes of acetone R and 85 volumes of methyl ethyl
ketone R. Allow the plate to dry in air and spray with dilute potassium iodobismuthate solution R. Any
30-70
intense than the spot in the chromatogram obtained with the reference solution (a) (0.5 per cent) and
at most one such spot is more intense than the spot in the chromatogram obtained with reference
at 100C to 105C.
ASSAY
Dissolve 0.300 g in 100 ml of 2-propanol R1. Titrate under a current of nitrogen with 0.1M
tetrabutylammonium hydroxide in 2-propanol. Determine the end-point potentiometrically (2.2.20),
using a glass electrode as indicator electrode and a calomel electrode containing a saturated solution
of tetramethylammonium chloride R in 2-propanol R1 as the comparison electrode. Carry out a blank
titration.
1 ml of 0.1M tetrabutylammonium hydroxide in 2-propanol is equivalent to 37.75 mg of
C18H23N3O4S.
STORAGE
IMPURITIES
A. N-(2-aminoethyl)-2-[(3-hydroxyphenyl)(4-methylphenyl)amino]acetamide.
_______________________________________________________________________________________________________________________ Ph Eur
30-71
Phenylalanine
H
NH2
COOH
C9H11NO2
165.2
63-91-2
Phenylalanine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0782].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Phenylalanine contains not less than 98.5 per cent and not more than the equivalent of 101.0 per
cent of (S)-2-amino-3-phenylpropanoic acid, calculated with reference to the dried substance.
PRODUCTION
When Phenylalanine is produced by a process involving fermentation steps, it complies with the
CHARACTERS
A white or almost white, crystalline powder or shiny, white flakes, sparingly soluble in water, very
slightly soluble in alcohol, practically insoluble in ether. It dissolves in dilute mineral acids and in
dilute solutions of alkali hydroxides.
IDENTIFICATION
obtained with phenylalanine CRS. Examine the substances prepared as discs.
D. To about 10 mg add 0.5 g of potassium nitrate R and 2 ml of sulphuric acid R. Heat in a water-bath
for 20 min. Allow to cool. Add 5 ml of a 50 g/l solution of hydroxylamine hydrochloride R and allow to
stand in iced water for 10 min. Add 9 ml of strong sodium hydroxide solution R. A violet-red to violetbrown colour develops.
TESTS
acid. The solution is clear (2.2.1) and not more intensely coloured than reference solution BY6
(Method II, 2.2.2).
substance.
gel plate R.
glacial acetic acid R and water R and dilute to 10 ml with the same mixture of solvents.
Test solution (b). Dilute 1 ml of test solution (a) to 50 ml with a mixture of equal volumes of glacial
acetic acid R and water R.
Reference solution (a). Dissolve 10 mg of phenylalanine CRS in a mixture of equal volumes of glacial
acetic acid R and water R and dilute to 50 ml with the same mixture of solvents.
glacial acetic acid R and water R.
Reference solution (c). Dissolve 10 mg of phenylalanine CRS and 10 mg of tyrosine CRS in a mixture of
equal volumes of glacial acetic acid R and water R and dilute to 25 ml with the same mixture of
solvents.
30-72
spots.
Chlorides (2.4.4). Dissolve 0.25 g in 3 ml of dilute nitric acid R and dilute to 15 ml with water R.
The solution complies with the limit test for chlorides, without any further addition of nitric acid
(200 ppm).
Sulphates (2.4.13). Dissolve 0.5 g in a mixture of 5 volumes of dilute hydrochloric acid R and 25
volumes of distilled water R and dilute to 15 ml with the same mixture of solvents. The solution
complies with the limit test for sulphates (300 ppm).
To the inner wall of the upper watch glass stick a piece of red litmus paper R 5 mm square and wetted
Briefly triturate with a glass rod. Immediately close the cell by putting the two watch glasses together.
(100 ppm NH4 ) R, 0.5 ml of water R and 0.30 g of heavy magnesium oxide R (200 ppm).
100C to 105C.
ASSAY
0.1M perchloric acid using 0.1 ml of naphtholbenzein solution R as indicator, until the colour changes
from yellow to green.
STORAGE
_______________________________________________________________________________________________________________________ Ph Eur
30-73
Phenylbutazone
O
N
Bun
H
Ph
N
Ph
O
C19H20N2O2
308.4
50-33-9
Phenylbutazone complies with the requirements of the 3rd edition of the European Pharmacopoeia [0422].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Phenylbutazone contains not less than 99.0 per cent and not more than the equivalent of 101.0 per
cent of 4-butyl-1,2-diphenylpyrazolidine-3,5-dione, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, practically insoluble in water, soluble in ether, sparingly
soluble in alcohol. It dissolves in alkaline solutions.
IDENTIFICATION
B. Dissolve 30.0 mg in 25 ml of methanol R, add 50 ml of 1M sodium hydroxide and dilute to 100.0 ml
with water R. Dilute 5.0 ml of the solution to 250.0 ml with water R. Examined between 240 nm and
the maximum is 650 to 700. Use as the compensation liquid a mixture of 0.5 ml of methanol R,
1.0 ml of 1M sodium hydroxide and 98.5 ml of water R.
obtained with phenylbutazone CRS.
D. To 0.1 g add 1 ml of glacial acetic acid R and 2 ml of hydrochloric acid R and heat the mixture
under a reflux condenser for 30 min. Cool, add 10 ml of water R and filter. To the filtrate add 3 ml of
a 7 g/l solution of sodium nitrite R. A yellow colour is produced. To 1 ml of the solution add a solution of 10 mg of -naphthol R in 5 ml of sodium carbonate solution R. A reddish-brown to reddish-violet
TESTS
Solution S Dissolve 1.0 g with shaking in 20 ml of dilute sodium hydroxide solution R and maintain the
solution at 25C for 3 h.
Acidity or alkalinity Heat to boiling 1.0 g in 50 ml of water R, cool with shaking in a closed flask
and filter. To 25 ml of filtrate add 0.5 ml of phenolphthalein solution R. The solution is colourless. Not
more than 0.5 ml of 0.01M sodium hydroxide is required to change the colour of the indicator. Add
0.6 ml of 0.01M hydrochloric acid and 0.1 ml of methyl red solution R; the solution becomes red or
orange.
Absorbance (2.2.25). The absorbance of solution S measured at 420 nm in a 4 cm cell is not greater
than 0.20.
coating substance. Spray the plate evenly with a 20 g/l solution of sodium metabisulphite R until
thoroughly wet, allow to dry in air for 15 min and heat at 120C for 30 min. Allow to cool before use.
Test solution. Dissolve 0.2 g of the substance to be examined in a mixture of equal volumes of chloroform R and ethanol R containing 0.2 g/l of butylhydroxytoluene R and dilute to 5 ml with the same
mixture of solvents. Prepare immediately before use.
Reference solution. Dilute 1 ml of the test solution to 200 ml with a mixture of equal volumes of chloroform R and ethanol R containing 0.2 g/l of butylhydroxytoluene R. Prepare immediately before use.
Apply separately to the plate without delay 5 l of each solution.
30-74
Develop without delay over a path of 10 cm using a mixture of 10 volumes of glacial acetic acid R, 40
volumes of cyclohexane R and 50 volumes of chloroform R. Dry the plate in a current of warm air for
10 min and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with the
test solution, apart from the principal spot, is not more intense than the spot in the chromatogram
80C for 4 h.
ASSAY
Dissolve 0.500 g in 25 ml of acetone R and add 0.5 ml of bromothymol blue solution R1. Titrate with
0.1M sodium hydroxide until a blue colour is obtained which persists for 15 s. Carry out a blank titration.
1 ml of 0.1M sodium hydroxide is equivalent to 30. 84 mg of C19H20N2O2.
STORAGE
_______________________________________________________________________________________________________________________ Ph Eur
30-75
Phenylephrine
H
HO
C9H13NO2
OH
NHMe
167.2
59-42-7
Phenylephrine complies with the requirements of the 3rd edition of the European Pharmacopoeia [1035].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Phenylephrine contains not less than 99.0 per cent and not more than the equivalent of 100.5 per
cent of (R)-1-(3-hydroxyphenyl)-2-(methylamino)ethanol, calculated with reference to the dried
substance.
CHARACTERS
A white or almost white, crystalline powder, slightly soluble in water, sparingly soluble in methanol,
slightly soluble in alcohol. It dissolves in dilute mineral acids and in dilute solutions of alkali
hydroxides.
IDENTIFICATION
obtained with phenylephrine CRS.
D. Dissolve about 10 mg in 1 ml of 1M hydrochloric acid, add 0.05 ml of copper sulphate solution R and
1 ml of a 200 g/l solution of sodium hydroxide R. A violet colour develops. Add 1 ml of ether R and
shake. The upper layer remains colourless.
TESTS
Appearance of solution Dissolve 1 g in 1M hydrochloric acid and dilute to 10 ml with the same acid.
2.2.2).
Specific optical rotation (2.2.7). Dissolve 1.250 g in 1M hydrochloric acid and dilute to 25.0 ml with
the same acid. The specific optical rotation is 53 to 57, calculated with reference to the dried
substance.
Absorbance Dissolve 0.50 g in 0.1M hydrochloric acid and dilute to 200 ml with the same solvent.
Measure the absorbance (2.2.25) at 315 nm using 0.1M hydrochloric acid as the compensation liquid.
The absorbance is not more than 0.15 (0.4 per cent, calculated as 1-(3-hydroxyphenyl)-2(methylamino)ethan-1-one).
coating substance.
Solvent mixture. Prepare a mixture of equal volumes of methylene chloride R and methanolic hydrochloric acid (hydrochloric acid R diluted to 10 volumes with methanol R).
Test solution. Dissolve 0.1 g of the substance to be examined in the solvent mixture and dilute to 5 ml
with the same solvent mixture.
Reference solution (a). Dissolve 20 mg of phenylephrine CRS in the solvent mixture and dilute to 1 ml
with the same solvent mixture.
Reference solution (b). Dilute 0.1 ml of the test solution to 20 ml with the solvent mixture.
Reference solution (c). Dilute 0.1 ml of the test solution to 50 ml with the solvent mixture.
30-76
Apply separately to the plate 10 l of each solution and develop over a path of 15 cm using a mixture
of 0.5 volumes of concentrated ammonia R, 25 volumes of methanol R and 70 volumes of methylene
chloride R. Dry the plate in a current of cold air. Examine in ultraviolet light at 254 nm. Spray with a
1 g/l solution of fast red B salt R in a 50 g/l solution of sodium carbonate R and examine in daylight.
more intense than the spot in the chromatogram obtained with reference solution (b) (0.5 per cent)
and at most two such spots are more intense than the spot in the chromatogram obtained with reference solution (c) (0.2 per cent).
100C to 105C.
ASSAY
STORAGE
IMPURITIES
O
HO
H
N
A. 1-(3-hydroxyphenyl)-2-(benzylamino)ethan-1-one,
H OH
HO
NHMe
B. (S)-1-(3-hydroxyphenyl)-2-(methylamino)ethanol,
O
HO
NHMe
C. 1-(3-hydroxyphenyl)-2-(methylamino)ethan-1-one.
_______________________________________________________________________________________________________________________ Ph Eur
30-77
Phenylephrine Hydrochloride
H
HO
OH
NHMe
,HCl
C9H13NO2,HCl
203.7
61-76-7
Phenylephrine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia [0632]. These requirements are reproduced after the heading Definition below.
Preparations
Phenylephrine Eye Drops
Phenylephrine Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Phenylephrine hydrochloride contains not less than 98.5 per cent and not more than the equivalent
of 101.0 per cent of (R)-2-methylamino-1-(3-hydroxyphenyl)ethanol hydrochloride, calculated with
CHARACTERS
A white or almost white, crystalline powder, freely soluble in water and in alcohol.
IDENTIFICATION
obtained with phenylephrine hydrochloride CRS. Examine the substances prepared as discs.
C. Dissolve 0.3 g in 3 ml of water R, add 1 ml of dilute ammonia R1 and initiate crystallisation by
scratching the wall of the tube with a glass rod. The crystals, washed with iced water R and dried at
105C for 2 h, melt (2.2.14) at 171C to 176C.
D. Dissolve about 10 mg in 1 ml of water R and add 0.05 ml of a 125 g/l solution of copper sulphate R
and 1 ml of a 200 g/l solution of sodium hydroxide R. A violet colour is produced. Add 1 ml of ether R
and shake; the ether layer remains colourless.
TESTS
sodium hydroxide. The solution is yellow. Not more than 0.4 ml of 0.01M hydrochloric acid is required
to change the colour of the indicator to red.
Related substances Examine by thin-layer chromatography (2.2.27), using silica gel H R as the
coating substance.
the same solvent.
Reference solution (a). Dilute 1 ml of the test solution to 200 ml with methanol R.
volumes of chloroform R, 15 volumes of ammonia R and 80 volumes of 2-propanol R. Dry the plate in a
current of cold air, spray with ninhydrin solution R and heat at 100C to 105C for 5 min to 10 min.
Examine in daylight. Any spot in the chromatogram obtained with the test solution, apart from the
30-78
principal spot, is not more intense than the spot in the chromatogram obtained with the reference
solution (a) (0.5 per cent) and at most two such spots are more intense than the spot in the chromatogram obtained with reference solution (b) (0.2 per cent).
Ketones Dilute 10.0 ml of solution S to 50.0 ml with 0.01M hydrochloric acid. The absorbance
(2.2.25), measured at 310 nm using 0.01M hydrochloric acid as the compensation liquid, is not greater
than 0.20.
100C to 105C.
ASSAY
potentiometric titration (2.2.20) using 0.1M ethanolic sodium hydroxide. Read the volume added
1 ml of 0.1M ethanolic sodium hydroxide is equivalent to 20.37 mg of C9H14ClNO2.
_______________________________________________________________________________________________________________________ Ph Eur
31-1
Phenylmercuric Borate
1/01
C6H7BHgO3.C6H6HgO 633.2
8017-88-7
Phenylmercuric Borate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Antiseptic; antimicrobial preservative.
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Phenylmercuric borate is a compound consisting of equimolecular proportions of phenylmercuric
orthoborate and phenylmercuric hydroxide (C12H13BHg2O4; Mr 633) or of the dehydrated form
(metaborate, C12H11BHg2O3; Mr 615) or a mixture of the two compounds. It contains not less than
64.5 per cent and not more than 66.0 per cent of Hg (Ar 200.6) and not less than the equivalent of
9.8 per cent and not more than the equivalent of 10.3 per cent of borates, expressed as H3BO3, both
CHARACTERS
A white or slightly yellowish, crystalline powder or colourless, shiny crystals, slightly soluble in water
and in alcohol.
IDENTIFICATION
spectrum of phenylmercuric borate. Examine the substance as a disc.
B. To 2 ml of solution S (see Tests) add 8 ml of water R and 0.1 ml of sodium sulphide solution R. A
white precipitate is formed that darkens slowly on heating.
C. Dissolve about 20 mg in 2 ml of methanol R. The solution is clear and colourless. Ignite; the
solution burns with a green-edged flame.
TESTS
Solution S Dissolve 0.25 g by sprinkling it on the surface of 25 ml of boiling water R, cool and dilute
to 25 ml with water R.
Ionised mercury To 10 ml of solution S add 2 ml of potassium iodide solution R and 3 ml of dilute
hydrochloric acid R. Filter. The filtrate is colourless. Wash the precipitate with 3 ml of water R.
Combine the filtrate and the washings, add 2 ml of dilute sodium hydroxide solution R and dilute to
20 ml with water R. 12 ml of this solution complies with limit test A for heavy metals (2.4.8). Prepare
the standard using a mixture of 2.5 ml of lead standard solution (2 ppm Pb) R and 7.5 ml of water R.
45C for 15 h ( 30 min).
ASSAY
Mercury Dissolve 0.300 g in 100 ml of water R and add 3 ml of nitric acid R. Titrate with 0.1M
ammonium thiocyanate, using 2 ml of ferric ammonium sulphate solution R2 as indicator, until a
persistent reddish-yellow colour is obtained.
1 ml of 0.1M ammonium thiocyanate is equivalent to 20.06 mg of Hg.
Borates Dissolve 0.600 g with heating in 25 ml of water R. Dissolve 10 g of sorbitol R in the hot
solution and cool. Titrate with 0.1M sodium hydroxide, using 0.5 ml of phenolphthalein solution R as
indicator, until a persistent pink colour is obtained. Carry out a blank titration.
1 ml of 0.1M sodium hydroxide is equivalent to 6.18 mg of H3BO3.
STORAGE
_______________________________________________________________________________ Ph Eur
31-2
Phenylmercuric Nitrate
55-68-5
Phenylmercuric Nitrate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Phenylmercuric nitrate is a mixture of phenylmercuric nitrate (C6H5HgNO3; Mr 339.7) and
phenylmercuric hydroxide (C6H5HgOH; Mr 294.7). It contains not less than 62.5 per cent and not
more than 64.0 per cent of Hg (Ar 200.6), calculated with reference to the dried substance.
CHARACTERS
A white or pale yellow powder, very slightly soluble in water and in alcohol, slightly soluble in hot
water. It dissolves in glycerol and in fatty oils.
IDENTIFICATION
A. To 5 ml of solution S (see Tests) add 8 ml of water R and 0.1 ml of sodium sulphide solution R. A
white precipitate is formed that darkens slowly on heating.
B. To 1 ml of a saturated solution of the substance to be examined add 1 ml of dilute hydrochloric
acid R. A white, flocculent precipitate is formed.
C. To 5 ml of solution S add 1 ml of dilute hydrochloric acid R, 2 ml of methylene chloride R and 0.2 ml
of dithizone solution R. Shake. The lower layer is orange-yellow.
D. About 10 mg gives the reaction of nitrates (2.3.1).
TESTS
Solution S To 0.1 g add 45 ml of water R and heat to boiling with shaking. Cool, filter and dilute to
50 ml with water R.
Appearance of solution Solution S is colourless (Method II, 2.2.2).
Inorganic mercuric compounds To 10 ml of solution S add 2 ml of potassium iodide solution R and
3 ml of dilute hydrochloric acid R. Filter. The filtrate is colourless. Wash the precipitate with 2 ml of
water R. Combine the filtrate and washings, add 2 ml of dilute sodium hydroxide solution R and dilute
to 20 ml with water R. 12 ml of the solution complies with limit test A for heavy metals (0.1 per cent)
(2.4.8). Prepare the standard using lead standard solution (1 ppm Pb) R.
24 h.
ASSAY
Dissolve 0.150 g in a mixture of 10 ml of dilute nitric acid R and 90 ml of water R, heating to boiling.
Cool to 15C to 20C. Titrate with 0.1M ammonium thiocyanate using 2 ml of ferric ammonium
sulphate solution R2 as indicator, until a persistent reddish-yellow colour is obtained. Carry out a
blank titration.
1 ml of 0.1M ammonium thiocyanate is equivalent to 20.06 mg of Hg.
STORAGE
_______________________________________________________________________________ Ph Eur
31-3
Phenylpropanolamine Hydrochloride
corrected 1/01
H
OH
CH 3
,HCl
H
NH2
and enantiomer
C9H13NO,HCl
187.7
154-41-6
Phenylpropanolamine Hydrochloride complies with the requirements of the 3rd edition of the European
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Phenylpropanolamine hydrochloride contains not less than 99.0 per cent and not more than the
equivalent of 101.5 per cent of (1RS,2SR)-2-amino-1-phenylpropanol hydrochloride, calculated with
CHARACTERS
A white or almost white, crystalline powder, freely soluble in water and in alcohol, practically
IDENTIFICATION
obtained with phenylpropanolamine hydrochloride CRS. Examine the substances prepared as discs
without recrystallisation.
D. Dissolve 50 mg in 5 ml of water R, add 0.2 ml of copper sulphate solution R and 0.3 ml of dilute
sodium hydroxide solution R. A violet colour develops. Add 2 ml of ether R and shake. A violet precipitate is formed between the two layers.
TESTS
sodium hydroxide. The solution is yellow. Add 0.4 ml of 0.01M hydrochloric acid. The solution is red.
coating substance.
Test solution (a). Dissolve 0.20 g of the substance to be examined in alcohol R and dilute to 10 ml
Reference solution (a). Dissolve 20 mg of phenylpropanolamine hydrochloride CRS in alcohol R and dilute
Reference solution (c). Dissolve 20 mg of norpseudoephedrine hydrochloride CRS in alcohol R, add 1 ml of
test solution (a) and dilute to 10 ml with alcohol R.
Reference solution (d). Dissolve 60 mg of ammonium chloride R in methanol R and dilute to 10 ml with
the same solvent.
31-4
Before applying the solutions, spray the plate with a 20 g/l solution of disodium tetraborate R, using
8 ml for a plate 100 mm by 200 mm and dry in a stream of cold air for 30 min. Apply separately to
the plate as bands about 10 mm by 3 mm 10 l of each solution. Develop over a path of 10 cm using
a mixture of 6 volumes of concentrated ammonia R, 24 volumes of alcohol R and 70 volumes of
butanol R. Dry the plate in a current of warm air until the odour of the solvents is no longer
perceptible, allow to cool, spray with a 2 g/l solution of ninhydrin R in alcohol R and heat at 110C for
15 min. Any spot in the chromatogram obtained with test solution (a) apart from the principal spot
and the spot corresponding to ammonium chloride is not more intense than the spot in the chromatogram obtained with reference solution (b) (1.0 per cent). The test is not valid unless the chromatogram obtained with reference solution (c) shows two clearly separated spots.
Phenylpropanonamine Dissolve 1.0 g in 0.01M hydrochloric acid and dilute to 50.0 ml with the
same acid. The absorbance (2.2.25) of the solution measured at 283 nm is not greater than 0.10.
100C to 105C.
ASSAY
Dissolve 0.1500 g in a mixture of 5 ml of 0.01M hydrochloric acid and 50 ml of alcohol R. Carry out a
1 ml of 0.1M sodium hydroxide is equivalent to 18.77 mg of C9H14ClNO.
_______________________________________________________________________________ Ph Eur
31-5
Phenytoin
Ph
H
N
Ph
NH
O
C15H12N2O2
252.3
57-41-0
Phenytoin complies with the requirements of the 3rd edition of the European Pharmacopoeia [1253]. These
Preparation
Phenytoin Oral Suspension
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Phenytoin contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of
5,5-diphenylimidazolidine-2,4-dione, calculated with reference to the dried substance.
CHARACTERS
alcohol, very slightly soluble in methylene chloride. It dissolves in dilute solutions of alkali
hydroxides.
IDENTIFICATION
A. Examine by infrared absorption, spectrophotometry (2.2.24), comparing with the spectrum
obtained with phenytoin CRS.
C. To about 10 mg add 1 ml of water R and 0.05 ml of ammonia R. Heat until boiling begins. Add
0.05 ml of a 50 g/l solution of copper sulphate R in dilute ammonia R2 and shake. A pink, crystalline
D. It complies with the test for sulphated ash (see Tests).
TESTS
Appearance of solution Dissolve 1.0 g in a mixture of 5 ml of 1M sodium hydroxide and 20 ml of
water R. The solution is clear (2.2.1) and not more intensely coloured than reference solution BY6
(Method II, 2.2.2).
Acidity or alkalinity To 1.0 g add 45 ml of water R and boil for 2 min. Allow to cool and filter.
Wash the filter with carbon dioxide-free water R and dilute the combined filtrate and washings to 50 ml
with the same solvent. To 10 ml of the solution add 0.15 ml of methyl red solution R. Not more than
0.5 ml of 0.01M hydrochloric acid is required to change the colour of the indicator to red. To 10 ml of
the solution add 0.15 ml of bromothymol blue solution R1. Not more than 0.5 ml of 0.01M sodium
hydroxide is required to change the colour of the indicator to blue.
Related substances. Examine by thin-layer chromatography (2.2.27), using as the coating substance
a suitable silica gel with a fluorescent indicator having an optimal intensity at 254 nm. Before use,
wash the plate with a mixture of 30 volumes of dioxan R and 75 volumes of hexane R. Allow the plate
to dry in air.
acetone R and methanol R and dilute to 10 ml with the same mixture of solvents.
Test solution (b). Dilute 1 ml of test solution (a) to 20 ml with a mixture of equal volumes of acetone R
and methanol R.
Reference solution (a). Dissolve 20 mg of phenytoin CRS in a mixture of equal volumes of acetone R
and methanol R and dilute to 10 ml with the same mixture of solvents.
Reference solution (b). Dissolve 8 mg of benzophenone R in a mixture of equal volumes of acetone R and
31-6
Reference solution (c). Dissolve 8 mg of benzil R in a mixture of equal volumes of acetone R and
Reference solution (d). Dilute 1 ml of test solution (a) to 100 ml with a mixture of equal volumes of
acetone R and methanol R.
Reference solution (e). Mix 1 ml of reference solution (b) and 1 ml of reference solution (c).
Apply separately to the plate 10 l of each solution and dry the plate in a stream of cold air for 2 min.
Develop over a path of 15 cm using a mixture of 30 volumes of dioxan R and 75 volumes of hexane R.
Allow the plate to dry in air and examine in ultraviolet light at 254 nm. In the chromatogram
obtained with test solution (a): any spot corresponding to benzophenone is not more intense than the
spot in the chromatogram obtained with reference solution (b) (0.2 per cent); any spot corresponding
to benzil is not more intense than the spot in the chromatogram obtained with reference solution (c)
(0.2 per cent) and any spot, apart from the principal spot and any spot corresponding to
benzophenone and benzil, is not more intense than the spot in the chromatogram obtained with
reference solution (d) (1 per cent). The test is not valid unless the chromatogram obtained with
reference solution (e) shows two clearly separated principal spots.
100C to 105C.
ASSAY
Dissolve 0.200 g in 50 ml of dimethylformamide R. Titrate with 0.1M sodium methoxide, determining
1 ml of 0.1M sodium methoxide is equivalent to 25.23 mg of C15H12N2O2.
STORAGE
IMPURITIES
O
A. diphenylmethanone (benzophenone),
O
B. diphenylethanedione (benzil).
_______________________________________________________________________________ Ph Eur
31-7
Phenytoin Sodium
Ph
H
N
ONa
Ph
N
O
C15H11N2NaO2
274.3
630-93-3
Phenytoin Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia [0521].
Preparations
Phenytoin Capsules
Phenytoin Injection
Phenytoin Tablets
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Phenytoin sodium contains not less than 98.5 per cent and not more than the equivalent of 100.5 per
cent of sodium 4-oxo-5,5-diphenyl-2-imidazolin-2-olate, calculated with reference to the anhydrous
substance.
CHARACTERS
A white, crystalline powder, slightly hygroscopic, soluble in water and in alcohol, practically insoluble
in ether and in methylene chloride.
IDENTIFICATION
A. Dissolve 0.1 g in 20 ml of water R. Acidify with dilute hydrochloric acid R and shake with three
quantities, each of 30 ml, of chloroform R. Wash the combined chloroform layers with water R,
evaporate to dryness and dry the residue at 100C to 105C (test residue). Repeat the operations
using 0.1 g of phenytoin sodium CRS (reference residue). Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with phenytoin sodium CRS. Examine as
discs prepared using potassium bromide R.
B. To about 10 mg add 1 ml of water R and 0.05 ml of ammonia R. Heat until boiling begins. Add
0.05 ml of a 50 g/l solution of copper sulphate R in dilute ammonia R2 and shake. A pink, crystalline
C. Ignite 1 g and cool. Add 2 ml of water R to the residue and neutralise the solution with hydrochloric
acid R. Filter and dilute the filtrate to 4 ml with water R. 0.1 ml of the solution gives reaction (b) of
sodium (2.3.1).
TESTS
Appearance of solution Suspend 1.0 g in 5 ml of water R and dilute to 20 ml with 0.1M sodium
hydroxide. The solution is clear (2.2.1) and not more intensely coloured than reference solution BY6
(Method II, 2.2.2).
a suitable silica gel containing a fluorescent indicator having an optimal intensity at 254 nm.
the same solvent.
Reference solution (b). Dissolve 20 mg of benzophenone R in methanol R and dilute to 100 ml with the
same solvent.
Apply separately to the plate 10 l of each solution and dry the plate in a stream of cold air for 2 min.
Develop over a path of 15 cm using a mixture of 10 volumes of concentrated ammonia R, 45 volumes
of chloroform R and 45 volumes of 2-propanol R. Dry the plate in an oven at 80C for 5 min and
examine in ultraviolet light at 254 nm. In the chromatogram obtained with the test solution, any spot
corresponding to benzophenone is not more intense than the spot in the chromatogram obtained
31-8
with reference solution (b) (0.5 per cent) and any spot, apart from the principal spot and the spot
corresponding to benzophenone, is not more intense than the spot in the chromatogram obtained
with reference solution (a) (1.0 per cent).
Free phenytoin Dissolve 0.30 g in 10 ml of a mixture of equal volumes of pyridine R and water R.
Add 0.5 ml of phenolphthalein solution R and 3 ml of silver nitrate solution in pyridine R. Not more than
1.0 ml of 0.1M sodium hydroxide is required to change the colour of the indicator to pink.
ASSAY
Suspend 0.180 g in 2 ml of water R. Add 8.0 ml of 0.05M sulphuric acid and heat gently for 1 min.
Add 30 ml of methanol R and cool. Carry out a potentiometric titration (2.2.20), using 0.1M sodium
hydroxide. After reaching the first point of inflexion, interrupt the addition of 0.1M sodium hydroxide,
add 5 ml of silver nitrate solution in pyridine R, mix and continue the titration. Read the volume of
0.1M sodium hydroxide added between the two points of inflexion.
1 ml of 0.1M sodium hydroxide is equivalent to 27.43 mg of C15H11N2NaO2.
STORAGE
_______________________________________________________________________________ Ph Eur
31-9
Pholcodine
O
N
O
O
H
N Me
H
HO
H
C23H30N2O4,H2O
416.5
509-67-1
Pholcodine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0522]. These
Action and use Cough suppressant.
Preparations
Pholcodine Linctus
Strong Pholcodine Linctus
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Pholcodine contains not less than 98.5 per cent and not more than the equivalent of 100.5 per cent
of (5R,6S)-4,5-epoxy-N-methyl-3-(2-morpholinoethoxy)morphin-7-en-6-ol, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder or colourless crystals, sparingly soluble in water, freely
soluble in acetone and in alcohol, slightly soluble in ether. It dissolves in dilute mineral acids.
IDENTIFICATION
spectrum of pholcodine.
B. Dissolve 0.100 g in water R and dilute to 100.0 ml with the same solvent. To 10.0 ml of this
solution add 75 ml of water R and 10 ml of 1M sodium hydroxide and dilute to 100.0 ml with water R.
Examined between 230 nm and 350 nm (2.2.25), the solution shows an absorption maximum at
284 nm. The specific absorbance at the maximum is 36 to 38.
C. Dissolve 50 mg in 1 ml of sulphuric acid R and add 0.05 ml of ammonium molybdate solution R. A
pale-blue colour is produced which becomes deep blue on gentle warming. Add 0.05 ml of dilute
nitric acid R. The colour becomes brownish-red.
TESTS
coating substance.
the same solvent.
Reference solution (a). Dilute 0.5 ml of the test solution to 50 ml with chloroform R.
Reference solution (b). Dilute 5 ml of reference solution (a) to 10 ml with chloroform R.
2.5 volumes of concentrated ammonia R, 32.5 volumes of acetone R, 35 volumes of alcohol R and 35
volumes of toluene R. Dry the plate in a current of air and spray with dilute potassium iodobismuthate
solution R. In the chromatogram obtained with the test solution, any spot apart from the principal
spot is not more intense than the spot in the chromatogram obtained with reference solution (a)
(1.0 per cent) and not more than one such spot situated above the principal spot is more intense than
the spot in the chromatogram obtained with reference solution (b) (0.5 per cent).
Morphine Dissolve 0.10 g in 0.1M hydrochloric acid and dilute to 5 ml with the same acid. Add 2 ml
31-10
of a 10 g/l solution of sodium nitrite R, allow to stand for 15 min and add 3 ml of dilute ammonia R1.
The solution is not more intensely coloured than reference solution B4 (Method II, 2.2.2) (about
0.13 per cent of morphine).
100C to 105C.
ASSAY
Dissolve 0.180 g in 50 ml of anhydrous acetic acid R, warming gently. Titrate with 0.1M perchloric acid
determining the end-point potentiometrically (2.2.20) at the second point of inflexion.
STORAGE
_______________________________________________________________________________ Ph Eur
31-11
Phosphoric Acid
H3PO4
98.0
7664-38-2
Phosphoric Acid complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Concentrated Phosphoric Acid [0004]. These requirements are reproduced after the heading Definition
below.
Preparation
Dilute Phosphoric Acid
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Concentrated phosphoric acid contains not less than 84.0 per cent m/m and not more than 90.0 per
cent m/m of H3PO4.
CHARACTERS
A clear, colourless, syrupy liquid, corrosive, miscible with water and with alcohol. When stored at a
low temperature it may solidify into a mass of colourless crystals which do not melt at a temperature
below 28C.
IDENTIFICATION
A. Dilute with water R. The solution is strongly acid (2.2.4).
B. Solution S (see Tests) neutralised with dilute sodium hydroxide solution R gives the reactions of
phosphates (2.3.1).
TESTS
Solution S Dilute 10.0 g to 150 ml with water R.
Substances precipitated with ammonia To 10 ml of solution S add 8 ml of dilute ammonia R1.
Any opalescence in the solution is not more intense than that in a mixture of 10 ml of solution S and
8 ml of water R.
Hypophosphorous acid and phosphorous acid To 5 ml of solution S add 2 ml of silver nitrate
solution R2 and heat on a water-bath for 5 min. The solution shows no change in appearance.
Sulphates (2.4.13). 1.5 g diluted to 15 ml with distilled water R complies with the limit test for
sulphates (100 ppm).
Arsenic (2.4.2). 7.5 ml of solution S complies with limit test A for arsenic (2 ppm).
Heavy metals (2.4.8). To 2.5 g add 4 ml of dilute ammonia R1 and dilute to 25 ml with water R.
12 ml of the solution complies with limit test A for heavy metals (10 ppm). Prepare the standard
using lead standard solution (1 ppm Pb) R.
(50 ppm).
ASSAY
To 1.000 g add a solution of 10 g of sodium chloride R in 30 ml of water R. Titrate with 1M sodium
hydroxide, using phenolphthalein solution R as indicator.
1 ml of 1M sodium hydroxide is equivalent to 49.00 mg of H3PO4.
STORAGE
Store in a well-closed, glass container.
_______________________________________________________________________________ Ph Eur
31-12
Dilute Phosphoric Acid

Dilute Phosphoric Acid complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Dilute phosphoric acid contains 9.5 per cent m/m to 10.5 per cent m/m of H3PO4 (Mr 98.0).
PREPARATION
To 115 g of concentrated phosphoric acid add 885 g of water R and mix.
IDENTIFICATION
A. It is strongly acid (2.2.4).
B. Solution S (see Tests), neutralised with dilute sodium hydroxide solution R, gives the reactions of
phosphates (2.3.1).
TESTS
Solution S Dilute 86 g to 150 ml with water R.
Substances precipitated with ammonia To 10 ml of solution S add 8 ml of dilute ammonia R1.
Any opalescence in the solution is not more intense than that in a mixture of 10 ml of solution S and
8 ml of water R.
Hypophosphorous acid and phosphorous acid To 5 ml of solution S add 2 ml of silver nitrate
solution R2 and heat on a water-bath for 5 min. The solution shows no change in appearance.
Sulphates (2.4.13). 15 ml of the substance to be examined complies with the limit test for sulphates
(10 ppm).
Arsenic (2.4.2). 7.5 ml of solution S complies with limit test A for arsenic (0.2 ppm).
Heavy metals (2.4.8). To 20 g of the substance to be examined add 4 ml of dilute ammonia R1 and
dilute to 25 ml with water R. 12 ml of the solution complies with limit test A for heavy metals
(1 ppm). Prepare the standard using a mixture of 8 ml of lead standard solution (1 ppm Pb) R and 2 ml
of water R.
(6 ppm).
ASSAY
To 8.60 g add a solution of 10 g of sodium chloride R in 30 ml of water R. Titrate with 1M sodium
hydroxide, using phenolphthalein solution R as indicator.
1 ml of 1M sodium hydroxide is equivalent to 49.00 mg of H3PO4.
_______________________________________________________________________________ Ph Eur
31-13
Phthalylsulfathiazole
COOH
H
N
H
N
S
O
C17H13N3O5S2
403.4
N
85-73-4
Phthalylsulfathiazole complies with the requirements of the 3rd edition of the European Pharmacopoeia
When phthalylsulphathiazole is prescribed or demanded, Phthalylsulfathiazole shall be dispensed or
supplied.
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Phthalylsulfathiazole contains not less than 98.5 per cent and not more than the equivalent of
101.5 per cent of 4-(thiazol-2-ylsulphamoyl)phthalanilic acid, calculated with reference to the dried
substance.
CHARACTERS
A white or yellowish-white, crystalline powder, practically insoluble in water and in ether, freely
soluble in dimethylformamide, slightly soluble in acetone and in alcohol.
IDENTIFICATION
obtained with phthalylsulfathiazole CRS.
B. To 1 g add 8.5 ml of dilute sodium hydroxide solution R and boil under a reflux condenser for
30 min. Cool and add 17.5 ml of dilute hydrochloric acid R. Shake vigorously and filter. Neutralise the
filtrate with dilute sodium hydroxide solution R. Filter, wash the precipitate with water R, recrystallise
from water R and dry the crystals at 100C to 105C. The crystals melt (2.2.14) at 200C to 203C.
C. To 0.1 g in a test-tube add 3 ml of dilute sulphuric acid R and 0.5 g of zinc powder R. Fumes are
evolved which produce a black stain on lead acetate paper R.
D. To 0.1 g add 0.5 g of resorcinol R and 0.3 ml of sulphuric acid R and heat on a water-bath until a
homogeneous mixture is obtained. Allow to cool. Add 5 ml of dilute sodium hydroxide solution R.
Dilute 0.1 ml of this brownish-red mixture to 25 ml with water R. An intense green fluorescence
appears which disappears on acidification.
E. Dissolve about 10 mg of the crystals obtained in identification test B in 200 ml of 0.1M hydrochloric
acid. 2 ml of the solution gives the reaction of primary aromatic amines (2.3.1) with formation of an
orange precipitate.
TESTS
Appearance of solution Dissolve 1.0 g in 1M sodium hydroxide and dilute to 20 ml with the same
solvent. The solution is clear (2.2.1) and not more intensely coloured than reference solution BY5
(Method II, 2.2.2).
Acidity To 2.0 g add 20 ml of water R, shake continuously for 30 min and filter. To 10 ml of the
filtrate add 0.1 ml of phenolphthalein solution R. Not more than 0.2 ml of 0.1M sodium hydroxide is
Sulfathiazole and other primary aromatic amines Dissolve 5 mg in a mixture of 3.5 ml of
water R, 6 ml of dilute hydrochloric acid R and 25 ml of alcohol R, previously cooled to 15C. Place
immediately in iced water and add 1 ml of a 2.5 g/l solution of sodium nitrite R. Allow to stand for
3 min, add 2.5 ml of a 40 g/l solution of sulphamic acid R and allow to stand for 5 min. Add 1 ml of a
4 g/l solution of naphthylethylenediamine dihydrochloride R and dilute to 50 ml with water R. Measured
at 550 nm, the absorbance (2.2.25) is not greater than that of a standard prepared at the same time
31-14
and in the same manner using a mixture of 1 ml of a solution containing in 100 ml; 10 mg of
sulfathiazole R and 0.5 ml of hydrochloric acid R, 2.5 ml of water R, 6 ml of dilute hydrochloric acid R
and 25 ml of alcohol R.
Loss on drying (2.2.32). Not more than 2 per cent, determined on 1.00 g by drying in an oven at
100C to 105C.
ASSAY
Dissolve 0.300 g in 40 ml of dimethylformamide R. Titrate with 0.1M sodium hydroxide until the colour
becomes blue using 0.2 ml of thymolphthalein solution R as indicator. Carry out a blank titration.
1 ml of 0.1M sodium hydroxide is equivalent to 20.17 mg of C17H13N3O5S2.
STORAGE
_______________________________________________________________________________ Ph Eur
31-15
Physostigmine Salicylate
Me
O
MeHN
COOH
NMe
,
OH
Me
C15H21N3O2,C7H6O3
413.5
57-64-7
Physostigmine Salicylate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Used in treatment of glaucoma.
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Physostigmine salicylate contains not less than 98.5 per cent and not more than the equivalent of
101.0 per cent of (3aS,8aR)-1,2,3,3a,8,8a-hexahydro-1,3a,8-trimethylpyrrolo[2,3-b]indol-5-yl
methylcarbamate salicylate, calculated with reference to the dried substance.
CHARACTERS
Colourless or almost colourless crystals, sparingly soluble in water, soluble in alcohol, very slightly
soluble in ether. The crystals gradually become red when exposed to air and light; the colour
develops more quickly when the crystals are also exposed to moisture. Aqueous solutions are
unstable.
IDENTIFICATION
obtained with physostigmine salicylate CRS.
C. Heat about 10 mg in a porcelain dish with a few drops of dilute ammonia R1. An orange colour
develops. Evaporate the solution to dryness. The residue dissolves in alcohol R giving a blue solution.
Add 0.1 ml of glacial acetic acid R. The colour becomes violet. Dilute with water R. An intense red
fluorescence appears.
D. Solution S (see Tests) gives reaction (a) of salicylates (2.3.1).
TESTS
Solution S Dissolve 0.900 g, without heating, in 95 ml of carbon dioxide-free water R prepared from
distilled water R and dilute to 100.0 ml with the same solvent. Prepare immediately before use.
coating substance.
the same solvent.
Test solution (b). Dilute 2.5 ml of test solution (a) to 50 ml with alcohol R.
Reference solution (a). Dissolve 10 mg of physostigmine salicylate CRS in alcohol R and dilute to 10 ml
2 volumes of concentrated ammonia R, 23 volumes of 2-propanol R and 100 volumes of cyclohexane R.
Dry the plate in a current of cold air and carry out a second development in the same direction.
31-16
Allow the plate to dry in air and spray with freshly prepared potassium iodobismuthate solution R and
then with dilute hydrogen peroxide solution R. Examine the plate within 2 min. Any spot in the chromatogram obtained with test solution (a), apart from the principal spot, is not more intense than the
Eseridine To 5 ml of solution S add a few crystals of potassium iodate R, 0.05 ml of dilute hydrochloric
acid R and 2 ml of chloroform R. Shake. No violet colour develops in the chloroform layer within
1 min.
Sulphates (2.4.13). 15 ml of solution S complies with the limit test for sulphates (0.1 per cent).
100C to 105C.
for loss on drying.
ASSAY
Dissolve 0.350 g in 50 ml of a mixture of equal volumes of anhydrous acetic acid R and chloroform R.
STORAGE
_______________________________________________________________________________ Ph Eur
31-17
Physostigmine Sulphate
Me
O
MeHN
NMe
O
N
Me
(C15H21N3O2)2,H2SO4 648.8
,H2SO4
H
2
64-47-1
Physostigmine Sulphate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Used in treatment of glaucoma.
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Physostigmine sulphate contains not less than 97.0 per cent and not more than the equivalent of
101.0 per cent of di[(3aS,8aR)-1,2,3,3a,8,8a-hexahydro-1,3a,8-trimethylpyrrolo[2,3-b]indol-5-yl
methylcarbamate] sulphate, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, hygroscopic, very soluble in water, freely soluble in
alcohol, practically insoluble in ether. It gradually becomes red when exposed to air and light; the
colour develops more quickly when the substance is also exposed to moisture. Aqueous solutions are
unstable.
IDENTIFICATION
obtained with physostigmine sulphate CRS. Examine the substances prepared as discs using potassium
bromide R.
C. Heat about 10 mg in a porcelain dish with 0.5 ml of dilute ammonia R1. An orange colour
develops. Evaporate the solution to dryness. The residue dissolves in alcohol R giving a blue solution.
Add 0.1 ml of glacial acetic acid R. The colour becomes violet. Dilute with water. An intense red
fluorescence appears.
D. It gives reaction (a) of sulphates (2.3.1).
TESTS
Solution S Dissolve 0.500 g without heating in carbon dioxide-free water R and dilute to 50.0 ml with
coating substance.
the same solvent.
Reference solution (a). Dissolve 30 mg of physostigmine sulphate CRS in alcohol R and dilute to 10 ml
Reference solution (b). Dilute 5 ml of test solution (b) to 100 ml with alcohol R.
2 volumes of concentrated ammonia R, 23 volumes of 2-propanol R and 100 volumes of cyclohexane R.
31-18
Dry the plate in a current of cold air and carry out a second development in the same direction.
Allow the plate to dry in air and spray with freshly prepared potassium iodobismuthate solution R and
then with dilute hydrogen peroxide solution R. Examine the plate within 2 min. Any spot in the chromatogram obtained with test solution (a), apart from the principal spot, is not more intense than the
Eseridine To 5 ml of solution S add a few crystals of potassium iodate R, 0.05 ml of dilute hydrochloric
acid R and 2 ml of chloroform R and shake. After 1 min, the chloroform layer is not more intensely
coloured than a reference solution prepared at the same time in the same manner using 5 ml of
water R instead of solution S.
100C to 105C.
for loss on drying.
ASSAY
Titrate with 0.1M perchloric acid determining the end-point potentiometrically (2.2.20) at the first
inflexion point.
STORAGE
Store in a well-filled, airtight glass container, protected from light.
_______________________________________________________________________________ Ph Eur
31-19
Phytomenadione
O
Me
O
H
Me
Me
Me
Me
Me
trans-phytomenadione
C31H46O2
450.7
84-80-0
Phytomenadione complies with the requirements of the 3rd edition of the European Pharmacopoeia [1036].
Action and use Vitamin K analogue.
Preparations
Phytomenadione Injection
Phytomenadione Tablets
When vitamin K1 is prescribed or demanded, Phytomenadione shall be dispensed or supplied.
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Phytomenadione is a mixture of 2-methyl-3-[(2E)-(7R,11R)-3,7,11,15-tetramethylhexadec-2enyl]naphthalene-1,4-dione (trans-phytomenadione), 2-methyl-3-[(2Z)-(7R,11R)-3,7,11,15tetramethylhexadec-2-enyl]naphthalene-1,4-dione (cis-phytomenadione) and 2,3-epoxy-2-methyl-3[(2E)-(7R,11R)-3,7,11,15-tetramethylhexadec-2-enyl]-2,3-dihydronaphthalene-1,4-dione (transepoxyphytomenadione). It contains not more than 4.0 per cent of trans-epoxyphytomenadione and
not less than 75.0 per cent of trans-phytomenadione. The total of the three components is not less
than 97.0 per cent and not more than the equivalent of 103.0 per cent.
CHARACTERS
A clear, intense yellow, viscous, oily liquid, practically insoluble in water, sparingly soluble in alcohol,
miscible with ether and with fatty oils.
It decomposes on exposure to actinic light.
The refractive index is about 1.526.
IDENTIFICATION
Carry out all operations as rapidly as possible avoiding exposure to actinic light.
A. Dissolve 10.0 mg in trimethylpentane R and dilute to 100.0 ml with the same solvent. Examined
between 275 nm and 340 nm (2.2.25), the solution shows an absorption maximum at 327 nm and an
absorption minimum at 285 nm. The specific absorbance at the maximum is 67 to 73. Dilute
10.0 ml of the solution to 50.0 ml with trimethylpentane R. Examined between 230 nm and 280 nm,
the solution shows four absorbance maxima, at 243 nm, 249 nm, 261 nm and 270 nm respectively.
B. Examine the chromatograms obtained in the test for menadione and other related substances. The
principal spot in the chromatogram obtained with test solution (b) is similar in position, colour and
size to the principal spot in the chromatogram obtained with reference solution (a).
C. Dissolve 50 mg in 10 ml of methanol R and add 1 ml of a 200 g/l solution of potassium hydroxide R
in methanol R. A green colour is produced which becomes violet-red on heating in a water-bath at
40C and then reddish-brown on standing.
TESTS
Appearance of solution Dissolve 2.5 g in trimethylpentane R and dilute to 25 ml with the same
solvent. The solution is clear (2.2.1).
Menadione and other related substances Examine by thin-layer chromatography (2.2.27), using a
TLC silica gel F254 plate R.
31-20
Test solution (a). Dissolve 0.40 g of the substance to be examined in cyclohexane R and dilute to 10 ml
Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with cyclohexane R.
Reference solution (a). Dissolve 40 mg of phytomenadione CRS in cyclohexane R and dilute to 10 ml
Reference solution (b). Dilute 1 ml of test solution (b) to 20 ml with cyclohexane R.
Reference solution (c). Dissolve 4.0 mg of menadione R in cyclohexane R and dilute to 50 ml with the
same solvent.
volumes of cyclohexane R and 80 volumes of toluene R. Allow the plate to dry in air for 5 min. Examine in ultraviolet light at 254 nm and spray with a 100 g/l solution of phosphomolybdic acid R in
ethanol R. Heat at 120C for 5 min. Examine in daylight. In the chromatogram obtained with test
solution (a): any spot corresponding to menadione is not more intense that the spot in the chromatogram obtained with reference solution (c) (0.2 per cent); any spot, apart from the principal spot and
any spot corresponding to menadione, is not more intense than the spot in the chromatogram
obtained with reference solution (b) (0.5 per cent). Disregard any spot below the principal spot,
which may not be completely separated from the principal spot.
ASSAY
Reference solution (a). Dissolve 15.0 mg of phytomenadione CRS in the mobile phase and dilute to
Reference solution (b). Dissolve 15.0 mg of phytomenadione CRS and 4.0 mg of trans-epoxyphytomenadione CRS in the mobile phase and dilute to 10.0 ml with the mobile phase.
a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with spherical silica
gel for chromatography R (5 m) with a porosity of 8 nm,
as mobile phase at a flow rate of 0.4 ml/min a mixture of 0.67 volumes of octanol R, 3.3 volumes
of di-isopropyl ether R and 1000 volumes of heptane R,
Inject reference solution (b). When using a recorder, adjust the sensitivity of the system so that the
height of the principal peak is at least 50 per cent of the full scale of the recorder. The test is not valid
unless the order of elution of the peaks is: trans-epoxyphytomenadione, cis-phytomenadione and
trans-phytomenadione. Carry out six replicate injections of reference solution (a). The assay is not
valid unless the relative standard deviation of the peak area of the trans-isomer is less than 1.0 per
cent and the resolution between the peaks corresponding to trans-phytomenadione and cis-phytomenadione is at least 2.5. Inject the test solution and reference solution (a) and calculate the percentage contents of trans-phytomenadione, cis-phytomenadione and trans-epoxyphytomenadione using
the following expressions:
trans -phytomenadione =
cis -phytomenadione =
m Atrans S trans
m S trans
m Acis S cis
m S cis
trans-epoxyphytomenadione =
m Aepoxy S epoxy
m S epoxy
m = mass of the reference substance in reference solution (a), in milligrams,

m = mass of the substance to be examined in the test solution, in milligrams,
A trans = percentage content of trans-phytomenadione in phytomenadione CRS,
A cis = percentage content of cis-phytomenadione in phytomenadione CRS,
A epoxy = percentage content of trans-epoxyphytomenadione in phytomenadione CRS,
S trans = area of the peak corresponding to the trans-isomer in the chromatogram obtained with the
test solution,
S cis = area of the peak corresponding to the cis-isomer in the chromatogram obtained with the
test solution,
S epoxy = area of the peak corresponding to trans-epoxyphytomenadione in the chromatogram
obtained with the test solution,
S trans = area of the peak corresponding to the trans-isomer in the chromatogram obtained with
reference solution (a),
31-21
S cis = area of the peak corresponding to the cis-isomer in the chromatogram obtained with
S epoxy = area of the peak corresponding to trans-epoxyphytomenadione in the chromatogram
STORAGE
IMPURITIES
O
Me
A. 2-methylnaphthalene-1,4-dione.
_________________________________________________________________
Ph Eur
31-22
Picotamide Monohydrate
H
N
N
,H2O
H
N
OMe
C21H20N4O3,H2O
394.4
80530-63-8
Picotamide Monohydrate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Antiplatelet agent.
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Picotamide monohydrate contains not less than 98.0 per cent and not more than 101.0 per cent of
4-methoxy-N,N-bis(pyridin-3-ylmethyl)benzene-1,3-dicarboxamide, calculated with reference to the
CHARACTERS
A white or almost white, crystalline powder, slightly soluble in water, soluble in ethanol and in
methylene chloride. It dissolves in dilute mineral acids.
IDENTIFICATION
Examine by infrared spectrophotometry (2.2.24), comparing with the spectrum obtained with
picotamide monohydrate CRS. If the spectra obtained in the solid state shows differences, dissolve the
substance to be examined and the reference substance separately in acetone R, evaporate to dryness
and record new spectra using the residues.
TESTS
2.2.2).
plate R.
the same solvent.
Reference solution (a). Dilute 1 ml of the test solution to 10 ml with methanol R. Dilute 1 ml of the
Reference solution (c). Dissolve 0.5 g of the substance to be examined and 5 mg of picotamide impurity
A CRS in methanol R and dilute to 10 ml with the same solvent.
Apply to the plate 5 l of each solution. Develop over a path of 15 cm using a mixture 0.8 volumes of
glacial acetic acid R, 1 volume of water R, 2.5 volumes of methanol R and 8 volumes of butanol R.
Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with the test solution, apart from the principal spot, is not more intense than the
principal spot in the chromatogram obtained with reference solution (a) (0.5 per cent) and not more
than one such spot is more intense than the spot in the chromatogram obtained with reference
solution (b) (0.25 per cent). The test is not valid unless the chromatogram obtained with reference
solution (c) shows two clearly separated principal spots.
Chlorides (2.4.4). Dissolve 0.25 g in a mixture of 2.5 ml of dilute nitric acid R and 12.5 ml of
water R. The solution complies with the limit test for chlorides (200 ppm).
Heavy metals (2.4.8). Dissolve 1.0 g by gently heating in a mixture of 15 volumes of water R and 85
volumes of methanol R and dilute to 20 ml with the same mixture of solvents. 12 ml of the solution
31-23
complies with limit test B for heavy metals (20 ppm). Prepare the standard, using lead standard
solution (1 ppm Pb) obtained by diluting lead standard solution (100 ppm Pb) R with a mixture of 15
volumes of water R and 85 volumes of methanol R.
ASSAY
IMPURITIES
COOH
COOH
OMe
A. 4-methoxybenzene-1,3-dicarboxylic acid,
H
N
COOH
OMe
B. 2-methoxy-5-[[(pyridin-3-ylmethyl)-amino]carbonyl]benzoic acid,
COOH
H
N
OMe O
C. 4-methoxy-3-[[(pyridin-3-ylmethyl)-amino]carbonyl]benzoic acid,
N
NH2
D. (pyridin-3-yl)methanamine.
_________________________________________________________________
Ph Eur
31-24
Pilocarpine Hydrochloride
N
O
O
,HCl
N
Me
C11H16N2O2,HCl
Et
244.7
54-71-7
Pilocarpine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Cholinergic.
Preparation
Pilocarpine Hydrochloride Eye Drops
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Pilocarpine hydrochloride contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of (3S,4R)-3-ethyl-4-[(1-methyl-1H-imidazol-5-yl)methyl]dihydrofuran-2(3H)-one
hydrochloride, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder or colourless crystals, hygroscopic, very soluble in water
and in alcohol.
IDENTIFICATION
obtained with pilocarpine hydrochloride CRS. If the substances are examined as discs, prepare using
potassium chloride R.
Test solution. Dissolve 10 mg in methanol R and dilute to 2 ml with the same solvent.
Reference solution. Dissolve 10 mg of pilocarpine hydrochloride CRS in methanol R and dilute to 2 ml
concentrated ammonia R, 14 volumes of methanol R and 85 volumes of methylene chloride R. Dry the
plate at 100C to 105C for 10 min, allow to cool and spray with dilute potassium iodobismuthate
solution R. The principal spot in the chromatogram obtained with the test solution is similar in
position, colour and size to the principal spot in the chromatogram obtained with the reference
solution.
D. Dilute 0.2 ml of solution S (see Tests) to 2 ml with water R. Add 0.05 ml of a 50 g/l solution of
potassium dichromate R, 1 ml of dilute hydrogen peroxide solution R and 2 ml of methylene chloride R and
shake. A violet colour develops in the organic layer.
TESTS
Specific optical rotation (2.2.7). +89 to +93, determined on solution S and calculated with
the same solvent.
Reference solution (a). Dilute 5.0 ml of the test solution to 100.0 ml with water R. Dilute 2.0 ml of the
31-25
Reference solution (b). Dissolve 10.0 mg of isopilocarpine nitrate CRS in water R and dilute to 10.0 ml
with the same solvent. Dilute 1.0 ml of the solution to 100.0 ml with water R.
Reference solution (c). Dissolve 1.0 mg of isopilocarpine nitrate CRS in 1.0 ml of the test solution and
Reference solution (d). To 5 ml of the test solution, add 0.1 ml of ammonia R and heat the solution in
an oven at 90C for 30 min, cool and dilute to 25 ml with water R. Take 3 ml of this solution and
dilute to 25 ml with water R. Mainly pilocarpic acid is formed.
as mobile phase at a flow rate of 1.2 ml/min a solution prepared as follows: mix 55 volumes of
methanol R, 60 volumes of acetonitrile R and 885 volumes of a 0.679 g/l solution of tetrabutylammonium dihydrogen phosphate R, previously adjusted to pH 7.7 with dilute ammonia R2,
Inject 20 l of each solution. Continue the chromatography for twice the retention time of the
principal component (about 40 min). When the chromatograms are recorded in the prescribed
conditions, the substances are eluted in the following sequence; pilocarpic acid, isopilocarpic acid,
isopilocarpine and pilocarpine. The test is not valid unless the resolution between the peaks corresponding to isopilocarpine and pilocarpine in the chromatogram obtained with reference solution (c)
is at least 1.6. In the chromatogram obtained with the test solution: the area of the peak corresponding to isopilocarpine is not greater than the area of the principal peak in the chromatogram obtained
with reference solution (b) (1 per cent); the sum of the areas of the peaks corresponding to
isopilocarpine and pilocarpic acid is less than three times the area of the principal peak in the chromatogram obtained with reference solution (a) (1.5 per cent); and the sum of the areas of any peaks
apart from the principal peak and the peaks corresponding to isopilocarpine and pilocarpic acid is not
greater than the principal peak in the chromatogram obtained with reference solution (a) (0.5 per
cent). Disregard any peak with an area less than 0.4 times the area of the principal peak in the
Iron (2.4.9). 10 ml of solution S complies with the limit test for iron (10 ppm). Prepare the standard
using 5 ml of iron standard solution (1 ppm Fe) R and 5 ml of water R.
100C to 105C.
ASSAY
Dissolve 0.200 g in 50 ml of alcohol R and add 5 ml of 0.01M hydrochloric acid. Titrate with 0.1M
sodium hydroxide, determining the end-point potentiometrically (2.2.20). Read the volume added
1 ml of 0.1M sodium hydroxide is equivalent to 24.47 mg of C11H17ClN2O2.
STORAGE
IMPURITIES
N
O
O
N
HH
Me
Et
A. (3R,4R)-3-ethyl-4-[(1-methyl-1H-imidazol-5-yl)methyl]-dihydrofuran-2(3H)-one
(isopilocarpine),
N
OH
H
COOH
H Et
Me
B. (2S,3R)-2-ethyl-3-(hydroxymethyl)-4-(1-methyl-1H-imidazol-5-yl)butanoic acid (pilocarpic

acid),
N
N
Me
OH
H
COOH
H Et
C. (2R,3R)-2-ethyl-3-(hydroxymethyl)4-(1-methyl-1H-imidazol-5-yl)butanoic acid
(isopilocarpic acid).
_________________________________________________________________
Ph Eur
31-26
Pilocarpine Nitrate
N
O
O
,HNO3
N
Me
C11H16N2O2,HNO3
Et
271.3
148-72-1
Pilocarpine Nitrate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0104].
Action and use Cholinergic.
Preparation
Pilocarpine Nitrate Eye Drops
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Pilocarpine nitrate contains not less than 98.5 per cent and not more than the equivalent of 101.0 per
cent of (3S,4R)-3-ethyl-4-[(1-methyl-1H-imidazol-5-yl)methyl]dihydrofuran-2(3H)-one nitrate,
CHARACTERS
A white or almost white, crystalline powder or colourless crystals, sensitive to light, freely soluble in
water, sparingly soluble in alcohol, practically insoluble in ether.
IDENTIFICATION
obtained with pilocarpine nitrate CRS.
C. Examine thin-layer chromatography (2.2.27), using a TLC silica gel G plate R.
same solvent.
Reference solution. Dissolve 10 mg of pilocarpine nitrate CRS in water R and dilute to 10 ml with the
same solvent.
of concentrated ammonia R, 14 volumes of methanol R and 85 volumes of methylene chloride R. Dry the
plate at 100C to 105C for 10 min, allow to cool and spray with potassium iodobismuthate solution R.
The principal spot in the chromatogram obtained with the test solution is similar in position, colour
and size to the principal spot in the chromatogram obtained with the reference solution.
D. Dilute 0.2 ml of solution S (see Tests) to 2 ml with water R. Add 0.05 ml of a 50 g/l solution of
potassium dichromate R, 1 ml of dilute hydrogen peroxide solution R and 2 ml of methylene chloride R and
shake. A violet colour develops in the organic layer.
E. It gives the reaction of nitrates (2.3.1).
TESTS
Prepare immediately before use.
the same solvent.
Reference solution (a). Dissolve 5.0 ml of the test solution to 100.0 ml with water R. Dilute 2.0 ml of
this solution to 20.0 ml with water R.
31-27
Reference solution (b). Dissolve 10.0 mg of isopilocarpine nitrate CRS in water R and dilute to 10.0 ml
with the same solvent. Dilute 1.0 ml of this solution to 100.0 ml with water R.
Reference solution (c). Dissolve 1.0 mg of isopilocarpine nitrate CRS in 1.0 ml of the test solution and
Reference solution (d). To 5 ml of the test solution, add 0.1 ml of ammonia R and heat the solution in
an oven at 90C for 30 min, cool and dilute to 25 ml with water R. Take 3 ml of this solution and
dilute to 25 ml with water R. Mainly pilocarpic acid is formed.
as mobile phase at a flow rate of 1.2 ml/min a solution prepared as follows: mix 55 volumes of
methanol R, 60 volumes of acetonitrile R and 885 volumes of a 0.679 g/l solution of tetrabutylammonium dihydrogen phosphate R, previously adjusted to pH 7.7 with diluted ammonia R2,
Inject 20 l of each solution. Continue the chromatography for twice the retention time of the
principal peak (about 40 min). When the chromatograms are recorded in the prescribed conditions,
the substances are eluted in the following sequence; pilocarpic acid, isopilocarpic acid, isopilocarpine
and pilocarpine. The test is not valid unless the resolution between the peaks corresponding to
isopilocarpine and pilocarpine in the chromatogram obtained with reference solution (c) is at least
1.6. In the chromatogram obtained with the test solution: the area of the peak corresponding to
isopilocarpine is not greater than the area of the principal peak in the chromatogram obtained with
reference solution (b) (1 per cent); the sum of the area of the peaks corresponding to isopilocarpine
and pilocarpic acid is less than three times the area of the principal peak in the chromatogram
obtained with reference solution (a) (1.5 per cent); and the sum of the areas of any peaks apart from
the principal peak and the peaks corresponding to isopilocarpine and pilocarpic acid is not greater
than the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent).
Disregard any peak with an area less than 0.4 times the area of the principal peak in the chromatogram obtained with reference solution (a) and any peak due to the nitrate ion with a relative retention
time relative to pilocarpine of about 0.3.
using 5 ml of iron standard solution (1 ppm Fe) R and 5 ml of water R.
100C to 105C.
ASSAY
STORAGE
IMPURITIES
N
O
O
N
HH
Et
A. (3R,4R)-3-ethyl-4-[(1-methyl-1H-imidazol-5-yl)methyl]-dihydrofuran-2(3H)-one
(isopilocarpine),
N
OH
H
COOH
N
e
H Et
B. (2S,3R)-2-ethyl-3-(hydroxymethyl)-4-(1-methyl-1H-imidazol-5-yl)butanoic acid (pilocarpic

acid),
N
N
e
OH
H
COOH
H Et
C. (2R,3R)-2-ethyl-3-(hydroxymethyl)4-(1-methyl-1H-imidazol-5-yl)butanoic acid
(isopilocarpic acid).
_________________________________________________________________
Ph Eur
31-28
Pimozide
F
O
NH
N
N
F
C28H29F2N3O
461.6
2062-78-4
Pimozide complies with the requirements of the 3rd edition of the European Pharmacopoeia [1254]. These
Action and use Antipsychotic.
Preparation
Pimozide Tablets
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Pimozide contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of
1-[1-[4,4-bis(4-fluorophenyl)butyl]piperidin-4-yl]-1,3-dihydro-2H-benzimidazol-2-one, calculated
CHARACTERS
A white or almost white powder, practically insoluble in water, soluble in methylene chloride, sparingly soluble in methanol, slightly soluble in alcohol.
IDENTIFICATION
obtained with pimozide CRS. Examine the substances prepared as discs.
substance.
Test solution. Dissolve 30 mg of the substance to be examined in a mixture of 1 volume of acetone R
Reference solution (a). Dissolve 30 mg of pimozide CRS in a mixture of 1 volume of acetone R and 9
volumes of methanol R and dilute to 10 ml with the same mixture of solvents.
Reference solution (b). Dissolve 30 mg of pimozide CRS and 30 mg of benperidol CRS in a mixture of 1
volume of acetone R and 9 volumes of methanol R and dilute to 10 ml with the same mixture of
solvents.
1 volume of acetone R and 9 volumes of methanol R. Allow the plate to dry in a current of warm air for
15 min and expose it to iodine vapour until the spots appear. The principal spot in the chromatogram
with reference solution (b) shows two clearly separated spots.
D. Mix about 5 mg with 45 mg of heavy magnesium oxide R and ignite in a crucible until an almost
white residue is obtained (usually less than 5 min). Allow to cool, add 1 ml of water R, 0.05 ml of
phenolphthalein solution R1 and about 1 ml of dilute hydrochloric acid R to render the solution colourless. Filter. To a freshly prepared mixture of 0.1 ml of alizarin S solution R and 0.1 ml of zirconyl
nitrate solution R, add 1.0 ml of the filtrate. Mix, allow to stand for 5 min and compare the colour of
the solution with that of a blank prepared in the same manner. The test solution is yellow and the
blank is red.
31-29
TESTS
Appearance of solution. Dissolve 0.2 g in methanol R and dilute to 20 ml with the same solvent.
2.2.2).
Related substances. Examine by liquid chromatography (2.2.29).
Reference solution (a). Dissolve 5.0 mg of pimozide CRS and 2.0 mg of mebendazole CRS in methanol R
as mobile phase at a flow rate of 2.0 ml per minute:
Mobile phase A. A solution containing 2.5 g/l of ammonium acetate R and 8.5 g/l of tetrabutylammonium hydrogen sulphate R,
Time
(min)
Mobile phase A
(per cent V/V)

(per cent V/V)
0 10
8070
70
80
2030
30
20
80
20
10 15
15 20
20 = 0
Linear gradient
Isocratic elution
Switch to initial
eluent composition
Re-start gradient

Equilibrate the column for at least 10 min at the initial elution composition.
Inject 10 l of reference solution (a). When the chromatogram is recorded in the prescribed conditions, the retention times are: mebendazole, about 7 min and pimozide, about 8 min. The test is not
valid unless the resolution between the peaks due to mebendazole and pimozide is at least 5.0. If
necessary, adjust the concentration of acetonitrile in the mobile phase or adjust the time programme
for the linear-gradient elution.
with reference solution (b) (0.5 per cent); the sum of the areas of all peaks, apart from the principal
peak, is not greater than 1.5 times the area of the principal peak in the chromatogram obtained with
reference solution (b) (0.75 per cent). Disregard any peak in the chromatogram obtained with the
blank run and any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (b).
100C to 105C.
ASSAY
Dissolve 0.300 g in 50 ml of a mixture of 1 volume of anhydrous acetic acid R and 7 volumes of methyl
ethyl ketone R and titrate with 0.1M perchloric acid, using 0.2 ml of naphtholbenzein solution R as
indicator.
1 ml 0.1M perchloric acid is equivalent to 46.16 mg of C28H29F2N3O.
STORAGE
31-30
IMPURITIES
O
NH
N
R=
Ar =
F
R
HN
A. 1-(piperidin-4-yl)-1,3-dihydro-2H-benzimidazol-2-one,
R
H
Ar
and enantiomer
B. 1-[1-[(4RS)-4-(4-fluorophenyl)-4-phenylbutyl]piperidin-4-yl]-1,3-dihydro-2Hbenzimidazol-2-one,
R
H
Ar
and enantiomer
N
F
C. 1-[1-[(4RS)-4-(2-fluorophenyl)-4-(4-fluorophenyl)butyl]-piperidin-4-yl]-1,3-dihydro-2Hbenzimidazol-2-one,
R
Ar
N
Ar
D. 1-[1-[4,4-bis(4-fluorophenyl)butyl]-1,2,3,6-tetrahydropyridin-4-yl]-1,3-dihydro-2Hbenzimidazol-2-one,
R
Ar
Ar
N
O
E. 1-[1-[4,4-bis(4-fluorophenyl)butyl]piperidin-4-yl 1-oxide]-1,3-dihydro-2H-benzimidazol2-one.
_______________________________________________________________________________ Ph Eur
31-31
Pindolol
H
HN
OH
NHPri
and enantiomer
C14H20N2O2
248.3
13523-86-9
Pindolol complies with the requirements of the 3rd edition of the European Pharmacopoeia [0634]. These
Preparation
Pindolol Tablets
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Pindolol contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of
(RS)-1-indol-4-yloxy-3-isopropylamino-2-propanol, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, practically insoluble in water, slightly soluble in
methanol. It dissolves in dilute mineral acids.
IDENTIFICATION
B. Dissolve 20.0 mg in a 0.085 per cent V/V solution of hydrochloric acid R in methanol R and dilute
to 100.0 ml with the same solution. Dilute 10.0 ml of the solution to 100.0 ml with a 0.085 per cent
V/V solution of hydrochloric acid R in methanol R. Examined between 230 nm and 320 nm (2.2.25),
the solution shows two absorption maxima, at 264 nm and at 287 nm, and a shoulder at 275 nm.
The specific absorbance at the maxima are 330 to 350 and 170 to 190, respectively.
obtained with pindolol CRS.
D. Examine in daylight the chromatograms on plate A obtained in the test for related substances.
The principal spot in the chromatogram obtained with test solution (b) is similar in position, colour
TESTS
Appearance of solution Dissolve 0.5 g in dilute acetic acid R and dilute to 10 ml with the same acid.
The solution is clear (2.2.1) and not more intensely coloured than reference solution BY5 or B5
(Method II, 2.2.2).
coating substance.
Carry out all operations as rapidly as possible, protected from light.
Test solution (a). Dissolve 0.10 g of the substance to be examined in a mixture of 1 volume of
anhydrous acetic acid R and 99 volumes of methanol R and dilute to 5 ml with the same mixture of
solvents. Prepare immediately before use and apply this solution to the plate last.
Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with a mixture of 1 volume of anhydrous
acetic acid R and 99 volumes of methanol R.
Reference solution (a). Dissolve 20 mg of pindolol CRS in a mixture of 1 volume of anhydrous acetic
acid R and 99 volumes of methanol R and dilute to 10 ml with the same mixture of solvents.
Reference solution (b). Dilute 1.5 ml of reference solution (a) to 50 ml with a mixture of 1 volume of
anhydrous acetic acid R and 99 volumes of methanol R.
A. Apply separately 5 l of each solution. Develop the plate without delay over a path of 10 cm using
a freshly prepared mixture of 4 volumes of concentrated ammonia R, 50 volumes of ethyl acetate R and
50 volumes of methanol R. Dry the plate briefly in a current of cold air. Spray the plate without delay
31-32
with dimethylaminobenzaldehyde solution R7 and heat to 50C for 20 min. Any spot in the chromatogram obtained with test solution (a), apart from the principal spot, is not more intense than the spot in
the chromatogram obtained with reference solution (b) (0.3 per cent).
B. Apply separately 10 l of each solution. Develop the plate without delay over a path of 10 cm
using a freshly prepared mixture of 4 volumes of concentrated ammonia R, 50 volumes of ethyl
acetate R and 50 volumes of methanol R. Dry the plate briefly in a current of cold air. Examine the
plate without delay in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with test
solution (a), apart from the principal spot and the spots detected on plate A, is not more intense than
100C to 105C.
ASSAY
Dissolve 0.200 g in 80 ml of methanol R. Titrate with 0.1M hydrochloric acid, determining the endpoint potentiometrically (2.2.20).
1 ml of 0.1M hydrochloric acid is equivalent to 24.83 mg of C14H20N2O2.
STORAGE
IMPURITIES
A. 1-[7-(2-hydroxy-3-isopropylaminopropyl)indol-4-yloxy]-3-isopropylamino-2-propanol,
B. 1-[1-(2-hydroxy-3-isopropylaminopropyl)indol-4-yloxy]-3-isopropylamino-2-propanol,
C. 3,3-bis(indol-4-yloxy)-N-isopropyl-1,1-imino-bis-(2-propanol)hydrogen malonate,
D. 4-(2,3-dihydroxypropoxy) indole,
E. 4-hydroxyindole,
F. 4-(2-hydroxy-3-chloropropoxy) indole.
_______________________________________________________________________________ Ph Eur
31-33
Piperacillin
Et
O H NH
C23H27N5O7S,H2O
COOH
Me
N
H
N
S
H
Me
535.6
66258-76-2
Piperacillin complies with the requirements of the 3rd edition of the European Pharmacopoeia [1169]. These
Preparation
Piperacillin Intravenous infusion
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Piperacillin contains not less than 96.0 per cent and not more than the equivalent of 101.0 per cent
of (2S,5R,6R)-6-[[(2R)-2-[[(4-ethyl-2,3-dioxopiperazin-1-yl)carbonyl]amino]-2-phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo-[3.2.0]heptane-2-carboxylic acid, calculated with
PRODUCTION
If manufactured by a process that may leave residues of N,N-dimethylaniline in the substance and/or
by a process using starting materials or intermediates which contain residues of N,N-dimethylaniline,
it complies with the following test:
N,N-Dimethylaniline Not more than 20 ppm, determined by gas chromatography (2.2.28), using a
suitable validated method.
CHARACTERS
A white or almost white powder, slightly soluble in water, freely soluble in methanol, slightly soluble
in ethyl acetate.
IDENTIFICATION
with piperacillin CRS.
TESTS
Solution S Dissolve 2.50 g in sodium carbonate solution R and dilute to 25 ml with the same solvent.
The absorbance of solution S measured at 430 nm (2.2.25) is not greater than 0.10.
Specific optical rotation (2.2.7). Dissolve 0.250 g in methanol R and dilute to 25.0 ml with the
same solvent. The specific optical rotation is +160 to +170, calculated with reference to the
Related substances Examine by liquid chromatography (2.2.29) as prescribed under Assay. Inject
20 l of reference solution (b) and elute isocratically with the chosen mobile phase. Inject 20 l of test
solution (b). Start the elution isocratically. Immediately after elution of the piperacillin peak start the
Time
(min)
Mobile phase A
(per cent V/V)

(per cent V/V)
030
3045
880
088
12100
10012
linear gradient
re-equilibration
In the chromatogram obtained with test solution (b), the area of any peak apart from the principal
31-34
peak, is not greater than twice the area of the principal peak in the chromatogram obtained with
reference solution (b) (2 per cent). Disregard any peak due to the solvent.
ASSAY
Solvent mixture. Mix 25 volumes of acetonitrile R and 75 volumes of a 31.2 g/l solution of sodium
dihydrogen phosphate R.
Test solution (a). Dissolve 25.0 mg of the substance to be examined in the solvent mixture and dilute
to 50.0 ml with the solvent mixture.
Test solution (b). Prepare the solution immediately before use. Dissolve 40.0 mg of the substance to be
examined in the solvent mixture and dilute to 20.0 ml with the solvent mixture.
Reference solution (a). Dissolve 25.0 mg of piperacillin CRS in the solvent mixture and dilute to
Reference solution (b). Dilute 1.0 ml of reference solution (a) to 25.0 ml with the solvent mixture.
Reference solution (c). Dissolve 10.0 mg of piperacillin CRS and 10.0 mg of anhydrous ampicillin CRS in
the solvent mixture and dilute to 50.0 ml with the solvent mixture.
Reference solution (d). Dilute 1.0 ml of reference solution (a) to 100.0 ml with the solvent mixture.
Dilute 1.0 ml of the solution to 50.0 ml with the solvent mixture.
as mobile phase at a flow rate of 1.0 ml/min a mixture of 88 volumes of mobile phase A and 12
volumes of mobile phase B:
Mobile phase A. Mix 576 ml of water R, 200 ml of a 31.2 g/l solution of sodium dihydrogen
phosphate R and 24 ml of an 80 g/l solution of tetrabutylammonium hydroxide R. If necessary,
adjust to pH 5.5 with dilute phosphoric acid R or dilute sodium hydroxide solution R; then add
200 ml of acetonitrile R,
Mobile phase B. Mix 126 ml of water R, 200 ml of a 31.2 g/l solution of sodium dihydrogen
phosphate R and 24 ml of an 80 g/l solution of tetrabutylammonium hydroxide R. If necessary,
adjust to pH 5.5 with dilute phosphoric acid R or dilute sodium hydroxide solution R; then add
650 ml of acetonitrile R,
resolution between the peaks corresponding to ampicillin and to piperacillin is at least ten (if
necessary, adjust the ratio A:B of the mobile phase) and the mass distribution ratio for the second
peak (piperacillin) is 2.0 to 3.0. Inject 20 l of reference solution (d). Adjust the sensitivity of the
system to obtain a peak with a signal-to-noise ratio of at least three. Inject reference solution (a) six
times. The test is not valid unless the relative standard deviation of the peak area of piperacillin is at
most 1.0 per cent. Inject alternately test solution (a) and reference solution (a).
IMPURITIES
A. (2S,5R,6R)-6-[[(2R)-2-amino-2-phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1azabicyclo[3.2.0]heptane-2-carboxylic acid (ampicillin),
Et
O
N
N
O H NH
H
N *
COOH
Me
HN
*
Me
COOH
B. (4S)-2-[carboxy[[(2R)-2-[[(4-ethyl-2,3-dioxopiperazin-1-yl)carbonyl]amino]-2phenylacetyl]amino]methyl]-5,5-dimethylthiazolidine-4-carboxylic acid (penicilloic acids of

piperacillin),
31-35
Et
N
N
O H NH
COOH
HN
H
N
Me
and epimer at C*
Me
C. (2RS,4S)-2-[[[(2R)-2-[[(4-ethyl-2,3-dioxopiperazin-1-yl)carbonyl]amino]-2-phenylacetyl]
amino]methyl]-5,5-dimethylthiazolidine-4-carboxylic acid (penilloic acids of piperacillin),
O
Et
H
N
COOH
H
N
HN
Me
S
Me
O H H
H
O
O H NH H
N
Me
S
Me
H H
D. (2S,5R,6R)-6-[[(2R)-2-[[[(2S,5R,6R)-6-[[(2R)-2-[[(4-ethyl-2,3-dioxopiperazin-1yl)carbonyl]amino]-2-phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]hept-2-yl]carbonyl]amino]-2-phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid (piperacillinylampicillin),

O
O
Et
N
NH
E. 1-ethylpiperazine-2,3-dione,
Et
O
N
N
O H NH
COOH
Me
AcN
H
N *
Me
COOH
F. (4S)-3-acetyl-2-[carboxy[[(2R)-2-[[(4-ethyl-2,3-dioxopiperazin-1-yl)carbonyl]amino]-2phenylacetyl]amino]methyl]-5,5-dimethylthiazolidine-4-carboxylic acid (acetylated

penicilloic acids of piperacillin).
_________________________________________________________________
Ph Eur
31-36
Piperacillin Sodium
Et
O H NH
COONa
Me
N
H
N
Me
H H
C23H26N5NaO7S
539.5
59703-84-3
Piperacillin Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia [1168].
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Piperacillin sodium contains not less than 95.0 per cent and not more than the equivalent of
101.0 per cent of sodium (2S,5R,6R)-6-[[(2R)-2-[[(4-ethyl-2,3-dioxopiperazin-1-yl)carbonyl]amino]-2-phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate,
PRODUCTION
If manufactured by a process that may leave residues of N,N-dimethylaniline in the substance and/or
by a process using starting materials or intermediates which contain residues of N,N-dimethylaniline,
it complies with the following test:
CHARACTERS
A white or almost white powder, hygroscopic, freely soluble in water and in methanol, practically
insoluble in ethyl acetate.
IDENTIFICATION
A. Dissolve 0.250 g in water R, add 0.5 ml of dilute hydrochloric acid R and 5 ml of ethyl acetate R; stir
and allow to stand for 10 min in iced water. Filter the crystals through a small sintered-glass filter
(40), applying suction. Wash with 5 ml of water R and 5 ml of ethyl acetate R and dry in an oven at
60C for 60 min. Examine the crystals by infrared absorption spectrophotometry (2.2.24), comparing
with the spectrum obtained with piperacillin CRS.
B. It gives reaction (a) of sodium (2.3.1).
TESTS
substance.
20 l of reference solution (b) and elute isocratically with the chosen mobile phase. Inject 20 l of test
solution (b). Start the elution isocratically. Immediately after elution of the piperacillin peak start the
Time
(min)
Mobile phase A
(per cent V/V)

(per cent V/V)
030
3045
880
088
12100
10012
linear gradient
re-equilibration
31-37
In the chromatogram obtained with test solution (b), the area of any peak, apart from the principal
reference solution (b) (2 per cent). Disregard any peak due to the solvent.
Sterility (2.6.1). If intended for use in the manufacture of a parenteral dosage forms without a
further appropriate sterilisation procedure, it complies with the test for sterility.
ASSAY
Solvent mixture. Mix 25 volumes of acetonitrile R and 75 volumes of a 31.2 g/l solution of sodium
dihydrogen phosphate R.
Test solution (b). Prepare the solution immediately before use. Dissolve 40.0 mg of the substance to be
examined in the solvent mixture and dilute to 20.0 ml with the solvent mixture.
Reference solution (a). Dissolve 25.0 mg of piperacillin CRS in the solvent mixture and dilute to
Reference solution (c). Dissolve 10.0 mg of piperacillin CRS and 10.0 mg of anhydrous ampicillin CRS in
the solvent mixture and dilute to 50.0 ml with the solvent mixture.
Reference solution (d). Dilute 1.0 ml of reference solution (a) to 100.0 ml with the solvent mixture.
Dilute 1.0 ml of the solution to 50.0 ml with the solvent mixture.
as mobile phase at a flow rate of 1.0 ml/min a mixture of 88 volumes of mobile phase A and 12
volumes of mobile phase B:
Mobile phase A. Mix 576 ml of water R, 200 ml of a 31.2 g/l solution of sodium dihydrogen
phosphate R and 24 ml of a 80 g/l solution of tetrabutylammonium hydroxide R. If necessary, adjust
to pH 5.5 with dilute phosphoric acid R or dilute sodium hydroxide solution R; then add 200 ml of
acetonitrile R,
Mobile phase B. Mix 126 ml of water R, 200 ml of a 31.2 g/l solution of sodium dihydrogen
phosphate R and 24 ml of a 80 g/l solution of tetrabutylammonium hydroxide R. If necessary, adjust
to pH 5.5 with dilute phosphoric acid R or dilute sodium hydroxide solution R; then add 650 ml of
acetonitrile R,
resolution between the peaks corresponding to ampicillin and to piperacillin is at least ten (if
necessary, adjust the A:B ratio of the mobile phase) and the mass distribution ratio for the second
peak (piperacillin) is 2.0 to 3.0. Inject 20 l of reference solution (d). Adjust the sensitivity of the
system to obtain a peak with a signal-to-noise ratio of at least 3. Inject reference solution (a) six
times. The test is not valid unless the relative standard deviation of the peak area of piperacillin is at
most 1.0 per cent. Inject alternately test solution (a) and reference solution (a).
Calculate the percentage content of piperacillin sodium multiplying the result by 1.042.
STORAGE
Store in an airtight container. If the substance is sterile, store in a sterile, airtight, tamper-proof
container.
LABELLING
The label states:
IMPURITIES
A. (2S,5R,6R)-6-[[(2R)-2-amino-2-phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1azabicyclo[3.2.0]heptane-2-carboxylic acid (ampicillin),
31-38
Et
N
N
O H NH
H
N *
COOH
Me
HN
*
Me
COOH
B. (4S)-2-[carboxy[[(2R)-2-[[(4-ethyl-2,3-dioxopiperazin-1-yl)carbonyl]amino]-2phenylacetyl]amino]methyl]-5,5-dimethylthiazolidine-4-carboxylic acid (penicilloic acids of

piperacillin),
Et
N
N
O H NH
COOH
HN
H
N
Me
and epimer at C*
Me
C. (2RS,4S)-2-[[[(2R)-2-[[(4-ethyl-2,3-dioxopiperazin-1-yl)carbonyl]amino]-2-phenylacetyl]
amino]methyl]-5,5-dimethylthiazolidine-4-carboxylic acid (penilloic acids of piperacillin),
O
Et
H
N
COOH
H
N
HN
Me
S
Me
O H H
H
O
O H NH H
N
Me
S
Me
H H
D (2S,5R,6R)-6-[[(2R)-2-[[[(2S,5R,6R)-6-[[(2R)-2-[[(4-ethyl-2,3-dioxopiperazin-1yl)carbonyl]amino]-2-phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]hept-2-yl]carbonyl]amino]-2-phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1azabicyclo[3.2.0]heptane-2-carboxylic acid (piperacillinylampicillin),

O
O
Et
N
NH
E. 1-ethylpiperazine-2,3-dione,
Et
O
N
N
O H NH
COOH
Me
AcN
H
N *
Me
COOH
F. (4S)-3-acetyl-2-[carboxy[[(2R)-2-[[(4-ethyl-2,3-dioxopiperazin-1-yl)carbonyl]amino]-2phenylacetyl]amino]methyl]-5,5-dimethylthiazolidine-4-carboxylic acid (acetylated

penicilloic acids of piperacillin).
_________________________________________________________________
Ph Eur
31-39
Piperazine Adipate
COOH
HN
NH
,[CH2] 4
COOH
C4H10N2,C6H10O4
232.3
142-88-1
Piperazine Adipate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0423].
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Piperazine adipate contains not less than 98.0 per cent and not more than the equivalent of 101.0 per
cent of piperazine adipate, calculated with reference to the anhydrous substance.
CHARACTERS
A white crystalline powder, soluble in water, practically insoluble in alcohol. It melts at about 250C,
with decomposition.
IDENTIFICATION
obtained with piperazine adipate CRS. Examine the substances prepared as discs.
B. Examine the chromatograms obtained in the test for related substances after spraying with the
ninhydrin solutions. The principal spot in the chromatogram obtained with test solution (b) is similar
in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a).
C. To 10 ml of solution S (see Tests) add 5 ml of hydrochloric acid R and shake with three quantities,
each of 10 ml, of ether R. Evaporate the combined ether layers to dryness. The residue, washed with
5 ml of water R and dried at 100C to 105C, melts (2.2.14) at 150C to 154C.
TESTS
Test solution (a). Dissolve 1.0 g of the substance to be examined in 6 ml of concentrated ammonia R
and dilute to 10 ml with ethanol R.
Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with a mixture of 2 volumes of ethanol R and
3 volumes of concentrated ammonia R.
Reference solution (a). Dissolve 0.1 g of piperazine adipate CRS in a mixture of 2 volumes of ethanol R
and 3 volumes of concentrated ammonia R and dilute to 10 ml with the same mixture of solvents.
Reference solution (b). Dissolve 25 mg of ethylenediamine R in a mixture of 2 volumes of ethanol R and
3 volumes of concentrated ammonia R and dilute to 100 ml with the same mixture of solvents.
Reference solution (c). Dissolve 25 mg of triethylenediamine R in a mixture of 2 volumes of ethanol R
Reference solution (d). Dissolve 12.5 mg of triethylenediamine R in 5.0 ml of test solution (a) and dilute
to 50 ml with a mixture of 2 volumes of ethanol R and 3 volumes of concentrated ammonia R.
prepared mixture of 20 volumes of concentrated ammonia R and 80 volumes of acetone R. Dry the
plate at 105C and spray successively with a 3 g/l solution of ninhydrin R in a mixture of 3 volumes of
anhydrous acetic acid R and 100 volumes of butanol R and a 1.5 g/l solution of ninhydrin R in
ethanol R. Dry the plate at 105C for 10 min. Any spot in the chromatogram obtained with test
solution (a), apart from the principal spot, is not more intense than the spot in the chromatogram
obtained with reference solution (b) (0.25 per cent). Spray the plate with 0.05M iodine and allow to
31-40
stand for about 10 min. Any spot corresponding to triethylenediamine in the chromatogram obtained
with test solution (a) is not more intense than the spot in the chromatogram obtained with reference
solution (c) (0.25 per cent). The test is not valid unless the chromatogram obtained with reference
solution (d) shows two clearly separated spots. Disregard any spots remaining on the starting line.
of water.
ASSAY
Dissolve 0.100 g in 10 ml of anhydrous acetic acid R with gentle heating and dilute to 70 ml with the
same acid. Titrate with 0.1M perchloric acid using 0.25 ml of naphtholbenzein solution R as indicator
until the colour changes from brownish-yellow to green.
STORAGE
_______________________________________________________________________________ Ph Eur
31-41
Piperazine Citrate
CH2COOH
HN
, HO C
NH
3
(C4H10N2)3,2C6H8O7
COOH
CH2COOH
643
41372-10-5
Piperazine Citrate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0424].
Preparation
Piperazine Citrate Elixir
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Piperazine citrate contains not less than 98.0 per cent and not more than the equivalent of 101.0 per
cent of tripiperazine bis(2-hydroxy-propane-1,2,3-tricarboxylate), calculated with reference to the
anhydrous substance. It contains a variable quantity of water.
CHARACTERS
A white granular powder, freely soluble in water, practically insoluble in alcohol and in ether.
After drying at 100C to 105C, it melts at about 190C.
IDENTIFICATION
obtained with piperazine citrate CRS. Dry the substance to be examined and the reference substance
at 120C for 5 h, powder the substances avoiding uptake of water, prepare discs and record the
spectra without delay.
C. Dissolve 0.5 g in water R and dilute to 5 ml with the same solvent. The solution gives the reaction
of citrates (2.3.1).
TESTS
Reference solution (a). Dissolve 0.1 g of piperazine citrate CRS in a mixture of 2 volumes of ethanol R
31-42
solution (d) shows two clearly separated spots. Disregard any spots remaining on the starting line.
ASSAY
Dissolve 0.100 g in 10 ml of anhydrous acetic acid R with gentle heating and dilute to 70 ml with the
STORAGE
_______________________________________________________________________________ Ph Eur
31-43
Piperazine Hydrate
HN
NH
C4H10N2,6H2O
,6 H 2O
194.2
142-63-2
Piperazine Hydrate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0425].
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Piperazine hydrate contains not less than 98.0 per cent and not more than the equivalent of
101.0 per cent of piperazine hexahydrate.
CHARACTERS
Colourless, deliquescent crystals, freely soluble in water and in alcohol, very slightly soluble in ether.
IDENTIFICATION
obtained with piperazine hydrate CRS. Dry the substance to be examined and the reference substance
over diphosphorus pentoxide R in vacuo for 48 h, powder the substances avoiding uptake of water,
prepare discs and record the spectra without delay.
C. Dissolve 0.5 g in 5 ml of dilute sodium hydroxide solution R. Add 0.2 ml of benzoyl chloride R and
mix. Continue to add benzoyl chloride R in portions of 0.2 ml until no further precipitate is formed.
Filter and wash the precipitate with a total of 10 ml of water R added in small portions. Dissolve the
precipitate in 2 ml of hot alcohol R and pour the solution into 5 ml of water R. Allow to stand for 4 h,
filter, wash the crystals with water R and dry at 100C to 105C. The crystals melt (2.2.14) at 191C
to 196C.
TESTS
Reference solution (a). Dissolve 0.1 g of piperazine hydrate CRS in a mixture of 2 volumes of ethanol R
31-44
solution (d) shows two clearly separated spots.
ASSAY
Dissolve 80.0 mg in 10 ml of anhydrous acetic acid R with gentle heating and dilute to 70 ml with the
1 ml of 0.1M perchloric acid is equivalent to 9.705 mg of C4H10N2,6H20.
STORAGE
Store in an airtight container, protected from light
_______________________________________________________________________________ Ph Eur
31-45
Piperazine Phosphate
HN
NH ,H3PO4
C4H10N2,H3PO4,H2O
202.1
18534-18-4
Definition Piperazine Phosphate contains not less than 98.5% and not more than 100.5% of
C4H10N2,H3PO4, calculated with reference to the anhydrous substance.
Sparingly soluble in water; practically insoluble in ethanol (96%).
Identification
A. Dissolve 0.1 g in 5 ml of water, add 0.5 g of sodium hydrogen carbonate, 0.5 ml of a 5% w/v solution
of potassium hexacyanoferrate(III) and 0.1 ml of mercury. Shake vigorously for 1 minute and allow to
stand for 20 minutes. A reddish colour is produced slowly.
B. Dissolve 0.2 g in 5 ml of 2M hydrochloric acid, add with stirring 1 ml of a 50% w/v solution of
sodium nitrite and cool in ice for 15 minutes, stirring if necessary to induce crystallisation. The melting
point of the crystals, after washing with 10 ml of iced water and drying at 105, is about 159,
Appendix V A.
C. A solution yields the reactions characteristic of phosphates, Appendix VI.
Heavy metals Dissolve 2.0 g in 20 ml of 2M acetic acid. 12 ml of the resulting solution complies with
limit test A for heavy metals, Appendix VII. Use lead standard solution (2 ppm Pb) to prepare the
standard (20 ppm).
Water 8.0 to 9.5% w/w, Appendix IX C. Use 0.25 g.
Assay Dissolve 0.2 g in a mixture of 3.5 ml of 0.5M sulphuric acid and 10 ml of water. Add 100 ml of
picric acid solution R1, heat on a water bath for 15 minutes and allow to stand for 1 hour. Filter
through a sintered-glass filter (BS porosity No. 4) and wash the residue with successive 10-ml
quantities of a mixture of equal volumes of a saturated solution of picric acid and water until the
washings are free from sulphate. Wash the residue with five 10-ml quantities of absolute ethanol and
dry to constant weight at 100 to 105. Each g of residue is equivalent to 0.3382 g of
C4H10N2,H3PO4.
Preparation
Piperazine Phosphate Tablets
31-46
Piretanide
1/01
N
COOH
O
SO2NH2
C17H18N2O5S
362.4
55837-27-9
Piretanide complies with the requirements of the 3rd edition of the European Pharmacopoeia [1556]. These
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Piretanide contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of
4-phenoxy-3-(pyrrolidin-1-yl)-5-sulphamoylbenzoic acid, calculated with reference to the dried
substance.
CHARACTERS
A yellowish-white to yellowish powder, very slightly soluble in water, sparingly soluble in ethanol.
IDENTIFICATION
with piretanide CRS. Examine the substances prepared as discs. If the spectra obtained show differences, dissolve the substance to be examined and the reference substance separately in acetone R,
TESTS
The solution is clear (2.2.1) and not more intensely coloured than reference solution GY4 (Method II,
2.2.2).
Test solution. Dissolve 20 mg of the substance to be examined in a mixture of 10 volumes of ethanol R,
45 volumes of acetonitrile R and 45 volumes of water R and dilute to 20.0 ml with the same mixture of
solvents.
Reference solution (a). Dissolve 10 mg of piretanide CRS and 3 mg of piretanide impurity A CRS in a
mixture of 10 volumes of ethanol R, 45 volumes of acetonitrile R and 45 volumes of water R and dilute
to 10.0 ml with the same mixture of solvents.
Reference solution (b). Dilute 0.3 ml of the test solution to 100.0 ml with a mixture of 10 volumes of
ethanol R, 45 volumes of acetonitrile R and 45 volumes of water R.
a stainless steel column 0.125 m long and 4 mm in internal diameter packed with octylsilyl silica
volumes of a solution prepared as follows: add 1 ml of trifluoroacetic acid R to 500 ml of water R,
add 1 ml of triethylamine R and dilute to 1000 ml with water R,
described the relative retention of impurity A is about 0.9. The test is not valid unless the resolution
between the peaks corresponding to impurity A and piretanide is not less than 2.
of the test solution for 5 times the retention time of the principal peak. In the chromatogram
31-47
cent); the sum of the areas of all the peaks, apart from the principal peak, is not greater than 3.33
100C to 105C for 4 h.
ASSAY
STORAGE
IMPURITIES
N
COOH
O
SO2NH2
A. 4-phenoxy-3-(1H-pyrrol-1-yl)-5-sulphamoylbenzoic acid,
COOMe
N
O
SO2N
NMe2
B. methyl-3-[[(dimethylamino)methylene]sulphamoyl]-4-phenoxy-5-(pyrrolidin-1yl)benzoate,
N
COOH
C. 4-(pyrrolidin-1-yl)dibenzo[b,d]furan-2-carboxylic acid.
___________________________________________________________________________________________________ Ph Eur
31-48
Piroxicam
1/01
O
O
S
NMe
H
N
OH
C15H13N3O4S
O
331.4
36322-90-4
Piroxicam complies with the requirements of the 3rd edition of the European Pharmacopoeia [0944]. These
Preparations
Piroxicam Capsules
Piroxicam Gel
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Piroxicam contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of
4-hydroxy-2-methyl-N-(pyridin-2-yl)-2H-1,2-benzothiazine-3-carboxamide 1,1-dioxide, calculated
CHARACTERS
A white or slightly yellow, crystalline powder, practically insoluble in water, soluble in methylene
chloride, slightly soluble in ethanol.
IDENTIFICATION
with piroxicam CRS. Examine the substances at discs prepared using potassium bromide R. If the
spectra obtained in the solid state show differences, dissolve the substance to be examined and the
reference substance separately in the minimum volume of methylene chloride R, evaporate to dryness
on a water-bath and record new spectra using the residues.
TESTS
Test solution. Dissolve 75 mg of the substance to be examined in acetonitrile R warming slightly if
necessary and dilute to 50.0 ml with the same solvent.
Reference solution (a). Dissolve 10 mg of piroxicam for system suitability in acetonitrile R and dilute to
Reference solution (b). Dilute 1.0 ml of the test solution to 10.0 ml with acetonitrile R. Dilute 1.0 ml of
the solution to 50.0 ml with acetonitrile R.
as mobile phase at a flow rate of 1 ml/min, a mixture of 40 volumes of acetonitrile R and 60
volumes of a 6.81 g/l solution of potassium dihydrogen phosphate R adjusted to pH 3.0 with
phosphoric acid R,
Inject 20 l of each solution and continue the chromatography for five times the retention time of
piroxicam. The test is not valid unless the chromatogram obtained with reference solution (a) has s
similar profile to the chromatogram supplied with piroxicam for system suitability CRS and shows a
peak due to impurity B with a relative retention of about 0.85 and a symmetry factor of at most 1.5.
solution (b) (0.2 per cent) and the sum of the areas of such peaks is not greater than twice times the
Disregard any peak with an area less than 0.1 times that of the principal peak in the chromatogram
31-49
100C to 105C for 4 h.
ASSAY
Dissolve 0.250 g in 60 ml of a mixture of equal volumes of acetic anhydride R and anhydrous acetic
acid R. Titrate with 0.1M perchloric acid, determining the end-point potentiometrically (2.2.20).
STORAGE
IMPURITIES
2N
A. pyridin-2-ylamine,
O
O
S
NH
H
N
OH
B. 4-hydroxy-N-(pyridin-2-yl)-2H-1,2-benzothiazine-3-carboxamide 1,1-dioxide,
Imp.A
The following type chromatograms are given for information and guidance only; they do not form a
mandatory part of the monograph.
0.0500
AU
0.0400
0.0300
0.0100
Imp.J
Imp.C
Imp.D
Imp.G
Imp.B
Piroxicam
0.0200
0.0000
0.00
10.00
20.00
30.00
Minutes
Fig. 0944-1 Sample of piroxicam spiked with the impurities common to the three routes of synthesis and the
impurities from the methyl route
40.00
Imp.A
31-50
0.0500
AU
0.0400
0.0300
Imp.K
Imp.E
Imp.H
Imp.B
0.0100
Piroxicam
Imp.C
0.0200
0.0000
0.00
10.00
20.00
30.00
40.00
Minutes
Imp.A
impurities from the ethyl route
0.0500
AU
0.0400
0.0300
Imp.L
Imp.I
Imp.F
Imp.B
0.0100
Piroxicam
Imp.C
0.0200
0.0000
0.00
10.00
20.00
30.00
Minutes
impurities from the isopropyl route
40.00
31-51
O
O
S
NH
NH2
OH
C. 4-hydroxy-2-methyl-2H-1,2-benzothiazine-3-carboxamide 1,1-dioxide,
O
O
S
COOR
O
D. R = CH3: methyl (3-oxo-2,3-dihydro-1,2-benzisothiazol-2-yl) acetate 1,1-dioxide,

E. R = C2H5: ethyl (3-oxo-2,3-dihydro-1,2-benzisothiazol-2-yl) acetate 1,1-dioxide,
F. R = CH(CH3)2: isopropyl (3-oxo-2,3-dihydro-1,2-benzisothiazol-2-yl)acetate 1,1-dioxide,
O
O
S
NR2
OR1
OH
G. R1 = CH3, R2 = H: methyl 4-hydroxy-2H-1,2-benzothiazine-3-carboxylate 1,1-dioxide,

H. R1 = C2H5, R2 = H: ethyl 4-hydroxy-2H-1,2-benzothiazine-3-carboxylate 1,1-dioxide,
I. R1 = CH(CH3) 2, R2 = H: isopropyl 4-hydroxy-2H-1,2-benzothiazine-3-carboxylate 1,1-dioxide,
J. R = CH3, R2 = CH3: methyl 4-hydroxy-2-methyl-2H-1,2-benzothiazine-3-carboxylate 1,1dioxide,
K. R = C2H5, R2 = CH3: ethyl 4-hydroxy-2-methyl-2H-1,2-benzothiazine-3-carboxylate 1,1dioxide,
L. R = CH(CH3) 2, R2 = CH3: isopropyl 4-hydroxy-2-methyl-2H-1,2-benzothiazine-3-carboxylate
1,1-dioxide.
___________________________________________________________________________________________________ Ph Eur
31-52
Pivampicillin
O
NH2
O
H
Me
N
H
N
S
H
CMe3
Me
O
C22H29N3O6S
463.5
33817-20-8
Pivampicillin complies with the requirements of the 3rd edition of the European Pharmacopoeia [0852]. These
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Pivampicillin contains not less than 95.0 per cent and not more than the equivalent of 101.0 per cent
of methylene (2S,5R,6R)-6-[[(2R)-2-amino-2-phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1azabicyclo[3.2.0]heptane-2-carboxylate 2,2-dimethylpropanoate, calculated with reference to the
CHARACTERS
A white or almost white, crystalline powder, practically insoluble in water, freely soluble in methanol,
soluble in ethanol. It dissolves in dilute acids.
IDENTIFICATION
obtained with pivampicillin CRS.
substance.
Reference solution (a). Dissolve 10 mg of pivampicillin CRS in 2 ml of methanol R.
Reference solution (b). Dissolve 10 mg of bacampicillin hydrochloride CRS, 10 mg of pivampicillin CRS
and 10 mg of talampicillin hydrochloride CRS in 2 ml of methanol R.
of a 272 g/l solution of sodium acetate R, adjusted to pH 5.0 with glacial acetic acid R, 40 volumes of
water R and 50 volumes of alcohol R. Dry the plate in a current of warm air, spray with ninhydrin
solution R1 and heat the plate at 60C for 10 min. The principal spot in the chromatogram obtained
obtained with reference solution (a). The test is not valid unless the chromatogram obtained with
reference solution (b) shows three clearly separated spots.
by swirling; the solution is almost colourless. Place the test-tube in a water-bath for 1 min; a dark
yellow colour develops.
TESTS
Appearance of solution Dissolve 50 mg in 12 ml of 0.1M hydrochloric acid. The solution is not more
opalescent than reference suspension II (2.2.1) and not more intensely coloured than reference
solution B7 (Method I, 2.2.2).
Specific optical rotation (2.2.7). Dissolve 0.100 g in 5.0 ml of alcohol R and dilute to 10.0 ml with
0.1M hydrochloric acid. The specific optical rotation is 208 to 222, calculated with reference to the
before use.
31-53
Test solution. Dissolve 50.0 mg of the substance to be examined in 10.0 ml of acetonitrile R and dilute
to 20 ml with a 1 g/l solution of phosphoric acid R.
Reference solution. Mix 2.0 ml of the test solution with 9.0 ml of acetonitrile R and 9.0 ml of a 1 g/l
solution of phosphoric acid R.
a column 0.125 m long and 4 mm in internal diameter packed with end-capped octylsilyl silica gel
for chromatography R,
as mobile phase at a flow rate of 1.5 ml per minute:
Mobile phase A. A mixture of 50 volumes of a 1.32 g/l solution of ammonium phosphate R,
adjusted to pH 2.5 with a 100 g/l solution of phosphoric acid R, and 50 volumes of acetonitrile R,
Mobile phase B. A mixture of 15 volumes of a 1.32 g/l solution of ammonium phosphate R,
adjusted to pH 2.5 with a 100 g/l solution of phosphoric acid R, and 85 volumes of acetonitrile R,
Interval
(min)
Mobile phase A
(per cent V/V)

(per cent V/V)
010
1012
1217
100
0
100
0
100
0
isocratic
isocratic
re-equilibration

Inject 50 l of the test solution and 50 l of the reference solution. The test is not valid unless the
ratio of the mass distribution ratio of pivampicillin dimer (which has a retention time of about 5 min)
to that of pivampicillin (principal peak) is at least twelve. In the chromatogram obtained with the test
solution, the sum of the areas of all the peaks, apart from the principal peak is not greater than 0.3
times the area of the principal peak in the chromatogram obtained with the reference solution (3 per
cent). Disregard any peak due to solvent and any peak with an area less than 0.01 times the area of
the principal peak in the chromatogram obtained with the reference solution.
Dimethylaniline Not more than 20 ppm, determined by gas chromatography (2.2.28), using
naphthalene R as the internal standard.
Internal standard solution. Dissolve 50.0 mg of naphthalene R in cyclohexane R and dilute to 50.0 ml
with the same solvent. Dilute 5.0 ml of this solution to 100.0 ml with cyclohexane R.
Test solution. To 1.00 g of the substance to be examined in a ground-glass-stoppered tube add 10 ml
of 0.5M sulphuric acid. Heat the tube for 10 min in a water-bath, cool and add 15 ml of 1M sodium
hydroxide and 1.0 ml of the internal standard solution. Stopper the tube and shake vigorously for
1 min. Centrifuge if necessary and use the upper layer.
Reference solution. To 50.0 mg of dimethylaniline R add 2 ml of hydrochloric acid R and 20 ml of
water R, shake to dissolve and dilute to 50.0 ml with water R. Dilute 5.0 ml of this solution to
250.0 ml with water R. To 1.0 ml of the solution in a ground-glass-stoppered tube add 5 ml of 1M
sodium hydroxide and 1.0 ml of the internal standard solution. Stopper the tube and shake vigorously
for 1 min. Centrifuge if necessary and use the upper layer.
a glass column 2 m long and 2 mm in internal diameter packed with silanised diatomaceous earth
for gas chromatography R impregnated with 3 per cent m/m of polymethylphenylsiloxane R,
detector at 150C. Inject 1 l of the test solution and 1 l of the reference solution.
Triethanolamine Examine by thin-layer chromatography (2.2.27), using silica gel H R as the coating
substance.
Test solution. Dissolve 0.100 g of the substance to be examined in 1.0 ml of a mixture of 1 volume of
water R and 9 volumes of acetonitrile R.
Reference solution. Dissolve 5.0 mg of triethanolamine R in a mixture of 1 volume of water R and 9
volumes of acetonitrile R and dilute to 100 ml with the same solvent.
5 volumes of methanol R, 15 volumes of butanol R, 24 volumes of phosphate buffer solution pH 5.8 R,
40 volumes of glacial acetic acid R and 80 volumes of butyl acetate R. Dry the plate at 110C for
10 min and allow to cool. Place in a chromatographic tank an evaporating dish containing a mixture
of 1 volume of hydrochloric acid R1, 1 volume of water R and 2 volumes of a 15 g/l solution of
potassium permanganate R. Close the tank and allow to stand for 15 min. Place the dried plate in the
tank and close the tank. Leave the plate in contact with the chlorine vapour in the tank for 15 min to
20 min. Remove the plate, allow it to stand in air for 2 min to 3 min and spray with tetramethyldiaminodiphenylmethane reagent R. Any spot corresponding to triethanolamine in the chromato-
31-54
gram obtained with the test solution is not more intense than the spot in the chromatogram obtained
with the reference solution (0.05 per cent).
of water.
ASSAY
Examine by liquid chromatography (2.2.29). Use the solutions within 2 h of preparation.
Reference solution (a). Dissolve 50.0 mg of pivampicillin CRS in the mobile phase and dilute to 50.0 ml
with the mobile phase. Dilute 10.0 ml of the solution with 50.0 ml with the mobile phase.
Reference solution (b). Dissolve 25.0 mg of propyl parahydroxybenzoate CRS in the mobile phase and
dilute to 50.0 ml with the mobile phase. Dilute 10.0 ml of the solution to 50.0 ml with the mobile
phase. Mix 5.0 ml of this solution with 5.0 ml of reference solution (a).
as mobile phase at a flow rate of 1.5 ml per minute a mixture of 40 volumes of acetonitrile R and
60 volumes of a 2.22 g/l solution of phosphoric acid R adjusted to pH 2.5 with triethylamine R,
Inject 20 l of reference solution (b). Adjust the sensitivity of the system so that the height of each
of the two principal peaks in the chromatogram obtained are at least 50 per cent of the full scale of
the recorder. The test is not valid unless the resolution between the first peak (pivampicillin) and the
second peak (propyl parahydroxybenzoate) is at least 5.0 and the symmetry factor for the pivampicillin peak is at most 2.0. Inject 20 l of reference solution (a) six times. The test is not valid unless
the relative standard deviation for the area of the principal peak is at most 1.0 per cent. Inject alternately the test solution and reference solution (a).
STORAGE
IMPURITIES
O
H NH2 H
N
O
H
HN
*
CMe3
Me
Me
COOH
A. 2-[[(2R)-2-amino-2-phenylacetyl]amino]-2-[(4S)-4-[[[(2,2-dimethylpropanoyl)oxy]methoxy]carbonyl]-5,5-dimethyl-thiazolidin-2-yl]acetic acid (penicilloic acids of

pivampicillin),
O
H
HN
H
N * *S
O
*
N
H
O
Me
Me
O
CMe3
B. methylene (4S)-5,5-dimethyl-2-(3,6-dioxo-5-phenylpiperazin-2-yl)thiazolidine-4carboxylate 2,2-dimethylpropanoate (diketopiperazines of pivampicillin),
31-55
HN
H
N
O
HN
O
NH
H
N
O
Me
O
CMe3
Me
*S
H
HN
H
N
O
Me
O
CMe3
Me
H H
O
C. co-oligomers of pivampicillin and of penicilloic acids of pivampicillin.
_______________________________________________________________________________ Ph Eur
31-56
Pivmecillinam Hydrochloride
O
Me
O
Me
O
N
N
S
H
C21H33N3O5S,HCl
O
Me
Me
,HCl
Me
476.0
32887-03-9
Pivmecillinam Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia [1359]. These requirements are reproduced after the heading Definition below.
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Pivmecillinam hydrochloride contains not less than 97.0 per cent and not more than the equivalent of
101.5 per cent of methylene 2,2-dimethylpropanoate (2S,5R,6R)-6-[[(hexahydro-1H-azepin-1yl)methylene]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate hydrochloride, calculated with reference to the anhydrous substance.
PRODUCTION
If manufactured by a process that may leave residues of dimethylaniline in the substance and/or by a
process using starting materials or intermediates which contain residues of dimethylaniline, it
complies with the following test:
CHARACTERS
A white or almost white, crystalline powder, freely soluble in water, in ethanol and in methanol,
slightly soluble in acetone.
IDENTIFICATION
obtained with pivmecillinam hydrochloride CRS. Examine the substances prepared as discs.
TESTS
than reference solution B8 (Method I, 2.2.2).
20 l of reference solution (b). Adjust the sensitivity of the system so that the height of the principal
peak in the chromatogram obtained is at least 50 per cent of the full-scale of the recorder. Inject 20 l
of test solution (b) and continue the chromatography for three times the retention time of the
principal peak. In the chromatogram obtained with test solution (b): the area of any peak, apart from
obtained with reference solution (b) (1.5 per cent); the sum of the areas of all the peaks, apart from
the principal peak, is not greater than three times the area of the principal peak in the chromatogram
obtained with reference solution (b) (3 per cent). Disregard any peak with an area less than 0.1 times
that of the principal peak in the chromatogram obtained with reference solution (b).
standard solution (1 ppm) R.
of water.
31-57
ASSAY
Examine by liquid chromatography (2.2.29). Prepare the test and reference solutions immediately before
use.
Solvent mixture. To 45 volumes of acetonitrile R add 55 volumes of a 13.5 g/l solution of potassium
dihydrogen phosphate R previously adjusted to pH 3.0 with dilute phosphoric acid R.
Test solution (b). Dissolve 25.0 mg of the substance to be examined in the solvent mixture and dilute
Reference solution (a). Dissolve 20.0 mg of pivmecillinam hydrochloride CRS in the solvent mixture and
Reference solution (c). Dissolve 5 mg of pivmecillinam hydrochloride CRS and 5 mg of pivmecillinam
impurity C CRS in the solvent mixture and dilute to 50 ml with the same solvent.
tetraethylammonium hydrogen sulphate R and 1.0 g of tetramethylammonium hydrogen sulphate R in
the solvent mixture and dilute to 1000 ml with the same solvent,
recorder. The test is not valid unless, in the chromatogram obtained, the resolution between the first
peak (pivmecillinam) and the second peak (impurity C) is at least 3.5. Inject reference solution (a) six
times. The test is not valid unless the relative standard deviation of the peak area for pivmecillinam is
at most 1.0 per cent. Inject alternately test solution (a) and reference solution (a).
STORAGE
Store protected from light, at a temperature of 2C to 8C.
IMPURITIES
H3C CH3
O
O
N
H2N
CH3
O
Me
Me
S
H H
A. methylene (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2carboxylate 2,2-dimethylpropanoate (pivaloyloxymethyl 6-aminopenicillanate),

H3C CH3
O
H
N
HN
*
CH3
O
Me
Me
COOH
B. 2-[[(hexahydro-1H-azepin-1-yl)methylene]amino]-2-[(4S)-4-[[[(2,2-dimethylpropanoyl)oxy]methoxy]carbonyl]-5,5-dimethylthiazolidin-2-yl]acetic acid (penicilloic acids of

pivmecillinam),
H3C CH3
O
H
N
HN
*
Me
S
CH3
O
and epimer at C*
Me
C. methylene 2,2-dimethylpropanoate (2RS,4S)-2-[[[(hexahydro-1H-azepin-1-yl)methylene]amino]methyl]-5,5-dimethylthiazolidin-4-carboxylate,
31-58
H3C CH3
O
H
O
H
N
N
HN
*
Me
S
CH3
O
Me
D. methylene 2,2-dimethylpropanoate (4S)-2-[1-(formylamino)-2-(hexahydro-1H-azepin-1yl)-2-oxoethyl]-5,5-dimethylthiazolidin-4-carboxylate.
_________________________________________________________________
Ph Eur
31-59
Pizotifen Malate
S
H
,
OH
COOH
HOOC
N
Me
C19H21NS,C4H6O5
429.5
5189-11-7
Definition Pizotifen Malate is 9,10-dihydro-4-(1-methylpiperidin-4-ylidene)-4H-benzo[4,5]cyclohepta[1,2-b]thiophene hydrogen malate. It contains not less than 98.5% and not more than 101.5%
of C19H21NS, C4H6O5, calculated with reference to the dried substance.
Characteristics A white or slightly yellowish white, crystalline powder; odourless or almost odourless.
Very slightly soluble in water; slightly soluble in chloroform and in ethanol (96%); sparingly soluble
in methanol.
Identification
pizotifen malate (RS 277).
C. Carry out the method for thin-layer chromatography, Appendix III A, using silica gel G as the coating substance and a mixture of 70 volumes of isopropyl ether, 25 volumes of anhydrous formic acid and
5 volumes of water as the mobile phase. Apply separately to the plate 5 l of each of the following
solutions. For solution (1) dissolve 30 mg of the substance being examined in 1 ml of ethanol (80%),
heating if necessary. Solution (2) contains 1% w/v of malic acid in ethanol (80%). After removal of the
plate, dry it at 100 for 30 minutes, cool, spray with 0.02M potassium permanganate and dry in a
current of warm air for about 1 minute. The chromatogram obtained with solution (1) exhibits a spot
corresponding in position, colour and size to the spot in the chromatogram obtained with solution
(2).
Clarity and colour of solution A 1.0% w/v solution in a mixture of equal volumes of ethanol (96%)
and water is clear, Appendix IV A, and not more intensely coloured than reference solution BY6,
Appendix IV B, Method II.
gel G as the coating substance and a mixture of 100 volumes of toluene, 60 volumes of butan-1-ol, 40
volumes of hexane and 3 volumes of 13.5M ammonia as the mobile phase. Apply separately to the
plate 5 l of each of five solutions in a mixture of 9 volumes of chloroform and 1 volume of methanol
containing (1) 2.0% w/v, (2) 0.20% w/v, (3) 0.010% w/v and (4) 0.0050% w/v of the substance
being examined and (5) 0.20% w/v of pizotifen malate BPCRS. After removal of the plate, dry it in a
current of cold air for 5 minutes, spray with a mixture of 1 volume of potassium iodobismuthate solution
and 10 volumes of 2M acetic acid and then with hydrogen peroxide solution (10 vol), cover immediately
with a glass plate and examine in daylight. Any secondary spot in the chromatogram obtained with
solution (1) is not more intense than the spot in the chromatogram obtained with solution (3) and
not more than one such spot is more intense than the spot in the chromatogram obtained with solution (4). Disregard any yellow spot or band remaining on the line of application.
Loss on drying When dried to constant weight at 100 to 105, loses not more than 0.5% of its
weight. Use 1 g.
Sulphated ash Not more than 0.2%, Appendix IX A, Method II. Use the residue obtained in the
test for Loss on drying.
Assay Dissolve 0.35 g in 60 ml of anhydrous acetic acid and carry out Method I for non-aqueous
titration, Appendix VIII A, determining the end point potentiometrically. Each l of 0.1M perchloric
acid VS is equivalent to 42.95 mg of C19H21NS,C4H6O5.
Storage Pizotifen Malate should be protected from light.
Action and use Prophylaxis of migraine.
Preparation
Pizotifen Tablets
31-60
Podophyllum Resin
Podophyllin
Definition Podophyllum Resin is the resin obtained from rhizomes and roots of Podophyllum
hexandrum Royle (P. emodi Wall.). It contains not less than 50.0% of total aryltetralin lignans, calculated as podophyllotoxin.
Characteristics An amorphous powder varying in colour from light brown to greenish yellow, or
brownish grey masses; odour, characteristic; caustic.
Partly soluble in hot water, from which it is precipitated on cooling, in chloroform, in ether and in 5M
ammonia.
Identification Carry out the method for thin-layer chromatography, Appendix III A, using a silica gel
precoated plate (Merck silica gel 60 plates are suitable) and a mixture of 1 volume of methanol and 25
volumes of chloroform as the mobile phase, but allowing the solvent front to ascend 10 cm above the
line of application. Apply separately to the plate, as bands 15 mm long and not more than 3 mm
wide, 10 l of each of two solutions in absolute ethanol containing (1) 1% w/v of the substance being
examined and (2) 0.5% w/v of podophyllotoxin. After removal of the plate, allow it to dry in air, spray
with methanolic sulphuric acid (50%) and heat at 130 for 10 minutes. The chromatogram obtained
with solution (1) exhibits a band corresponding in position and colour to the principal band in the
chromatogram obtained with solution (2). It does not show greyish bands in the middle third of the
chromatogram but other coloured bands may be present.
Matter insoluble in ethanol (96%) Shake 1 g, finely powdered, with 20 ml of ethanol (96%) for 5
minutes, filter through a sintered-glass crucible (BS porosity No. 2), wash the filter with ethanol
(96%) and dry at 105. The residue weighs not more than 25 mg.
Matter insoluble in 5M ammonia Shake 0.5 g, finely powdered, with 30 ml of 5M ammonia for 30
minutes at about 20; filter through a sintered-glass crucible (BS porosity No. 2) and wash the flask
and filter with 30 ml of water, the time taken for filtering and washing being not more than 10
minutes. Dry the filter and residue to constant weight at 105. The residue weighs not less than
0.18 g and not more than 0.30 g.
1 g.
Assay Dissolve 0.5 g in sufficient ethanol (96%) to produce 100 ml. To 10 ml of this solution in a
separating funnel add 190 ml of water and extract with six 30-ml quantities of dichloromethane.
Combine the dichloromethane layers, extract with 10 ml of 0.2M sodium hydroxide followed by five
10-ml quantities of water and wash each of the six aqueous layers separately with the same 20-ml
quantity of dichloromethane. Combine the dichloromethane solutions, filter through absorbent cotton
and evaporate the filtrate to dryness. Dissolve the residue in sufficient ethanol (96%) to produce
100 ml, dilute 10 ml of this solution to 50 ml with ethanol (96%) and measure the absorbance of the
resulting solution at the maximum at 292 nm, Appendix II B. Calculate the content of total
aryltetralin lignans expressed as podophyllotoxin, taking 105.4 as the value of A(1%, 1 cm) at the
maximum at 292 nm.
Storage Podophyllum Resin should be kept in a well-closed container and protected from light. On
exposure to light, or to temperatures above 25, it becomes darker in colour.
Labelling The label states the botanical source.
Action and use Used in treatment of warts.
Preparation
Compound Podophyllin Paint
31-61
Poldine Metilsulfate
Me
Ph
Ph
Me
+
N
CH3SO4
O
HO
H
O
and enantiomer
C22H29NO7S
451.5
545-80-2
Definition Poldine Metilsulfate is (RS)-2-benziloyloxymethyl-1,1-dimethylpyrrolidinium methyl

sulphate. It contains not less than 98.5% and not more than 100.5% of C22H29NO7S, calculated
Freely soluble in water; soluble in ethanol (96%); slightly soluble in chloroform.
Identification
A. The infrared absorption spectrum, Appendix II A, is concordant with the reference spectrum of poldine
metilsulfate (RS 278).
B. The light absorption, Appendix II B, in the range 250 to 350 nm of a 0.1% w/v solution exhibits
maxima at 252 nm and 258 nm and inflections at 262, 264 and 268 nm. The absorbance at the
maximum at 252 nm is about 0.85 and at the maximum at 258 nm is about 0.99.
C. Dissolve 2 mg in 10 ml of water, add 20 ml of ammonium cobaltothiocyanate solution and 5 ml of
chloroform and shake well. The chloroform layer becomes blue.
Acidity or alkalinity pH of a 1% w/v solution, 5.0 to 7.0, Appendix V L.
chloroform, 30 volumes of methanol, 5 volumes of formic acid and 5 volumes of water as the mobile
phase. Apply separately to the plate 20 l of each of two solutions of the substance being examined in
a mixture of 5 volumes of methanol and 1 volume of formic acid containing (1) 1.0% w/v and (2)
0.010% w/v. After removal of the plate, allow it to dry in air, spray with sulphuric acid (50%) and heat
at 110 for 10 minutes. Any pink secondary spot in the chromatogram obtained with solution (1) is not
more intense than the spot in the chromatogram obtained with solution (2).
1 g.
Assay Dissolve 0.5 g in 10 ml of water in a stoppered flask, add 0.05 ml of cresol red solution and, if
necessary, sufficient 0.05M sodium hydroxide to produce a pale pink colour. Add 20 ml of 0.1M sodium
hydroxide VS, stopper the flask and allow to stand for 10 minutes at 25. Titrate with 0.1M hydrochloric acid VS using cresol red solution as indicator. Repeat the operation without the substance being
examined. The difference between the titrations represents the amount of alkali required. Each ml of
0.1M sodium hydroxide VS is equivalent to 45.15 mg of C22H29NO7S.
Preparation
Poldine Tablets
When poldine methylsulphate is prescribed or demanded, Poldine Metilsulfate shall be dispensed or
supplied.
31-62
Poloxamers
9003-11-6
Poloxamers comply with the requirements of the 3rd edition of the European Pharmacopoeia [1464]. These
Ph Eur ____________________________________________________________________________________________________
DEFINITION
Poloxamer is a synthetic block copolymer of ethylene oxide and propylene oxide. It is represented by
the following general formula:
Me
O
HO
O
O
Poloxamer Ethylene
type
oxide units
(a)
Propylene
oxide units
(b)
Content of
oxyethylene
(per cent)
Average molecular
mass
124
10 to 15
18 to 23
44.8 to 48.6
2090 to 2360
182
6 to 10
27 to 33
26.9 to 30.7
2200 to 2800
184
10 to 15
27 to 33
37.5 to 41.3
2600 to 3200
188
75 to 85
25 to 30
79.9 to 83.7
7680 to 9510
237
60 to 68
35 to 40
70.5 to 74.3
6840 to 8830
331
338
407
5 to 10
137 to 146
95 to 105
52 to 57
42 to 47
54 to 60
14.5 to 18.3
81.4 to 84.9
71.5 to 74.9
3400 to 4200
12,700 to 17,400
9840 to 14 600
It may contain an antioxidant, for example, butylhydroxytoluene.

CHARACTERS
Poloxamer 124, poloxamer 182, poloxamer 184 and poloxamer 331 are colourless or almost colourless liquids. Poloxamer 188, poloxamer 237, poloxamer 338 and poloxamer 407 are white or almost
white, waxy powders, microbeads or flakes having a melting point of about 50C.
All poloxamers, with the exception of poloxamer 331, are very soluble in water; all poloxamers are
very soluble in alcohol and practically insoluble in light petroleum (50C to 70C).
IDENTIFICATION
obtained with the corresponding chemical reference substance of the Ph. Eur.
B. It complies with the test for average molecular mass (see Tests).
TESTS
Solution S
Poloxamer 331. Dissolve 10.0 g in a mixture of 1 volume of carbon dioxide-free water R and 2 volumes
of alcohol R and dilute to 100 ml with the same mixture of solvents.
All other poloxamers. Dissolve 10.0 g in carbon dioxide-free water R and dilute to 100 ml with the same
solvent.
Residual ethylene oxide, propylene oxide and dioxan Determined by head-space gas chromatography (2.2.28), not more than 1 ppm of ethylene oxide and not more than 5 ppm each of propylene
oxide and dioxan.
Test preparation. Place 0.100 g of the substance to be examined in a suitable headspace injection vial.
Reference preparation. Place about 100 g of poloxamer 124 CRS in a suitable four-necked flask fitted
with a stirrer, a thermometer, a gas inlet tube, a dry ice trap and a vacuum outlet. Evacuate the flask
carefully to a pressure below 0.2 kPa, applying the vacuum slowly while observing the contents for
excessive foaming. After the foaming has subsided, pass nitrogen R through the flask, increasing the
pressure to 2 kPa, and heat the flask to 130C while increasing the pressure to 12 kPa. After 4 h, cool
the flask to room temperature, return the flask to atmospheric pressure while maintaining the flow of
31-63
nitrogen R; store the preparation under nitrogen R. To 50 g of this preparation in a suitable vial add
suitable quantities of propylene oxide R and dioxan R, determining the weights added by subtraction.
Add ethylene oxide R to this mixture in the following manner: transfer about 5 ml of ethylene oxide R at
2C to 5C to a beaker cooled in ice and transfer a suitable amount to the poloxamer mixture using a
chilled, gas-tight chromatographic syringe, seal the vial and shake; determine the amount of ethylene
oxide added by weight difference. Dilute this material with the prepared poloxamer to give preparations containing suitable concentrations between 1 ppm and 20 ppm each of ethylene oxide,
propylene oxide and dioxan (for example, 1, 5, 10 and 20 ppm). Place 0.100 g of each preparation in
a suitable headspace injection vial and seal.
a column 50 m long and 0.32 mm in internal diameter coated with a layer of poly(dimethyl)(diphenyl)siloxane R (film thickness 5m),
a flame ionisation detector,
maintaining the temperature of the column at 70C, then raising the temperature at a rate of 10C
per minute to 250C, maintaining the temperature of the injection port at 140C and that of the
detector at 250C.
Inject a suitable volume of the headspace gas above the test preparation and the reference preparation, maintaining the vials at 110C for 30 min before testing. When the chromatograms, are
recorded in the prescribed conditions, the relative retention times of ethylene oxide, propylene oxide
and dioxan are about 1.0, 1.3 and 3.1 respectively.
The test is not valid unless the resolution factor between the peaks corresponding to ethylene oxide
and propylene oxide in the chromatograms obtained with the test preparation is greater than 2.0.
Plot calibration curves as area against parts per million for each of ethylene oxide, propylene oxide
and dioxan using the chromatograms obtained from the reference preparations; no point should
deviate from the calculated straight line by more than 10 per cent. Calculate the content of ethylene
oxide, propylene oxide and dioxan in the substance to be examined from the areas of the peaks in the
chromatogram obtained with the test preparation and from the three calibration curves.
Average molecular mass Weigh 15 g (m g) of the substance to be examined into a 250 ml groundglass-stoppered flask, add 25.0 ml of phthalic anhydride solution R and a few glass beads and swirl to
dissolve. Boil gently under a reflux condenser for 1 h, allow to cool and add two quantities, each of
10 ml, of pyridine R through the condenser. Add 10 ml of water R, mix and allow to stand for 10 min.
Add 70.0 ml of 0.5M sodium hydroxide and 0.5 ml of a 10 g/l solution of phenolphthalein R in
pyridine R. Titrate with 0.5M sodium hydroxide to a light pink endpoint that persists for 15 s and
record the volume of sodium hydroxide used (S). Prepare a blank in the same manner but omitting
the substance to be examined. Record the volume of sodium hydroxide used (B).
Calculate the average molecular mass using the expression:
4000 m
BS
Oxypropylene : oxyethylene ratio Carry out the method for nuclear magnetic resonance spectrometry (2.2.33), using a 100 g/l solution of the substance to be examined in deuterated chloroform R.
Record the average area of the doublet appearing at about 1.08 ppm due to the methyl groups of the
oxypropylene units (A1) and the average area of the composite band from 3.2 to 3.8 ppm due to
CH2O groups of both the oxyethylene and oxypropylene units and the CHO groups of the
oxypropylene units (A2) with reference to the internal standard.
Calculate the percentage of oxyethylene, by weight, in the sample being examined using the
following expression:
3300
33 + 58
where =
A2
A1
of water.
STORAGE
LABELLING
The label states:
the poloxamer type,
the name and concentration of any added antioxidant.
___________________________________________________________________________________________________ Ph Eur
32-1
Polyacrylate Dispersion (30 per cent)

corrected 1/01
Polyacrylate Dispersion (30 per cent) complies with the requirements of the 3rd edition of the European
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Polyacrylate dispersion 30 per cent is a dispersion in water of a copolymer of ethyl acrylate and
methyl methacrylate having a mean relative molecular mass of about 800,000. It may contain a
suitable emulsifier. The residue on evaporation is not less than 28.5 per cent m/m and not more than
31.5 per cent m/m.
CHARACTERS
An opaque, white, slightly viscous liquid, miscible with water, soluble in acetone, in ethanol and in
2-propanol.
IDENTIFICATION
spectrum of polyacrylate.
B. To 1 g add 5 ml of water R and mix; the mixture remains opaque. Take three 1 g portions and mix
separately with 5 g each of ethanol R, acetone R and 2-propanol R. Transparent solutions are obtained.
C. To 1 g add 10 ml of 0.1M sodium hydroxide. The mixture remains opaque.
D. It complies with the test for appearance of a film (see Tests).
E. Dry 4 g in a Petri dish at 60C in an oven for 4 h and transfer the resulting clear film to a small
test-tube (100 mm 2 mm). Heat over a flame and collect the fumes that evolve in a second testtube held over the mouth of the first tube. The condensate gives the reaction of esters (2.3.1).
TESTS
Relative density (2.2.5). 1.037 to 1.047.
Apparent viscosity Determine the viscosity (2.2.10) using a rotating viscometer at 20C. At a shear
rate of 10 sl, the apparent viscosity is not more than 50 mPas.
Appearance of a film Pour 1 ml on a glass plate and allow to dry. A clear elastic film is formed.
Particulate matter Filter 100.0 g through a tared stainless steel sieve (90). Rinse with water R until
a clear filtrate is obtained and dry at 80C to constant mass. The mass of the residue is not more than
0.500 g.
Residual monomers Not more than 100 ppm, determined by liquid chromatography (2.2.29).
Test solution. Dissolve 1.00 g of the substance to be examined in tetrahydrofuran R and dilute to
50.0 ml with the same solvent. To 5.0 ml of a 35 g/l solution of sodium perchlorate R add 10.0 ml of
the solution dropwise whilst stirring continuously. Centrifuge and filter the clear supernatant liquid.
Dilute 5.0 ml to 10.0 ml with water R.
Reference solution. Dissolve 10 mg each of ethyl acrylate R and methyl methacrylate R in tetrahydrofuran R and dilute to 50.0 ml with the same solvent. Dilute 1.0 ml of this solution to 100.0 ml with
tetrahydrofuran R. To 10.0 ml of the final solution add 5.0 ml of a 35 g/l solution of sodium
perchlorate R and mix. Dilute 5.0 ml of the mixture to 10.0 ml with water R.
chromatography R (5 m to 10 m),
volumes of water R,
Inject separately equal volumes (about 50 l) of each solution.
reference solution.
32-2
Microbial contamination Total viable aerobic count (2.6.12) not more than 103 micro-organisms per
gram, determined by plate count.
ASSAY
Dry 1.000 g at 110C for 3 h and weigh the mass of the residue.
STORAGE
Store at a temperature of 5C to 25C. Protect from freezing. Handle the substance so as to minimise
microbial contamination.
LABELLING
The label states, where applicable, the name and concentration of any added emulsifier.
__________________________________________________________________________________________________________ Ph Eur
32-3
Polymyxin B Sulphate
RCODbuThrDbuDbuDbuD-PheLeu
DbuThr, H2SO4
Dbu =
L-2,4-diaminobutyric
acid
Polymyxin R
B1 -CH2[CH2]5CHMeCH3
B2 -CH2[CH2]4CHMeCH3
C56H98N16O13,H2SO4
1301
1405-20-5
Polymyxin B Sulphate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Polymyxin and Bacitracin Eye Ointment
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Polymyxin B sulphate is a mixture of the sulphates of polypeptides produced by the growth of certain
strains of Bacillus polymyxa or obtained by any other means. The potency is not less than 6500 I.U.
per milligram, calculated with reference to the dried substance.
CHARACTERS
A white or almost white powder, hygroscopic, soluble in water, slightly soluble in alcohol.
IDENTIFICATION
A. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance.
Test solution. Dissolve 5 mg of the substance to be examined in 1 ml of a mixture of equal volumes of
hydrochloric acid R and water R. Heat at 135C in a sealed tube for 5 h. Evaporate to dryness on a
water-bath and continue the heating until the odour of hydrochloric acid has been completely
removed. Dissolve the residue in 0.5 ml of water R.
Reference solution (a). Dissolve 20 mg of leucine R in water R and dilute to 10 ml with the same
solvent.
Reference solution (b). Dissolve 20 mg of threonine R in water R and dilute to 10 ml with the same
solvent.
Reference solution (c). Dissolve 20 mg of phenylalanine R in water R and dilute to 10 ml with the same
solvent.
Reference solution (d). Dissolve 20 mg of serine R in water R and dilute to 10 ml with the same solvent.
Carry out the following procedures protected from light.
Apply separately to the plate as 10 mm bands, 5 l of each solution. Place the plate in the chromatographic tank so that it is not in contact with the mobile phase; which consists of a mixture of 25
volumes of water R and 75 volumes of phenol R. Leave the plate to become impregnated with the
vapour of the solvent for at least 12 h. Develop over a path of 12 cm using the same mobile phase.
Dry the plate at 100C to 105C and spray with ninhydrin solution R1. Heat at 110C for 5 min. The
chromatogram obtained with the test solution shows bands corresponding to those in the chromatograms obtained with reference solutions (a), (b) and (c), but shows no band corresponding to that in
the chromatogram obtained with reference solution (d). The chromatogram obtained with the test
solution also shows a band with a very low Rf value (2,4-diaminobutyric acid).
B. Dissolve about 2 mg in 5 ml of water R and add 5 ml of a 100 g/l solution of sodium hydroxide R.
Shake and add dropwise 0.25 ml of a 10 g/l solution of copper sulphate R, shaking after each addition.
A reddish-violet colour develops.
C. It gives reaction (a) of sulphates (2.3.1).
TESTS
Phenylalanine 9 per cent to 12 per cent, calculated with reference to the dried substance. Dissolve
32-4
0.375 g (m g) of the substance to be examined in 0.1M hydrochloric acid and dilute to 100.0 ml with
the same solvent. Measure the absorbances (2.2.25) at the maxima at 264 nm (A264), 258 nm (A258)
and 252 nm (A252) and the absorbances at 300 nm (A300) and 280 nm (A280). Calculate the percentage content of phenylalanine from the expression:
9.4787
( A258 0.5 A252 + 0.5 A264 18
. A280 + 0.8 A300)
m
ammonia R.
Add 10.0 ml of 0.1M barium chloride and about 0.5 mg of phthalein purple R. Titrate with 0.1M
sodium edetate, adding 50 ml of alcohol R when the colour of the solution begins to change and
continuing the titration until the violet-blue colour disappears.
Sterility (2.6.1). If intended for parenteral administration, it complies with the test for sterility.
Pyrogens (2.6.8). If intended for parenteral administration, it complies with the test for pyrogens.
Inject per kilogram of the rabbits mass 1 ml of a solution in water for injections R containing 1.5 mg
per millilitre of the substance to be examined.
ASSAY
Carry out the microbiological assay of antibiotics (2.7.2).
The potency is not less than 6500 I.U. per milligram, calculated with reference to the dried
substance.
STORAGE
Store in an airtight container, protected from light. If the substance is sterile and intended for
parenteral administration, store in a sterile, airtight, tamper-proof container.
LABELLING
The label states:
where applicable, that the substance is apyrogenic.
__________________________________________________________________________________________________________________________________ Ph Eur
32-5
Polysorbate 20
Polyoxyethylene 20 Sorbitan Monolaurate
H
H O[C H 2C H 2O] w
[OC H 2C H 2] y OH
CH2 [OC H 2C H 2] z OC O[C H 2] 10C H 3
[OC H 2C H 2] x OH
w + x + y + z = 20
9005-64-5
Polysorbate 20 complies with the requirements of the 3rd edition of the European Pharmacopoeia [0426].
Action and use Non-ionic surface-active agent.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Polysorbate 20 is a mixture of partial lauric acid esters of sorbitol and its anhydrides copolymerised
with approximately 20 moles of ethylene oxide for each mole of sorbitol and sorbitol anhydrides. The
lauric acid used for the esterification may contain other fatty acids.
CHARACTERS
An oily, yellowish or brownish-yellow, clear or slightly opalescent liquid, miscible with water, with
ethanol, with ethyl acetate and with methanol, practically insoluble in fatty oils and in liquid paraffin.
IDENTIFICATION
A. Dissolve 0.5 g in water R at about 50C and dilute to 10 ml with the same solvent. The solution
produces a copious foam on shaking. Add 0.5 g of sodium chloride R and heat the solution to boiling.
The resulting cloudiness disappears during cooling to about 50C.
B. To 4 g add 40 ml of a 50 g/l solution of potassium hydroxide R and boil under a reflux condenser in
a water-bath for 30 min. Allow to cool to about 80C, add 20 ml of dilute nitric acid R and boil under
a reflux condenser for about 10 min to break the emulsion. Fatty acid separates on the surface as an
oily liquid. Allow to cool to room temperature. Transfer the fatty acid to a separating funnel with the
aid of 50 ml of light petroleum R, avoiding vigorous shaking. Wash the organic layer with three quantities, each of 5 ml, of water R. Evaporate the organic layer to dryness on a water-bath. The acid value
(2.5.1) of the residue is 245 to 300, determined on 0.30 g.
C. Dissolve 0.1 g in 5 ml of chloroform R. Add 0.1 g of potassium thiocyanate R and 0.1 g of cobalt
nitrate R. Stir with a glass rod. The solution becomes blue.
TESTS
Hydroxyl value (2.5.3). 96 to 108, determined on 2.0 g (Method A).
Saponification value (2.5.6). 40 to 50, determined on 2.0 g. Use 15.0 ml of 0.5M alcoholic
potassium hydroxide and dilute with 50 ml of alcohol R before carrying out the titration.
Reducing impurities Dissolve 2.00 g in 25 ml of hot water R and add 25 ml of dilute sulphuric
acid R and 0.1 ml of ferroin R. Titrate with 0.01M ammonium and cerium nitrate, shaking continuously,
until the colour change from red to greenish-blue persists for 30 s. Carry out a blank titration. Not
more than 2.0 ml of 0.01M ammonium and cerium nitrate is required.
of water.
Sulphated ash (2.4.14). Not more than 0.2 per cent. To 2.00 g in a silica or platinum crucible add
0.5 ml of sulphuric acid R and heat on a water bath for 2 h. Carefully ignite at a low temperature until
32-6
thoroughly charred. Add to the carbonised mass 2 ml of nitric acid R and 0.25 ml of sulphuric acid R,
cautiously heat until white fumes are evolved and ignite at 600C until all black particles have
disappeared. Allow to cool, weigh and repeat the ignition for periods of 15 min to constant mass.
STORAGE
__________________________________________________________________________________________________________________________________ Ph Eur
32-7
Polysorbate 60
1/01
Polyoxyethylene 20 Sorbitan Monostearate

H
H O[C H 2C H 2O] w
O
[OC H 2C H 2] y OH
CH2
[OC H 2C H 2] z OC O[C H 2] 16C H 3
[OC H 2C H 2] x OH
w + x + y + z = 20
9005-67-8
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Polysorbate 60 is a mixture of partial stearic acid esters of sorbitol and its anhydrides copolymerised
with approximately 20 moles of ethylene oxide for each mole of sorbitol and sorbitol anhydrides. The
stearic acid used for the esterification may contain other fatty acids, especially palmitic acid.
PRODUCTION
CHARACTERS
A yellowish-brown gelatinous mass which becomes a clear liquid at temperatures above 25C,
miscible with water, with ethanol, with ethyl acetate and with methanol, practically insoluble in fatty
oils and in liquid paraffin.
IDENTIFICATION
B. To 4 g add 40 ml of a 50 g/l solution of potassium hydroxide R and boil under a reflux condenser
for 60 min. Allow to cool to about 80C, add 20 ml of dilute nitric acid R and boil under a reflux
condenser for about 10 min to break the emulsion. Fatty acid separates on the surface as an oily
liquid. Allow to cool to room temperature. Transfer the fatty acid to a separating funnel with the aid
of 100 ml of light petroleum R, avoiding vigorous shaking. Wash the organic layer with three quantities, each of 10 ml, of water R. Evaporate the organic layer to dryness on a water-bath. Dry the
residue in an oven at 100C to 105C for 2 h to eliminate the residual solvents. The acid value
C. Dissolve 0.1 g in 5 ml of chloroform R. Add 0.1 g of potassium thiocyanate R and 0.1 g of cobalt
nitrate R. Stir with a glass rod. A blue colour is produced.
TESTS
Hydroxyl value (2.5.3, Method A). 81 to 96, determined on 2.0 g.
Saponification value (2.5.6). 45 to 55, determined on 2.0 g. Use 15.0 ml of 0.5M alcoholic potassium
hydroxide and dilute with 50 ml of alcohol R before carrying out the titration.
Reducing impurities Dissolve 2.00 g in 25 ml of hot water R and add 25 ml of dilute sulphuric
acid R and 0.1 ml of ferroin R. Titrate with 0.01M ammonium and cerium nitrate, shaking continuously,
until the colour change from red to greenish-blue persists for 30 s. Carry out a blank titration. Not
more than 2.0 ml of 0.01M ammonium and cerium nitrate is required.
32-8
of water.
Sulphated ash (2.4.14). Not more than 0.2 per cent. To 2.00 g in a silica or platinum crucible add
0.5 ml of sulphuric acid R and heat on a water bath for 2 h. Carefully ignite at a low temperature until
cautiously heat until white fumes are evolved and ignite at 600C until all black particles have
STORAGE
__________________________________________________________________________________________________________________________________ Ph Eur
32-9
Polysorbate 80
1/01
Polyoxyethylene 20 Sorbitan Mono-oleate

H
H O[C H 2 C H 2 O] w
[OC H 2 C H 2 ] y OH
O
C H 2 [OC H 2 C H 2 ] z OC O[C H 2 ] 7
[OC H 2 C H 2 ] x OH
H3C[CH2]7
w + x + y + z = 20
9005-65-6
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Polysorbate 80 is a mixture of partial esters of various fatty acids, mainly oleic acid, and sorbitol and
its anhydrides copolymerised with approximately 20 moles of ethylene oxide for each mole of sorbitol
and sorbitol anhydrides. The fatty-acid fraction can be of vegetable or animal origin. It contains not
less than 60.0 per cent of oleic acid and not less than 90.0 per cent and not more than 110.0 per cent
of the content stated on the label.
PRODUCTION
2-Chloroethanol, ethylene glycol and diethylene glycol Not more than 10 ppm of 2-chloroethanol and not more than a total of 0.25 per cent for ethylene glycol and diethylene glycol, determined by head-space gas chromatography (2.2.28), using the method of standard additions.
Test solution. Place 50 mg of the substance to be examined in a 20 ml vial, add 2.0 l of 2-propanol R
and close immediately. Allow to stand for about 2 min and in any case not more than 5 min.
Reference solution. Place 50 mg of the substance to be examined in a 20 ml vial, add 2.0 l of a
solution containing, in 2-propanol R, 0.25 mg/ml of 2-chloroethanol R, 31.25 mg ml of ethylene glycol R
and 31.25 mg/ml of diethylene glycol R and close immediately. Allow to stand for about 2 min and in
any case not more than 5 min.
a purge and trap head space sampler, assembled before the chromatographic system using a
stainless steel precolumn 13.6 mm long and 4 mm in internal diameter packed with
ethylvinylbenzene-divinylbenzene copolymer R as trap column and helium for chromatography R as
the carrier gas at a flow rate of 20 ml/min and an additional 20 ml/min as auxiliary gas. Place the
vials separately on a water-bath at 110C and begin purging within 5 minutes, whilst maintaining the trap column at 50C; continue purging for 40 min. Then raise the temperature of the
trap column to 210C and inject for 5 min in the chromatographic system,
a fused-silica column 30 m long and 0.53 mm in internal diameter, the internal wall of which is
coated with macrogol 20,000 for chromatography R (film thickness 1 m),
helium for chromatography R as the carrier gas at a linear velocity of 60 cm/s,
32-10
Time
(min)
Column
Temperature
(C)
06
60
6 16 60 110
16 31 110 230
31 36 230
Injection port
150
Detector
260
Rate (C
per min)
5
8
Comment
isothermal
linear gradient
linear gradient
isothermal
Calculate the content of 2-chloroethanol, ethylene glycol and diethylene glycol from the areas of
the peaks and the concentration of the solutions.
CHARACTERS
An oily, yellowish or brownish-yellow, clear liquid, miscible with water, with ethanol, with ethyl
acetate and with methanol, practically insoluble in fatty oils and in liquid paraffin.
It has a relative density of about 1.08. It has a viscosity of about 400 mPas at 25C.
IDENTIFICATION
Second identification: A, D, E.
B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the Ph.Eur. reference
spectrum of polysorbate 80 at the following wavelengths: 720 cm-1, 1110 cm-1, 1250 cm-1, 1300 cm-1,
1350 cm-1, 1640 cm-1, 1740 cm-1, 2850 cm-1, 2920 cm-1 and 3480 cm-1.
C. It complies with the limits of the assay.
D. To 2 ml of a 50 g/l solution, add 0.5 ml of bromine water R. The mixture is decolorised.
E. Dissolve 0.1 g in 5 ml of methylene chloride R. Add 0.1 g of potassium thiocyanate R and 0.1 g of
cobalt nitrate R. Stir with a glass rod. The solution becomes blue.
TESTS
Hydroxyl value (2.5.3, Method A). 65 to 80, determined on 2.0 g.
Peroxide value (2.5.5). Not more than 10.
Saponification value (2.5.6). 45 to 55, determined on 2.0 g. Use 15.0 ml of 0.5M alcoholic
potassium hydroxide and dilute with 50 ml of water R before carrying out the titration.
Residual ethylene oxide and dioxan (2.4.25, System A). Not more than 1 ppm of residual ethylene
oxide and not more than 10 ppm of residual dioxan. When determining the residual dioxan content,
apply a correction factor of 1/5 to the calculation.
Sulphated ash Not more than 0.25 per cent. To 2.00 g in a silica or platinum crucible, add 0.5 ml
of sulphuric acid R and heat on a water-bath for 2 h. Carefully ignite at a low temperature until
then cautiously heat until white fumes are evolved and ignite at 600C until all black particles have
Pyrogens (2.6.8). If intended for use in the manufacture of parenteral dosage forms without a
further appropriate procedure for the removal of pyrogens, it complies with the test for pyrogens.
Inject per kilogram of the rabbits mass 5.0 ml of a solution containing 2 mg/ml of the substance to
be examined in a 9 g/l solution of sodium chloride R.
ASSAY
Test solution. Dissolve 0.10 g of the substance to be examined in 2 ml of methanolic sodium hydroxide
solution R1 in a 25 ml conical flask and boil under a reflux condenser for 30 min. Add 2.0 ml of a
140 g/l solution of boron trifluoride R in methanol R through the condenser and boil for 30 min. Add
4 ml of heptane R through the condenser and boil for 5 min. Cool and add 10.0 ml of a saturated
32-11
solution of sodium chloride R, shake for about 15 s and add a quantity of a saturated solution of sodium
chloride R such that the upper layer is brought into the neck of the flask. Collect 1 ml of the upper
layer and dry it over anhydrous sodium sulphate R.
Reference solution. Dissolve 0.02 g of methyl oleate R in heptane R and dilute to 10 ml with the same
solvent. Dilute 1 ml of the solution to 50.0 ml with heptane R.
a fused-silica column 30 m long and 0.32 mm in internal diameter, the internal wall of which is
coated with macrogol 20,000 for chromatography R (film thickness 0.5 m),
helium for chromatography R as the carrier gas at a linear velocity of 50 cm/s,
Time
(min)
Column
Injection port
Temperature
(C)
04
70
4 38 70 240
38 53 240
Rate (C
per min)
5
Comment
isothermal
linear gradient
isothermal
280
Inject 0.1 l of the test solution and 0.1 l of the reference solution. The retention time of the
methyl ester of oleic acid is about 35 min. Continue the chromatography for 1.5 times the retention
time of the principal peak. Calculate the percentage content of oleic acid from the areas of the peaks
in the chromatogram obtained with the test solution, by the normalisation procedure. Disregard any
peak with an area less than that of the peak in the chromatogram obtained with the reference solution
(0.16 per cent).
STORAGE
LABELLING
The label states:
the content of oleic acid in the fatty-acid fraction,
where applicable, that the substance is free from pyrogens.
__________________________________________________________________________________________________________________________________ Ph Eur
32-12
Polythiazide
O
O
S
O
S
H2N
NMe
N
H H
Cl
CF3
and enantiomer
C11H13ClF3N3O4S3
439.9
346-18-9
Definition Polythiazide is (RS)-3,4-dihydro-2-methyl-3-(2,2,2-trifluoroethylthiomethyl)-1,2,4benzothiadiazine-7-sulphonamide 1,1-dioxide. It contains not less than 97.0% and not more than
102.0% of C11H13ClF3N3O4S3, calculated with reference to the dried substance.
Characteristics A white or almost white, crystalline powder; odour, alliaceous.
Practically insoluble in water; sparingly soluble in ethanol (96%); practically insoluble in chloroform.
Identification
A. The infrared absorption spectrum, Appendix II A, is concordant with the reference spectrum of polythiazide (RS 280).
methanol exhibits a maximum at 268 nm and a less well-defined maximum at 317 nm. The absorbance
at 268 nm is about 1.0.
C. Carry out the method for thin-layer chromatography, Appendix III A, using silica gel GF254 as the
coating substance and a mixture of 50 volumes of toluene, 30 volumes of ether and 20 volumes of
acetone as the mobile phase. Apply separately to the plate 20 l of each of two solutions in methanol
containing (1) 0.02% w/v of the substance being examined and (2) 0.02% w/v of polythiazide
BPCRS. After removal of the plate, allow it to dry in air until the solvent has evaporated, examine
under ultraviolet light (254 nm) and then treat the plate by Method I and examine again. The principal
spot in the chromatogram obtained with solution (1) corresponds in colour and intensity to that in
the chromatogram obtained with solution (2).
gel G as the coating substance and a mixture of 10 volumes of methanol and 90 volumes of chloroform
as the mobile phase. Apply separately to the plate 5 l of solution (1) and quantities of 1, 2, 3, 4 and
5 l of solution (2). Solutions (1) and (2) are solutions of the substance being examined in acetone
containing (1) 1.0% w/v and (2) 0.020% w/v. After removal of the plate, allow it to dry in air and
reveal the spots using Method I. Assess the intensity of any secondary spot in the chromatogram
obtained with solution (1) by reference to the spots in the chromatograms obtained with the applications of solution (2). The sum of the intensities so assessed does not exceed 3% and no such spot is
more intense than the spot in the chromatogram obtained with 5 l of solution (2) (2%).
1 g.
Assay Dissolve 0.1 g in sufficient methanol to produce 250 ml, dilute 5 ml to 200 ml with methanol
and measure the absorbance of the resulting solution at the maximum at 268 nm, Appendix II B.
Calculate the content of C11H13ClF3N3O4S3 taking 500 as the value of A(1%, 1 cm) at the maximum at 268 nm.
Preparation
Polythiazide Tablets
32-13
Potassium Acetate
C2H3O2K
98.14
127-08-2
Potassium Acetate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1139].
Action and use Used in solutions for dialysis.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Potassium acetate contains not less than 99.0 per cent and not more than the equivalent of 101.0 per
cent of C2H3KO2, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder or colourless crystals, deliquescent, very soluble in water, freely soluble in
alcohol.
IDENTIFICATION
A. It gives reaction (a) of acetates (2.3.1).
B. It gives reaction (a) of potassium (2.3.1).
TESTS
Reducing substances Dilute 10 ml of solution S to 100 ml with water R. Add 5 ml of dilute sulphuric
acid R and 0.5 ml of a 0.32 g/l solution of potassium permanganate R. Mix and boil gently for 5 min.
The solution remains pink.
Chlorides (2.4.4). Dilute 2.5 ml of solution S to 15 ml with water R. The solution complies with the
Sulphates (2.4.13). Dilute 7.5 ml of solution S to 15 ml with distilled water R. The solution complies
haemofiltration solutions or haemodialysis solutions, it complies with the test for aluminium.
Dissolve 2.0 g in 50 ml of water R and add 5 ml of acetate buffer solution pH 6.0 R. The solution
complies with the limit test for aluminium (1 ppm). Use as a reference solution a mixture of 1 ml of
aluminium standard solution (2 ppm Al) R, 5 ml of acetate buffer solution pH 6.0 R and 49 ml of water R.
To prepare the blank use a mixture of 5 ml of acetate buffer solution pH 6.0 R and 50 ml of water R.
Iron (2.4.9). Dilute 5 ml of solution S to 10 ml with water R. The solution complies with the limit
test for iron (20 ppm).
Sodium Not more than 0.5 per cent of Na, determined by atomic emission spectrometry (2.2.22).
diluted as necessary with water R.
at 100C to 105C.
ASSAY
Dissolve 80.0 mg in 20 ml of anhydrous acetic acid R. Add 0.2 ml of naphtholbenzein solution R. Titrate
with 0.1M perchloric acid. Carry out a blank titration.
1 ml of 0.1M perchloric acid is equivalent to 9.81 mg of C2H3KO2.
STORAGE
Store in a well-closed container, protected from moisture.
__________________________________________________________________________________________________________________________________ Ph Eur
32-14
Potassium Bicarbonate
KHCO3
100.1
298-14-6
Potassium Bicarbonate complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Potassium Hydrogen Carbonate [1141]. These requirements are reproduced after the heading Definition
below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Potassium hydrogen carbonate contains not less than 99.0 per cent and not more than the equivalent
of 101.0 per cent of KHCO3.
CHARACTERS
A white, crystalline powder or colourless crystals, freely soluble in water, practically insoluble in
alcohol. When heated in the dry state or in solution, it is gradually converted to potassium carbonate.
IDENTIFICATION
A. To 5 ml of solution S (see Tests) add 0.1 ml of phenolphthalein solution R. A pale pink colour is
produced. Heat; gas is evolved and the colour becomes red.
B. It gives the reaction of carbonates and bicarbonates (2.3.1).
C. 1 ml of solution S gives reaction (b) of potassium (2.3.1).
TESTS
Solution S. Dissolve 5.0 g in 90 ml of carbon dioxide-free water R prepared from distilled water R and
Appearance of solution. Solution S is clear (2.2.1) and colourless (Method II, 2.2.2).
Carbonates. The pH (2.2.3) of freshly prepared solution S is not more than 8.6.
Chlorides (2.4.4). Dilute 7 ml of solution S to 15 ml with dilute nitric acid R. The solution complies
with the limit test for chlorides (150 ppm).
Sulphates (2.4.13). Dilute 10 ml of solution S to 15 ml with acetic acid R. The solution complies
with the limit test for sulphates (150 ppm). Prepare the standard using a mixture of 7.5 ml of sulphate
standard solution (10 ppm SO4) R and 7.5 ml of distilled water R.
Ammonium (2.4.1). 10 ml of solution S diluted to 15 ml with water R complies with the limit test
for ammonium (20 ppm). Prepare the standard using a mixture of 5 ml of water R and 10 ml of
ammonium standard solution (1 ppm NH4 ) R.
Calcium (2.4.3). Dilute 10 ml of solution S to 15 ml with acetic acid R. The solution complies with
the limit test for calcium (100 ppm). Prepare the standard using 5 ml of calcium standard solution
(10 ppm Ca) R and 10 ml of distilled water R.
Heavy metals (2.4.8). Dissolve 2.0 g in a mixture of 2 ml of hydrochloric acid R and 18 ml of
water R. 12 ml of the solution complies with limit test A for heavy metals (10 ppm). Prepare the
Sodium Not more than 0.5 per cent of Na, determined by atomic emission spectrometry (Method II,
2.2.22).
ASSAY
Dissolve 0.800 g in 50 ml of carbon dioxide-free water R. Add 0.1 ml of methyl orange solution R.
Titrate with 1M hydrochloric acid until the yellow colour begins to change to yellowish-pink. Heat
cautiously and boil for at least 2 min. The solution becomes yellow. Cool and titrate until a
yellowish-red colour is obtained.
1 ml of 1M hydrochloric acid is equivalent to 0.1001 g of KHCO3.
STORAGE
__________________________________________________________________________________________________________________________________ Ph Eur
32-15
Potassium Bromide
KBr
119.0
7758-02-3
Potassium Bromide complies with the requirements of the 3rd edition of the European Pharmacopoeia [0184].
Action and use Sedative.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Potassium bromide contains not less than 98.0 per cent and not more than the equivalent of
100.5 per cent of KBr, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder or colourless crystals, freely soluble in water and in glycerol, slightly
soluble in alcohol.
IDENTIFICATION
A. It gives the reactions of bromides (2.3.1).
B. Solution S (see Tests) gives the reactions of potassium (2.3.1).
TESTS
Acidity or alkalinity To 10 ml of solution S add 0.1 ml of bromothymol blue solution R1. Not more
than 0.5 ml of 0.01M hydrochloric acid or 0.01M sodium hydroxide is required to change the colour of
the indicator.
Bromates To 10 ml of solution S add 1 ml of starch solution R, 0.1 ml of a 100 g/l solution of
potassium iodide R and 0.25 ml of 0.5M sulphuric acid and allow to stand protected from light for
5 min. No blue or violet colour develops.
Chlorides In a conical flask, dissolve 1.000 g in 20 ml of dilute nitric acid R. Add 5 ml of strong
hydrogen peroxide solution R and heat on a water-bath until the solution is completely decolorised.
Wash down the sides of the flask with a little water R and heat on a water-bath for 15 min. Allow to
cool, dilute to 50 ml with water R and add 5.0 ml of 0.1M silver nitrate and 1 ml of dibutyl phthalate R.
Shake and titrate with 0.1M ammonium thiocyanate, using 5 ml of ferric ammonium sulphate solution R2
as indicator. Not more than 1.7 ml of 0.1M silver nitrate is used (0.6 per cent). Note the volume of
0.1M silver nitrate used (see Assay).
Iodides To 5 ml of solution S add 0.15 ml of ferric chloride solution R1 and 2 ml of chloroform R.
Shake and allow to separate. The chloroform layer is colourless (Method I, 2.2.2).
Barium To 5 ml of solution S add 5 ml of distilled water R and 1 ml of dilute sulphuric acid R. After
15 min, any opalescence in the solution is not more intense than that in a mixture of 5 ml of solution
S and 6 ml of distilled water R.
(20 ppm).
Magnesium and alkaline-earth metals (2.4.7). 10.0 g complies with the limit test for magnesium
and alkaline-earth metals. The volume of 0.01M sodium edetate used does not exceed 5.0 ml
(200 ppm, calculated as Ca).
100C to 105C for 3 h.
32-16
ASSAY
Dissolve 2.000 g in water R and dilute to 100.0 ml with the same solvent. To 10.0 ml of the solution
add 50 ml of water R, 5 ml of dilute nitric acid R, 25.0 ml of 0.1M silver nitrate and 2 ml of dibutyl
phthalate R. Shake. Titrate with 0.1M ammonium thiocyanate, using 2 ml of ferric ammonium sulphate
solution R2 as indicator and shaking vigorously towards the end-point. Correct for the amount of
chloride present, as determined in the test for chlorides.
1 ml of 0.1M silver nitrate is equivalent to 11.90 mg of KBr.
__________________________________________________________________________________________________________________________________ Ph Eur
32-17
Potassium Carbonate
1/01
K2CO3
138.2
584-08-7
Potassium Carbonate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ________________________________________________________________________________________________________________________
DEFINITION
Potassium carbonate contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of K2CO3, calculated with reference to the dried substance.
CHARACTERS
A white granular powder, hygroscopic, freely soluble in water, practically insoluble in alcohol.
IDENTIFICATION
A. Dissolve 1 g in 10 ml of water R. The solution is strongly alkaline (2.2.4).
B. 2 ml of the solution prepared for identification test A gives the reaction of carbonates and
bicarbonates (2.3.1).
C. 1 ml of the solution prepared for identification test A gives reaction (b) of potassium (2.3.1).
TESTS
Solution S Dissolve 10.0 g in 25 ml of distilled water R. Slowly add 14 ml of hydrochloric acid R.
When the effervescence has ceased, boil for a few minutes. Allow to cool and dilute to 50 ml with
distilled water R.
Chlorides (2.4.4). Dissolve 0.50 g in 10 ml of water R. Carefully add dropwise 1 ml of nitric acid R.
Boil. Cool, add 5 ml of dilute nitric acid R and dilute to 15 ml with water R. The solution complies
with the limit test for chlorides (100 ppm).
Sulphates (2.4.13). Dilute 7.50 ml of solution S to 15 ml with distilled water R. The solution
Calcium (2.4.3). To 5 ml of solution S, add 1 ml of concentrated ammonia R. Boil. Cool. Dilute to
15 ml with distilled water R. The solution complies with the limit test for calcium (100 ppm).
(10 ppm).
120C to 125C for 5 h.
ASSAY
Dissolve 0.500 g in 50 ml of carbon dioxide-free water R. Carry out a potentiometric titration (2.2.20),
using 1M hydrochloric acid. Read the volume added at the second point of inflexion.
1 ml of 1M hydrochloric acid is equivalent to 69.1 mg of K2CO3.
STORAGE
_______________________________________________________________________________________________________________________ Ph Eur
32-18
Potassium Chloride
KCl
74.6
7447-40-7
Potassium Chloride complies with the requirements of the 3rd edition of the European Pharmacopoeia [0185].
Action and use Used in prevention and treatment of potassium deficiency and electrolyte
imbalance.
Preparations
Bumetanide and Slow Potassium Tablets
Oral Rehydration Salts
Sterile Potassium Chloride Concentrate
Potassium Chloride and Glucose Intravenous Infusion
Potassium Chloride and Sodium Chloride Intravenous Infusion
Potassium Chloride, Sodium Chloride and Glucose Intravenous Infusion
Slow Potassium Chloride Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Potassium chloride contains not less than 99.0 per cent and not more than the equivalent of
100.5 per cent of KCl, calculated with reference to the dried substance.
CHARACTERS
ethanol.
IDENTIFICATION
TESTS
the indicator.
Bromides Dilute 1.0 ml of solution S to 50 ml with water R. To 5.0 ml of the solution add 2.0 ml of
phenol red solution R2 and 1.0 ml of chloramine solution R1 and mix immediately. After exactly 2 min
add 0.15 ml of 0.1M sodium thiosulphate, mix and dilute to 10.0 ml with water R. The absorbance
(2.2.25) of the solution measured at 590 nm, using water R as the compensation liquid, is not greater
than that of a standard prepared at the same time and in the same manner using 5 ml of a 3.0 mg/l
solution of potassium bromide R (0.1 per cent).
Iodides Moisten 5 g by the dropwise addition of a freshly prepared mixture of 0.15 ml of sodium
nitrite solution R, 2 ml of 0.5M sulphuric acid, 25 ml of iodide-free starch solution R and 25 ml of water R.
After 5 min, examine in daylight. The substance shows no blue colour.
test for sulphates (300 ppm).
(20 ppm).
100C to 105C for 3 h.
Sodium If intended for use in the manufacture of parenteral dosage forms or haemodialysis solu-
32-19
tions, it complies with the test for sodium. Not more than 0.1 per cent of Na, determined by atomic
emission spectrometry (2.2.22).
the same solvent.
Reference solutions. Dissolve in water R 0.5084 g of sodium chloride R, previously dried at 100C to
105C for 3 h, and dilute to 1000.0 ml with the same solvent (200 g of Na per millilitre). Dilute as
required.
Aluminium (2.4.17), If intended for use in the manufacture of haemodialysis solutions, it complies
with the test for aluminium. Dissolve 4 g in 100 ml of water R and add 10 ml of acetate buffer solution
pH 6.0 R. The solution complies with the limit test for aluminium (1 ppm). Use as the reference
solution a mixture of 2 ml of aluminium standard solution (2 ppm Al) R, 10 ml of acetate buffer solution
pH 6.0 R and 98 ml of water R. To prepare the blank use a mixture of 10 ml of acetate buffer solution
pH 6.0 R and 100 ml of water R.
ASSAY
solution R2 as indicator and shaking vigorously towards the end-point.
1 ml of 0.1M silver nitrate is equivalent to 7.46 mg of KCl.
LABELLING
The label states:
forms,
where applicable, that the substance is suitable for use in the manufacture of haemodialysis
solutions.
__________________________________________________________________________________________________________________________________ Ph Eur
32-20
Potassium Citrate
corrected 1/01
CH2COOK
HO
COOK
CH2COOK
C6H5K3O7,H2O
324.4
6100-05-6
Potassium Citrate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0400].
Action and use Systemic alkalinising substance.
Preparation
Potassium Citrate Mixture
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Potassium citrate contains not less than 99.0 per cent and not more than the equivalent of 101.0 per
cent of tripotassium 2-hydroxypropane-1,2,3-tricarboxylate, calculated with reference to the anhydrous substance.
CHARACTERS
A white, granular powder or transparent crystals, hygroscopic, very soluble in water, practically
insoluble in alcohol.
IDENTIFICATION
A. To 1 ml of solution S (see Tests) add 4 ml of water R. The solution gives the reaction of citrates
(2.3.1).
B. 0.5 ml of solution S gives reaction (b) of potassium (2.3.1).
TESTS
indicator.
manner using 4 ml of a 0.05 g/l solution of oxalic acid R (300 ppm).
2.2.22).
Test solution. To 10 ml of solution S add 1 ml of dilute hydrochloric acid R and dilute to 100 ml with
distilled water R.
Reference solutions. Dilute as necessary with distilled water R a solution of sodium chloride R containing
1 mg of Na per millilitre.
32-21
Water (2.5.12). 4.0 per cent to 7.0 per cent, determined on 0.500 g by the semi-micro determination of water. After adding the substance to be examined, stir for 15 min before titrating.
ASSAY
Dissolve 0.150 g in 20 ml of anhydrous acetic acid R, heating to about 50C. Allow to cool. Titrate
with 0.1M perchloric acid using 0.25 ml of naphtholbenzein solution R as indicator until a green colour is
obtained.
1 ml of 0.1M perchloric acid is equivalent to 10.21 mg of C6H5K3O7.
STORAGE
__________________________________________________________________________________________________________________________________ Ph Eur
32-22
Potassium Clavulanate
H
COOK
H
N
O
CH2OH
H
C8H8KNO5
237.3
61177-45-5
Potassium Clavulanate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Beta-lactamase inhibitor.
Preparation
Co-amoxiclav Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Potassium clavulanate is the potassium salt of a substance produced by the growth of certain strains
of Streptomyces clavuligerus or obtained by any other means. It contains not less than 96.5 per cent
and not more than the equivalent of 100.5 per cent of potassium (Z)-(2R,5R)-3-(2-hydroxyethylidene)-7-oxo-4-oxa-1-azabicyclo[3.2.0]heptane-2-carboxylate, calculated with reference to the anhydrous substance.
CHARACTERS
A white or almost white, crystalline powder, hygroscopic, freely soluble in water, slightly soluble in
alcohol, very slightly soluble in acetone.
IDENTIFICATION
spectrum of potassium clavulanate.
B. It gives reaction (b) of potassium (2.3.1).
TESTS
solvent.
pH (2.2.3). Dilute 5 ml of solution S in carbon dioxide-free water R and dilute to 10 ml with the same
solvent. The pH of solution is 5.5 to 7.5.
Absorbance (2.2.25). Dissolve 50.0 mg in 0.1M phosphate buffer solution pH 7.0 R and dilute to
50.0 ml with the same solution. The absorbance measured immediately at 278 nm is not greater than
0.40.
Test solution. Dissolve 0.250 g of the substance to be examined in mobile phase A and dilute to
Reference solution (a). Dilute 1.0 ml of the test solution to 100.0 ml with mobile phase A.
Reference solution (b). Dissolve 10 mg of lithium clavulanate CRS and 10 mg of amoxicillin trihydrate
CRS in mobile phase A and dilute to 100 ml with mobile phase A.
as the mobile phase at a flow rate of 2 ml per minute, a mixture of a 7.8 g/l solution of sodium
dihydrogen phosphate R, adjusted to pH 4.0 with dilute phosphoric acid R (mobile phase A), and
methanol R1 (mobile phase B) with the following gradient of elution as described in the Table,
32-23
Time
(min)
0-4
Mobile phase A
(per cent V/V)
100
Mobile phase B
(per cent V/V)
0
4 - 15
15 - 18
10050
50
050
50

maintaining the temperature of the column at 40C. Inject 20 l of reference solution (b). Adjust the
sensitivity of the system so that height of the first peak (clavulanate) in the chromatogram obtained is
not less than 70 per cent of the full scale of the recorder. The test is not valid unless, in the chromatogram obtained, the resolution between the first peak (clavulanate) and the second peak
(amoxicillin) is at least ten. Inject 20 l of the test solution and 20 l of reference solution (a).
Continue the chromatography for ten times the retention time of the principal peak. In the chromatogram obtained with the test solution, the area of any peaks, apart from the principal peak, is not
greater than the area of the principal peak in the chromatogram obtained with reference solution (a)
twice the area of the principal peak in the chromatogram obtained with reference solution (a) (2.0 per
cent). Disregard any peak with an area less than 0.02 times that of the principal peak in the chromatogram obtained with reference solution (a).
1,1-Dimethylethylamine Not more than 0.2 per cent, determined by gas chromatography (2.2.28).
Test solution. Dissolve 0.20 g of the substance to be examined in 3.0 ml of a 4 g/l solution of sodium
hydroxide R and shake with 5.0 ml of methylene chloride R. Use the lower layer.
Reference solution. Shake 5.0 ml of a 80 mg/l solution of 1,1-dimethylethylamine R in methylene
chloride R with 3.0 ml of a 4 g/l solution of sodium hydroxide R. Use the lower layer.
a stainless steel column 4 m long and 3 mm in internal diameter, packed with diatomaceous earth
for gas chromatography R (135 to 175 m) impregnated with 8 per cent m/m of macrogol 20,000 R
and 2 per cent m/m of potassium hydroxide R,
the detector at 180C.
Potassium clavam-2-carboxylate Not more than 0.01 per cent, determined by liquid chromatography (2.2.29).
the same solvent.
Reference solution. Prepare a solution containing about 1.9 g/ml of potassium clavam-2-carboxylate
CRS.
as mobile phase at a flow rate of 0.5 ml per minute a 15.6 g/l solution of sodium dihydrogen
phosphate R adjusted to pH 4.0 with dilute phosphoric acid R,
Inject 20 l of the reference solution. Adjust the sensitivity of the system so that a measurable peak
is obtained with a retention time of about 11 min. Inject 20 l of the test solution. Calculate the
content of potassium clavam-2-carboxylate from the areas of the principal peak in the chromatogram
obtained with the reference solution and any corresponding peak in the chromatogram obtained with
the test solution.
without a further appropriate procedure for the removal of bacterial endotoxins, it contains not more
than 0.03 I.U. of endotoxin per milligram.
ASSAY
Examine by liquid chromatography (2.2.29). Prepare the solutions immediately before use.
Test solution. Dissolve 50.0 mg of the substance to be examined in a 4.1 g/l solution of sodium
acetate R previously adjusted to pH 6.0 with glacial acetic acid R, and dilute to 50.0 ml with the same
solution.
32-24
Reference solution (a). Dissolve 50.0 mg of lithium clavulanate CRS in a 4.1 g/l solution of sodium
acetate R previously adjusted to pH 6.0 with glacial acetic acid R and dilute to 50.0 ml with the same
solution.
Reference solution (b). Dissolve 50.0 mg of lithium clavulanate CRS and 50.0 mg of amoxicillin
trihydrate CRS in a 4.1 g/l solution of sodium acetate R previously adjusted to pH 6.0 with glacial acetic
acid R and dilute to 50.0 ml with the same solution.
as mobile phase with a flow rate of 1 ml per minute a mixture of 5 volumes of methanol R1 and
95 volumes of a 15 g/l solution of sodium dihydrogen phosphate R previously adjusted to pH 4.0
with dilute phosphoric acid R,
Inject 20 l of reference solution (b). The assay is not valid unless the resolution between the first
peak (clavulanate) and the second peak (amoxicillin) is at least 3.5 and the symmetry of the peak
corresponding to lithium clavulanate is at most 1.5. Inject reference solution (a) six times. The assay
is not valid unless the relative standard deviation of the peak area for clavulanate is at most 1.0 per
cent.
Inject 20 l of the test solution and reference solution (a).
1 mg of C8H9NO5 is equivalent to 1.191 mg of C8H8KNO5.
STORAGE
Store in an airtight container. If the substance is sterile, store in an sterile, airtight, tamper-proof
container.
LABELLING
The label states:
IMPURITIES
O
N
COOH
O
A. (3S,5S)-7-oxo-4-oxa-1-azabicyclo[3.2.0]heptane-2-carboxylic acid (clavam-2-carboxylic

acid),
N
HO
OH
R
B. R = H: pyrazine-2,5-diyl(ethanol),
C. R = CH2CH2CO2H: 3-[3,6-di(2-hydroxyethyl)pyrazin-2-yl]propionic acid,
D. R = CH2CH3: 3-ethylpyrazin-2,5-diyl(di-ethanol),
H
N
HO
COOH
E. 4-(2-hydroxyethyl)pyrrole-3-carboxylic acid.
__________________________________________________________________________________________________________________________________ Ph Eur
32-25
Potassium Dihydrogen Phosphate

KH2PO4
136.1
7778-77-0
Potassium Dihydrogen Phosphate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0920]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Potassium dihydrogen phosphate contains not less than 98.0 per cent and not more than the equivalent of 100.5 per cent of KH2PO4, calculated with reference to the dried substance.
CHARACTERS
alcohol.
IDENTIFICATION
A. Solution S (see Tests) is slightly acid (2.2.4).
B. Solution S gives reaction (b) of phosphates (2.3.1).
C. 0.5 ml of solution S gives reaction (b) of potassium (2.3.1).
TESTS
pH (2.2.3). To 5 ml of solution S add 5 ml of carbon dioxide-free water R. The pH of the solution is
4.2 to 4.5.
Reducing substances To 5 ml of solution S add 5 ml of dilute sulphuric acid R and 0.25 ml of 0.02M
potassium permanganate. Heat on a water-bath for 5 min. The colour of the permanganate is not
completely discharged.
Sulphates (2.4.13). To 5 ml of solution S add 0.5 ml of hydrochloric acid R and dilute to 15 ml with
Sodium If intended for use in the manufacture of parenteral dosage forms, it complies with the test
for sodium. Not more than 0.1 per cent of Na, determined by atomic emission spectrometry (Method
I, 2.2.22).
the same solvent.
Reference solution. Dissolve in water R 0.5084 g of sodium chloride R, previously dried at 100C to
105C for 3 h, and dilute to 1000.0 ml with the same solvent (200 g of Na per millilitre). Dilute as
necessary.
125C to 130C.
ASSAY
Dissolve 1.000 g in 50 ml of carbon dioxide-free water R. Titrate with 1M carbonate-free sodium
hydroxide, determining the end-point potentiometrically (2.2.20).
1 ml of 1M sodium hydroxide is equivalent to 0.1361 g of KH2PO4.
STORAGE
LABELLING
parenteral dosage forms.
__________________________________________________________________________________________________________________________________ Ph Eur
32-26
Potassium Hydrogen Tartrate

Cream of Tartar
H
OH
COOK
HOOC
H
C4H5KO6
OH
188.2
868-14-4
Definition Potassium Hydrogen Tartrate is monopotassium hydrogen (2R,3R)-tartrate. It contains

not less than 99.5% and not more than 100.5% of C4H5KO6, calculated with reference to the dried
substance.
Characteristics A white, crystalline powder or white or colourless crystals.
Slightly soluble in water; practically insoluble in ethanol (96%) and in ether.
Identification
A. A saturated solution in water yields reaction B characteristic of potassium salts, Appendix VI.
B. A saturated solution in water yields the reactions characteristic of tartrates, Appendix VI.
C. To 5 ml of a saturated solution in water add 0.1 ml of methyl red solution. The solution is red.
Clarity of solution To 1 g add 40 ml of water and boil until dissolution is complete. When examined immediately after preparation, the solution is not more opalescent than reference suspension II,
Appendix IV A.
Free tartaric acid Powder 2 g and shake with 20 ml of ethanol (96%) for 1 minute, filter, evaporate
10 ml of the filtrate to dryness and dry at 100 to 105. The residue weighs not more than 2 mg
(0.2%).
Chloride Dissolve 1 g in a mixture of 3 ml of 2M nitric acid and 50 ml of water with heating and add
sufficient water to produce 100 ml. Dilute 10 ml of this solution to 15 ml with water. The solution
complies with the limit test for chlorides, Appendix VII (500 ppm).
Sulphate To 0.3 g add 0.3 ml of 2M hydrochloric acid and sufficient distilled water to produce 15 ml
and heat to dissolve. The resulting solution complies with the limit test for sulphates, Appendix VII
(500 ppm).
Arsenic 0.5 g complies with limit test A for arsenic, Appendix VII (2 ppm).
Barium To 0.5 g add 1.5 ml of 2M hydrochloric acid and 8.5 ml of water and heat to dissolve. Cool
and add 1 ml of 1M sulphuric acid. The solution remains clear for at least 15 minutes.
Heavy metals 2.0 g complies with limit test C for heavy metals, Appendix VII (10 ppm). Use 2 ml of
weight. Use 1 g.
Assay Dissolve 0.17 g in 100 ml of water at 100. Titrate while hot with 0.1M sodium hydroxide VS
using 0.3 ml of phenolphthalein solution as indicator to a stable pink colour. Each ml of 0.1M sodium
hydroxide VS is equivalent to 18.82 mg of C4H5KO6.
32-27
Potassium Hydroxide
Caustic Potash
KOH
56.11
1310-58-3
Potassium Hydroxide complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Potassium Hydroxide Solution
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Potassium hydroxide contains not less than 85.0 per cent and not more than the equivalent of
100.5 per cent of total alkali, calculated as KOH.
CHARACTERS
White, crystalline, hard masses, supplied as sticks, pellets or irregularly shaped pieces, deliquescent in
air, hygroscopic, absorbing carbon dioxide, very soluble in water, freely soluble in alcohol.
IDENTIFICATION
A. Dissolve 0.1 g in 10 ml of water R (this solution is used for identification test B). Dilute 1 ml of
the solution to 100 ml with water R. The pH (2.2.3) of this solution is not less than 10.5.
B. 1 ml of the initial solution prepared in identification test A gives reaction (b) of potassium (2.3.1).
TESTS
Solution S1 Dissolve 2.5 g in 10 ml of water R. Carefully add 2 ml of nitric acid R, with cooling, and
dilute to 25 ml with dilute nitric acid R.
Solution S2 Dissolve 10 g in 15 ml of distilled water R. Carefully add 12 ml of hydrochloric acid R,
with cooling, and dilute to 50 ml with dilute hydrochloric acid R.
Appearance of solution Dissolve 5 g in carbon dioxide-free water R and dilute to 50 ml with the same
solvent. The solution is clear (2.2.1) and colourless (Method II, 2.2.2).
Carbonates Not more than 2.0 per cent, calculated as K2CO3, as determined in the assay.
Chlorides (2.4.4). Dilute 10 ml of solution S1 to 15 ml with water R. The solution complies with the
Phosphates (2.4.11). Dilute 5 ml of solution S1 to 100 ml with water R. The solution complies with
the limit test for phosphates (20 ppm).
Sulphates (2.4.13). 15 ml of solution S2 complies with the limit test for sulphates (50 ppm).
Aluminium (2.4.17). If intended for use in the manufacture of haemodialysis solutions, it complies
with the test for aluminium. Dissolve 20 g in 100 ml of water R and add 10 ml of acetate buffer solution
pH 6.0 R. The solution complies with the limit test for aluminium (0.2 ppm). Use as the reference
pH 6.0 R and 98 ml of water R. To prepare the blank use a mixture of 10 ml of acetate buffer solution
pH 6.0 R and 100 ml of water R.
Heavy metals (2.4.8). Dilute 10 ml of solution S2 to 20 ml with water R. 12 ml of the solution
complies with limit test A for heavy metals (10 ppm). Prepare the standard using lead standard solution (1 ppm Pb) R.
Iron (2.4.9). Dilute 5 ml of solution S2 to 10 ml with water R. The solution complies with the limit
Sodium Not more than 1.0 per cent of Na, determined by atomic absorption spectrometry (Method
II, 2.2.23).
Test solution. Dissolve 1.00 g in 50 ml of water R, add 5 ml of sulphuric acid R and dilute to 100.0 ml
with water R. Dilute 1.0 ml of the solution to 10.0 ml with water R.
Measure the absorbance at 589 nm using a sodium hollow-cathode lamp as a source of radiation and
an air-acetylene flame.
ASSAY
Dissolve 2.000 g in 25 ml of carbon-dioxide free water R. Add 25 ml of freshly prepared barium chloride
solution R1 and 0.3 ml of phenolphthalein solution R. Add slowly with shaking 25.0 ml of 1M hydro-
32-28
chloric acid and continue the titration with 1M hydrochloric acid until the colour changes from pink to
colourless. Add 0.3 ml of bromophenol blue solution R and continue the titration with 1M hydrochloric
acid until the colour changes from violet-blue to yellow.
1 ml of 1M hydrochloric acid used in the second part of the titration is equivalent to 69.11 mg of
K2CO3.
1 ml of 1M hydrochloric acid used in the combined titrations is equivalent to 56.11 mg of total alkali,
calculated as KOH.
STORAGE
Store in an airtight, non-metallic container.
LABELLING
haemodialysis solutions.
__________________________________________________________________________________________________________________________________ Ph Eur
32-29
Potassium Hydroxyquinoline Sulphate

Definition Potassium Hydroxyquinoline Sulphate is an equimolecular mixture of quinolin-8-ol
sulphate monohydrate, (C9H7NO)2,H2SO4,H2O, and potassium sulphate, K2SO4. It contains not
less than 50.6% and not more than 52.6% of quinolin-8-ol, C9H7NO, and not less than 29.5% and
not more than 32.5% of K2SO4, calculated with reference to the anhydrous substance.
Characteristics A pale yellow, microcrystalline powder; odourless or almost odourless. It partly
liquefies between 172 and 184.
Freely soluble in water; insoluble in ether. On extraction with hot absolute ethanol a residue of
potassium sulphate and a solution of quinolin-8-ol sulphate are obtained.
Identification
A. To 5 ml of a 5% w/v solution add dropwise, with shaking, 5M sodium hydroxide until a heavy
precipitate is produced. Filter, wash with water and dry at a pressure not exceeding 0.7 kPa for 3
hours. The infrared absorption spectrum of the residue, Appendix II A, is concordant with the reference
spectrum of quinolin-8-ol (RS 310).
B. To 5 ml of a 5% w/v solution add 0.5 ml of iron(III) chloride solution R1. A dark green colour is
produced.
C. Yields reaction A characteristic of potassium salts, Appendix VI.
D. Yields reaction A characteristic of sulphates, Appendix VI.
Assay
For quinolin-8-ol Dissolve 0.35 g in 50 ml of water and 20 ml of hydrochloric acid, add 50 ml of
0.05M bromine VS, stopper the flask and shake for 15 minutes. Allow to stand for 15 minutes, add
80 ml of water and 10 ml of dilute potassium iodide solution and titrate with 0.1M sodium thiosulphate VS
using starch mucilage, added towards the end of the titration, as indicator. Repeat the operation
without the substance being examined. The difference between the titrations represents the amount
of bromine required. Each ml of 0.05M bromine VS is equivalent to 3.629 mg of C9H7NO.
For potassium sulphate Prepare a solution of suitable concentration with water. Carry out the
method for atomic emission spectrophotometry, Appendix II D, measuring at 766.5 nm and using
potassium standard solution (600 ppm K), suitably diluted with water, to prepare the standard solutions.
Each mg of potassium is equivalent to 2.2284 mg of K2SO4.
Action and use Used in treatment of acne.
Preparation
Potassium Hydroxyquinoline Sulphate and Benzoyl Peroxide Cream.
32-30
Potassium Iodate
KIO3
214.0
7758-05-6
Definition Potassium Iodate contains not less than 99.0% and not more than 101.0% of KIO3,
Characteristics A white crystalline powder; odour, slight.
Slowly soluble in water; insoluble in ethanol (96%).
Dissolve 10 g of the substance being examined in sufficient water to produce 200 ml (solution S1).
Add 25 ml of hydrochloric acid to 6 g of the substance being examined, evaporate to dryness and repeat.
Heat until iodine is removed. Dissolve the residue in 2.5 ml of a 25% v/v solution of hydrochloric acid and
dilute to 50 ml with water (solution S2).
Identification
A. 1 ml of solution S1 yields reaction B characteristic of potassium salts, Appendix VI.
B. Dissolve 0.1 g in 5 ml of water. Add 1 ml of silver nitrate solution followed by 1 ml of sulphur dioxide
solution. A yellow precipitate is produced immediately.
Acidity or alkalinity pH of solution S1, 5.0 to 8.0, Appendix V L.
Clarity and colour of solution Solution S1 is clear, Appendix IV A, and colourless, Appendix IV B,
Method II.
Chloride, chlorate, bromide, bromate Dilute 5 ml of solution S1 to 15 ml with water, add 20 ml
of sulphur dioxide solution and heat on a water bath for 30 minutes. Heat to boiling, cool, add 10 ml of
18M ammonia and 20 ml of silver nitrate solution R2 and dilute to 70 ml with water. Filter, transfer
35 ml of the filtrate to a Nessler cylinder and acidify with 6 ml of nitric acid. After 5 minutes, any
opalescence, when viewed vertically, is not greater than that produced by treating 5 ml of a
0.00165% w/v solution of sodium chloride at the same time and in the same manner (0.02%).
Iodide Add 1 ml of 1.8M sulphuric acid to 25 ml of solution S1 and shake with 1 ml of chloroform.
Any violet colour produced is not more intense than that of a solution prepared at the same time and
in the same manner but using 5 ml of solution S1 and 2 ml of a solution containing 1.31% w/v of
potassium iodide (20 ppm).
Sulphate Add 1 ml of a 25% w/v solution of barium chloride to 1.5 ml of sulphate standard solution
(10 ppm SO4), shake and allow to stand for 1 minute. Add 12.5 ml of solution S2 diluted to 15 ml
with distilled water and 0.5 ml of 5M acetic acid and allow to stand for 5 minutes. Any opalescence
produced is not more intense than that of a standard prepared in the same manner but using 7.5 ml
of sulphate standard solution (10 ppm SO4) diluted to 15 ml with distilled water in place of the solution
being examined (50 ppm).
Heavy metals Adjust the pH of 20 ml of solution S2 to about 5 with 5M ammonia. The solution
complies with limit test A for heavy metals, Appendix VII. Use 10 ml of lead standard solution (2 ppm
Pb) to prepare the standard (20 ppm).
Loss on drying When dried at 130 for 1 hour, loses not more than 0.5% of its weight. Use 1 g.
Assay To 1.5 g add sufficient water to produce 250 ml. To 25 ml of the resulting solution in an
iodine flask add 3 g of potassium iodide, 100 ml of water and 10 ml of hydrochloric acid. Close the flask
and stand in the dark for 5 minutes. Titrate the solution with 0.1M sodium thiosulphate VS to a light
straw colour and then complete the titration to a colourless end point using starch mucilage as
indicator. Each ml of 0.1M sodium thiosulphate VS is equivalent to 3.567 mg of KIO3.
Action and use Iodine supplement for emergency use.
Preparation
Potassium Iodate Tablets
32-31
Potassium Iodide
KI
166.0
7681-11-0
Potassium Iodide complies with the requirements of the 3rd edition of the European Pharmacopoeia [0186].
Action and use Antithyroid.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Potassium iodide contains not less than 99.0 per cent and not more than the equivalent of 100.5 per
cent of KI, calculated with reference to the dried substance.
CHARACTERS
A white powder or colourless crystals, very soluble in water, freely soluble in glycerol, soluble in
alcohol.
IDENTIFICATION
A. Solution S (see Tests) gives the reactions of iodides (2.3.1).
B. Solution S gives the reactions of potassium (2.3.1).
TESTS
Alkalinity To 12.5 ml of solution S add 0.1 ml of bromothymol blue solution R 1. Not more than
0.5 ml of 0.01M hydrochloric acid is required to change the colour of the indicator.
Iodates To 10 ml of solution S add 0.25 ml of iodide-free starch solution R and 0.2 ml of dilute
sulphuric acid R and allow to stand protected from light for 2 min. No blue colour develops.
Thiosulphates To 10 ml of solution S add 0.1 ml of starch solution R and 0.1 ml of 0.005M iodine. A
blue colour is produced.
(20 ppm).
Loss on drying (2.2.32). Not more than 1.0 per cent, determined on 1.00 g of previously powdered
substance by drying in an oven at 100C to 105C for 3 h.
ASSAY
add 40 ml of hydrochloric acid R and titrate with 0.05M potassium iodate until the colour changes from
red to yellow. Add 5 ml of chloroform R and continue the titration, shaking vigorously, until the
chloroform layer is decolourised.
1 ml of 0.05M potassium iodate is equivalent to 16.60 mg of KI.
STORAGE
__________________________________________________________________________________________________________________________________ Ph Eur
32-32
Potassium Nitrate
KNO3
101.1
7757-79-1
Potassium Nitrate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1465].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Potassium nitrate contains not less than 99.0 per cent and not more than the equivalent of 101.0 per
cent of KNO3, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder or colourless crystals, freely soluble in water, very soluble in boiling
water, practically insoluble in alcohol.
IDENTIFICATION
A. It gives the reaction of nitrates (2.3.1).
TESTS
the indicator.
Reducible substances To 10 ml of solution S, add 0.5 ml of dilute sulphuric acid R and 2 ml of zinc
iodide and starch solution R. The solution does not become blue within 2 min.
Chlorides (2.4.4). If intended for ophthalmic use, it complies with the test for chlorides. Dissolve
2.5 g in water R and dilute to 15 ml with the same solvent. The solution complies with the limit test
for chlorides (20 ppm).
Ammonium (2.4.1). 1 ml of solution S complies with the limit test (A) for ammonium (100 ppm).
If intended for ophthalmic use, not more than 50 ppm of ammonium.
Calcium (2.4.3). Dilute 10 ml of solution S to 15 ml with distilled water R. The solution complies
with the limit test for calcium (100 ppm). If intended for ophthalmic use, not more than 50 ppm of
calcium.
test for iron (20 ppm). If intended for ophthalmic use, not more than 10 ppm of iron.
2.2.22).
100C to 105C.
ASSAY
Prepare a chromatography column 0.3 m long and 10 mm in internal diameter and filled with 10 g of
strongly acidic cation-exchange resin R covered with carbon dioxide-free water R. Maintain a 1 cm layer of
liquid above the resin at all times. Allow 100 ml of dilute hydrochloric acid R to run through the
column at a flow rate of about 5 ml/min. Wash the column (with the tap completely open) with
carbon dioxide-free water R until neutral to blue litmus paper R. Dissolve 0.200 g of the substance to be
examined in 2 ml of carbon dioxide-free water R in a beaker and transfer it to the column reservoir,
allow the solution to run through the column at a flow rate of about 3 ml/min and collect the eluate.
32-33
Wash the beaker with 10 ml of carbon dioxide-free water R and transfer this solution at the same flow
rate to the column before it runs dry. Finally wash the column with 200 ml of carbon dioxide-free
water R (with the tap completely open) until neutral to blue litmus paper R. Titrate the combined
eluate and washings with 0.1M sodium hydroxide, using 1 ml of phenolphthalein solution R as indicator.
1 ml of 0.1M sodium hydroxide is equivalent to 10.11 mg of KNO3.
LABELLING
The label states:
where applicable, that the substance is suitable for ophthalmic use.
__________________________________________________________________________________________________________ Ph Eur
32-34
Potassium Permanganate
KMnO4
158.0
7722-64-7
Potassium Permanganate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Antiseptic.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Potassium permanganate contains not less than 99.0 per cent and not more than the equivalent of
100.5 per cent of KMnO4.
CHARACTERS
A dark purple or brownish-black, granular powder or dark purple or almost black crystals, usually
having a metallic lustre, soluble in cold water, freely soluble in boiling water. It decomposes on
contact with certain organic substances.
IDENTIFICATION
A. Dissolve about 50 mg in 5 ml of water R and add 1 ml of alcohol R and 0.3 ml of dilute sodium
hydroxide solution R. A green colour develops. Heat to boiling. A dark brown precipitate is formed.
B. Filter the mixture obtained in identification test A. The filtrate gives reaction (b) of potassium
(2.3.1).
TESTS
Solution S Dissolve 0.75 g in 25 ml of distilled water R, add 3 ml of alcohol R and boil for 2 min to
3 min. Cool, dilute to 30 ml with distilled water R and filter.
Substances insoluble in water Dissolve 0.5 g in 50 ml of water R and heat to boiling. Filter
through a tared sintered-glass filter (16). Wash with water R until the filtrate is colourless and collect
the residue on the filter. The residue, dried in an oven at 100C to 105C, weighs not more than
5 mg (1.0 per cent).
ASSAY
add 20 ml of water R, 1 g of potassium iodide R and 10 ml of dilute hydrochloric acid R. Titrate the
liberated iodine with 0.1M sodium thiosulphate, using 1 ml of starch solution R as indicator.
1 ml of 0.1M sodium thiosulphate is equivalent to 3.160 mg of KMnO4.
__________________________________________________________________________________________________________________________________ Ph Eur
32-35
Potassium Sorbate
H
H3C
COOK
H
C6H7KO2
H
150.2
590-00-1
Potassium Sorbate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0618].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Potassium sorbate contains not less than 99.0 per cent and not more than the equivalent of 101.0 per
cent of potassium (E,E)-hexa-2,4-dienoate, calculated with reference to the dried substance.
CHARACTERS
White or almost white powder or granules, very soluble in water, slightly soluble in alcohol.
IDENTIFICATION
A. Dissolve 50.0 mg in water R and dilute to 250.0 ml with the same solvent. Dilute 2.0 ml of this
solution to 200.0 ml with 0.1M hydrochloric acid. Examined between 230 nm and 350 nm (2.2.25),
the solution shows a maximum at 264 nm. The specific absorbance at the maximum is 1650 to 1900.
obtained with potassium sorbate CRS.
C. Dissolve 1.0 g in 50 ml of water R, add 10 ml of dilute hydrochloric acid R and shake. Filter the
crystalline precipitate, wash with water R and dry in a vacuum over sulphuric acid R for 4 h. The
residue obtained melts (2.2.14) at 132C to 136C.
D. Dissolve 0.2 g in 2 ml of water R and add 2 ml of dilute acetic acid R. Filter. The solution gives
reaction (b) of potassium (2.3.1).
TESTS
indicator.
Aldehydes Dissolve 1.0 g in a mixture of 30 ml of water R and 50 ml of 2-propanol R, adjust the
solution to pH 4 with 1M hydrochloric acid and dilute to 100 ml with water R. To 10 ml of the solution
add 1 ml of decolorised fuchsin solution R and allow to stand for 30 min. Any colour in the solution is
not more intense than that in a standard prepared at the same time by adding 1 ml of decolorised
fuchsin solution R to a mixture of 1.5 ml of acetaldehyde standard solution (100 ppm C2H4O) R, 4 ml of
2-propanol R and 4.5 ml of water R (0.15 per cent, calculated as C2H4O).
100C to 105C for 3 h.
ASSAY
Dissolve 0.120 g in 20 ml of anhydrous acetic acid R. Titrate with 0.1M perchloric acid using 0.1 ml of
crystal violet solution R as indicator until the colour changes from violet to bluish-green.
1 ml of 0.1M perchloric acid is equivalent to 15.02 mg of C6H7KO2.
STORAGE
__________________________________________________________________________________________________________________________________ Ph Eur
32-36
Potato Starch
Potato Starch complies with the requirements of the 3rd edition of the European Pharmacopoeia [0355].
or used.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Potato starch is obtained from the tuber of Solanum tuberosum L.
CHARACTERS
A very fine, white powder which creaks when pressed between the fingers, practically insoluble in
cold water and in alcohol. Potato starch does not contain starch grains of any other origin. It may
contain a minute quantity, if any, of fragments of the tissue of the original plant.
IDENTIFICATION
A. Examined under a microscope using a mixture of equal volumes of glycerol R and water R, it
presents granules, irregularly shaped, ovoid or pear-shaped, 30 m to 100 m in size or rounded,
10 m to 35 m in size. There are occasional compound granules having two to four components.
The ovoid and pear-shaped granules have an eccentric hilum and the rounded granules acentric or
slightly eccentric hilum. All granules show clearly visible concentric striations. Between crossed nicol
prisms, the granules show a distinct black cross intersecting at the hilum.
B. Suspend 1 g in 50 ml of water R, boil for 1 min and cool. A thick, opalescent mucilage is formed.
C. To 1 ml of the mucilage obtained in identification test B, add 0.05 ml of iodine solution R1. A
dark-blue colour is produced which disappears on heating and reappears on cooling.
TESTS
pH (2.2.3). Shake 5.0 g with 25.0 ml of carbon dioxide-free water R for 60 s. Allow to stand for
15 min. The pH of the solution is 5.0 to 8.0.
Iron (2.4.9). Shake 1.5 g with 15 ml of dilute hydrochloric acid R. Filter. The filtrate complies with the
Foreign matter (2.8.2). Examined under a microscope using a mixture of equal volumes of
glycerol R and water R, not more than traces of cell walls and of cytoplasmatic residues are present.
Total protein Not more than 0.1 per cent of total protein (corresponding to 0.017 per cent N2,
conversion factor: 5.7) determined on 6.0 g by sulphuric acid digestion (2.5.9) modified as follows:
wash any adhering particles from the neck into the flask with 25 ml of sulphuric acid R; continue the
heating until a clear solution is obtained; add 45 ml of strong sodium hydroxide solution R.
Oxidising substances (2.5.30). It complies with the test for oxidising substances.
Microbial contamination Not more than 103 bacteria and not more than 102 fungi per gram,
determined by plate-count (2.6.12). It complies with the test for E. coli (2.6.13).
at 130C for 90 min.
STORAGE
__________________________________________________________________________________________________________________________________ Ph Eur
32-37
Povidone
H
CH
N
CH2
O
n
(C6H9NO)n
(111.1)n
9003-39-8
Povidone complies with the requirements of the 3rd edition of the European Pharmacopoeia [0685]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Povidone is poly[1-(2-oxo-1-pyrrolidinyl)ethylene] and consists of linear polymers of 1-vinylpyrrolidin-2-one. It contains not less than 11.5 per cent and not more than 12.8 per cent of nitrogen,
calculated with reference to the anhydrous substance. The different types of povidone are
characterised by their viscosity in solution, expressed as a K-value. The K-value of povidone having a
stated K-value of 15 or less is not less than 85.0 per cent and not more than 115.0 per cent of the
stated value. The K-value of povidone having a stated K-value or a stated K-value range with an
average of more than 15 is not less than 90.0 per cent and not more than 108.0 per cent of the stated
value or of the average of the stated range.
CHARACTERS
White or yellowish-white powder or flakes, hygroscopic, freely soluble in water, in alcohol and in
methanol, slightly soluble in acetone, practically insoluble in ether.
IDENTIFICATION
obtained with povidone CRS, both previously dried at 105C for 6 h. Record the spectra using 4 mg
of substance.
B. To 0.4 ml of solution S1 (see Tests) add 10 ml of water R, 5 ml of dilute hydrochloric acid R and
2 ml of potassium dichromate solution R. An orange-yellow precipitate is formed.
C. To 1 ml of solution S1 add 0.2 ml of dimethylaminobenzaldehyde solution R1 and 0.1 ml of sulphuric
acid R. A pink colour is produced.
D. To 0.1 ml of solution S1 add 5 ml of water R and 0.2 ml of 0.05M iodine. A red colour is
produced.
E. It is freely soluble in water R.
TESTS
Add the substance to be examined to the water in small portions with magnetic stirring.
Solution S1 Dissolve 2.5 g in carbon dioxide-free water R and dilute to 25 ml with the same solvent.
Add the substance to be examined to the water in small portions with magnetic stirring.
solution B6, BY6 or R6 (Method II, 2.2.2).
pH (2.2.3). The pH of solution S is 3.0 to 5.0 for povidone having a stated K-value of at most 30.
The pH of solution S is 4.0 to 7.0 for povidone having a stated K-value of more than 30.
Aldehydes Not more than 500 ppm, expressed as acetaldehyde.
Test solution. Dissolve 1.0 g in phosphate buffer solution pH 9.0 R and dilute to 100.0 ml with the same
solvent. Stopper the flask and heat at 60C for 1 h. Allow to cool.
Reference solution. Dissolve 0.100 g of freshly distilled acetaldehyde R in water R at 4C and dilute to
200.0 ml with the same solvent. Allow to stand at 4C for 20 h. Dilute 1.0 ml of this solution to
100.0 ml with phosphate buffer solution pH 9.0 R.
Into three identical spectrophotometric cells with a path length of 1 cm, introduce separately 0.5 ml
of the test solution, 0.5 ml of the reference solution and 0.5 ml of water R (blank). To each cell, add
2.5 ml of phosphate buffer solution pH 9.0 R and 0.2 ml of nicotinamide-adenine dinucleotide solution R.
32-38
Mix and stopper tightly. Allow to stand at 22C 2C for 2 min to 3 min and measure the absorbance (2.2.25) of each solution at 340 nm, using water R as the compensation liquid. To each cell, add
0.05 ml of aldehyde dehydrogenase solution R, mix and stopper tightly. Allow to stand at 22C 2C
for 5 min. Measure the absorbance of each solution at 340 nm using water R as compensation liquid.
Determine the content of aldehydes using the expression:
( A t 2 A t1) ( A b2 A b1) 100,000 C
( A s 2 A s1) ( A b2 A b1)
m
At1 = absorbance obtained with the test solution before the addition of aldehyde
dehydrogenase,
At2 = absorbance obtained with the test solution after the addition of aldehyde dehydrogenase,
As1 = absorbance obtained with the reference solution before the addition of aldehyde
dehydrogenase,
As2 = absorbance obtained with the reference solution after the addition of aldehyde
dehydrogenase,
Ab1 = absorbance obtained with the blank before the addition of aldehyde dehydrogenase,
Ab2 = absorbance obtained with the blank after the addition of aldehyde dehydrogenase,
m = mass of povidone, in grams, calculated with reference to the dried substance,
C = concentration, in mg per ml, of acetaldehyde in the reference solution.
Peroxides Dissolve 2.0 g in 50 ml of water R. To 25 ml of this solution, add 2 ml of titanium
trichloride-sulphuric acid reagent R. Allow to stand for 30 min. The absorbance (2.2.25) of the solution,
measured at 405 nm using a mixture of 25 ml of a 40 g/l solution of the substance to be examined
and 2 ml of a 13 per cent V/V solution of sulphuric acid R as the compensation liquid, is not greater
than 0.35 (400 ppm, expressed as H2O2).
Hydrazine Examine by thin-layer chromatography (2.2.27), using silanised silica gel H R as the
coating substance.
Use freshly prepared solutions.
Test solution. Dissolve 2.5 g of the substance to be examined in 25 ml of water R. Add 0.5 ml of a
50 g/l solution of salicylaldehyde R in methanol R, mix and heat in a water-bath at 60C for 15 min.
Allow to cool, add 2.0 ml of toluene R, shake for 2 min and centrifuge. Use the clear supernatant
layer.
Reference solution. Dissolve 9 mg of salicylaldehyde azine R in toluene R and dilute to 100 ml with the
same solvent. Dilute 1 ml of this solution to 10 ml with toluene R.
1 volume of water R and 2 volumes of methanol R. Allow to dry in air and examine in ultraviolet light
at 365 nm. Any spot corresponding to salicylaldehyde azine in the chromatogram obtained with the
test solution is not more intense than the spot in the chromatogram obtained with the reference
solution (1 ppm of hydrazine).
Vinylpyrrolidone Examine by liquid chromatography (2.2.29).
Test solution. Dissolve 0.25 g in the mobile phase and dilute to 10.0 ml with the mobile phase.
Reference solution (a). Dissolve 50 mg of 1-vinylpyrrolidin-2-one R in methanol R and dilute to 100.0 ml
with the same solvent. Dilute 1.0 ml of the solution to 100.0 ml with methanol R. Dilute 5.0 ml of the
Reference solution (b). Dissolve 10 mg of 1-vinylpyrrolidin-2-one R and 0.5 g of vinyl acetate R in
methanol R and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml of the solution to 100.0 ml
a stainless steel column, 0.25 m long and 4 mm in internal diameter and a precolumn 0.025 m
long and 4 mm in internal diameter, both packed with octylsilyl silica gel for chromatography R
(5 m),
as the mobile phase, a mixture of 1 volume of methanol R and 4 volumes of water R,
Inject 50 l of reference solution (a). Adjust the flow rate so that the retention time of the peak
corresponding to 1-vinylpyrrolidin-2-one is about 10 min. Adjust the sensitivity of the detector so
that the height of the peak corresponding to 1-vinylpyrrolidin-2-one in the chromatogram obtained
with the reference solution (a) is not less than 70 per cent of the full scale of the recorder. Inject 50 l
of reference solution (b). The test is not valid unless: in the chromatogram obtained with reference
solution (b), the resolution between the peaks corresponding to 1-vinylpyrrolidin-2-one and vinyl
acetate is at least 2.0. Inject 50 l of reference solution (a) five times. The test is not valid unless: the
relative standard deviation of the peak area of 1-vinylpyrrolidin-2-one is at most 2.0 per cent. Inject
50 l of the test solution. After each injection of the test solution, wash the precolumn by passing the
mobile phase backward, at the same flow rate applied in the test, for 30 min.
32-39
Calculate the content of 1-vinylpyrrolidin-2-one from the areas of the peaks. The content of
1-vinylpyrrolidin-2-one is not more than 10 ppm.
Viscosity, expressed as K-value For povidone having a stated value of 18 or less, use a 50 g/l
solution. For povidone having a declared value of more than 18, use a 10 g/l solution. For povidone
having a declared value of more than 95, use a 1.0 g/l solution. Allow to stand for 1 h and determine
the viscosity (2.2.9) of the solution at 25C, using viscometer No.1 with a minimum flow time of
100 s. Calculate the K-value from the expression:
300c log + ( c + 15
. c log )2
15
. log 1
+
0.15 + 0.003c
0.15c + 0.003c2
where c = concentration (g/l00 ml) of the substance to be examined, calculated with reference to the
dried substance,
= viscosity of the solution relative to that of water R.
ASSAY
Place 100.0 mg of the substance to be examined (m mg) in a combustion flask, add 5 g of a mixture
of 33 g of dipotassium sulphate R, 1 g of cupric sulphate R and 1 g of titanium dioxide R, and three glass
beads. Wash any adhering particles from the neck into the flask with a small quantity of water R. Add
7 ml of sulphuric acid R, allowing it to run down the sides of the flask, and mix the contents by rotation. Close the mouth of the flask loosely, for example by means of a glass bulb with a short stem, to
avoid excessive loss of sulphuric acid. Heat gradually at first, then increase the temperature until
there is vigorous boiling with condensation of sulphuric acid in the neck of the flask; precautions are
to be taken to prevent the upper part of the flask from becoming overheated. Continue the heating
for 45 min. Cool, dissolve the solid material by cautiously adding to the mixture 20 ml of water R,
cool again and place in a steam-distillation apparatus. Add 30 ml of strong sodium hydroxide solution R
through the funnel, rinse cautiously the funnel with 10 ml of water R and distil immediately by passing steam through the mixture. Collect about 80 ml to 100 ml of distillate in a mixture of 30 ml of a
40 g/l solution of boric acid R and 3 drops of bromocresol green-methyl red solution R and enough water R
to cover the tip of the condenser. Towards the end of the distillation lower the receiver so that the tip
of the condenser is above the surface of the acid solution and rinse the end part of the condenser with
a small quantity of water R. Titrate the distillate with 0.025M sulphuric acid until the color of the
solution changes from green through pale greyish-blue to pale greyish-red-purple (n1 ml of 0.025M
sulphuric acid).
Repeat the test using about 100 mg of glucose R in place of the substance to be examined (n2 ml of
0.025M sulphuric acid).
STORAGE
LABELLING
The label indicates the nominal K-value.
__________________________________________________________________________________________________________________________________ Ph Eur
32-40
Iodinated Povidone
Iodinated Povidone complies with the requirements of the 3rd edition of the European Pharmacopoeia [1142].
Preparations
PovidoneIodine Mouthwash
PovidoneIodine Solution
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Povidone, iodinated is a complex of iodine and povidone. It contains not less than 9.0 per cent and
not more than 12.0 per cent of available iodine, calculated with reference to the dried substance.
PRODUCTION
It is produced by using povidone that complies with the monograph on Povidone (685).
CHARACTERS
A yellowish-brown or reddish-brown amorphous powder. It is soluble in water and in alcohol. It is
practically insoluble in acetone.
IDENTIFICATION
spectrum of iodinated povidone.
B. Dissolve 10 mg in 10 ml of water R and add 1 ml of starch solution R; a deep blue colour is
produced.
C. Dissolve 0.1 g in 5 ml of water R and add dropwise a 10 g/l aqueous solution of sodium sulphite R
until the solution becomes colourless. Add 2 ml of potassium dichromate solution R and 1 ml of hydrochloric acid R. A light brown precipitate is formed.
TESTS
5.0.
Iodide Not more than 6.0 per cent, calculated with reference to the dried substance. Dissolve
0.500 g in 100 ml of water R. Add sodium metabisulphite R until the colour of iodine has disappeared.
Add 25.0 ml of 0.1M silver nitrate, 10 ml of nitric acid R and 5 ml of ferric ammonium sulphate solution R2. Titrate with 0.1M ammonium thiocyanate. Carry out a blank titration.
1 ml of 0.1M silver nitrate is equivalent to 12.69 mg of total iodine. From the percentage of total
iodine, calculated with reference to the dried substance, subtract the percentage of available iodine as
determined in the Assay to obtain the percentage of iodide.
Loss on drying (2.2.32). Not more than 8.0 per cent determined on 0.500 g by drying at 100C to
105C for 3 h.
ASSAY
Transfer 1.00 g of the substance to be examined into a ground-glass stoppered flask containing
150 ml of water R and stir for 1 hour. Add 0.1 ml of dilute acetic acid R and titrate with 0.1M sodium
thiosulphate using starch solution R as indicator.
1 ml of 0.1M sodium thiosulphate is equivalent to 12.69 mg of available iodine.
STORAGE
__________________________________________________________________________________________________________________________________ Ph Eur
32-41
Prazepam
corrected 1/01
Cl
C19H17ClN2O
324.8
2955-38-6
Prazepam complies with the requirements of the 3rd edition of the European Pharmacopoeia [1466]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Prazepam contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of
7-chloro-1-(cyclopropylmethyl)-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one, calculated with
CHARACTERS
A white or almost white, crystalline powder, practically insoluble in water, freely soluble in methylene
chloride, sparingly soluble in ethanol.
IDENTIFICATION
A. Dissolve 30.0 mg in alcohol R and dilute to 100.0 ml with the same solvent. Dilute 20.0 ml of the
solution to 100.0 ml (solution A) and 2.0 ml of the solution to 100.0 ml (solution B) with the same
solvent. Examined between 300 nm and 350 nm (2.2.25), solution A shows an absorption maximum
at 312 nm. Examined between 210 nm and 300 nm, solution B shows an absorption maximum at
228 nm and a point of inflexion at about 252 nm. The specific absorbance at the maximum at
228 nm is 900 to 940. The specific absorbance at the maximum at 312 nm is 59 to 63.
obtained with prazepam CRS.
chromatogram obtained with test solution (b) is similar in position, fluorescence at 365 nm and size
to the principal spot in the chromatogram obtained with reference solution (b).
TESTS
a suitable silica gel with a fluorescent indicator having an optimal intensity at 254 nm.
the same solvent.
Reference solution (a). Dilute 1 ml of test solution (b) to 10 ml with acetone R.
Reference solution (b). Dissolve 10 mg of prazepam CRS in acetone R and dilute to 10 ml with the same
solvent.
Reference solution (c). Dissolve 15 mg of nordazepam CRS in acetone R and dilute to 50 ml with the
same solvent.
32-42
Reference solution (d). To 1 ml of reference solution (a) add 1 ml of reference solution (c) and mix.
Apply to the plate 5 l of each solution. Develop over a path of 10 cm using a freshly prepared
mixture of 50 volumes of ethyl acetate R and 50 volumes of heptane R. Dry the plate in air and
examine in ultraviolet light at 254 nm and 365 nm. In the chromatogram obtained with test solution
(a): any spot corresponding to nordazepam is not more intense than the spot in the chromatogram
obtained with reference solution (c) (0.3 per cent); not more than four additional spots are present,
none of which are more intense than the spot in the chromatogram obtained with reference solution
(a) (0.1 per cent). The test is not valid unless the chromatogram obtained with reference solution (d)
100C to 105C.
ASSAY
STORAGE
IMPURITIES
O
H
N
Cl
A. 7-chloro-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one (nordazepam),
NH
Cl
B. [5-chloro-2-[(cyclopropylmethyl)amino]phenyl]phenylmethanone,
NH2
Cl
C. (2-amino-5-chlorophenyl)phenylmethanone.
__________________________________________________________________________________________________________ Ph Eur
32-43
Praziquantel
O
H
N
N
O
and enantiomer
C19H24N2O2
312.4
55268-74-1
Praziquantel complies with the requirements of the 3rd edition of the European Pharmacopoeia [0855]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Praziquantel contains not less than 98.0 per cent and not more than the equivalent of 103.0 per cent
of (RS)-2-cyclohexylcarbonyl-1,2,3,6,7,11b-hexahydro-4H-pyrazino[2,1-a]isoquinolin-4-one, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, very slightly soluble in water, freely soluble in alcohol
IDENTIFICATION
obtained with praziquantel CRS. Examine the substances prepared as discs.
Test solution. Dissolve 50 mg of the substance to be examined in alcohol R and dilute to 5 ml with the
same solvent.
Reference solution (a). Dissolve 50 mg of praziquantel CRS in alcohol R and dilute to 5 ml with the
same solvent.
Reference solution (b). Dissolve 10 mg of praziquantel impurity A CRS in alcohol R and dilute to 10 ml
with the same solvent. Dilute 1 ml of the solution to 2 ml with reference solution (a).
15 cm using a mixture of 15 volumes of methanol R and 85 volumes of toluene R. Allow the plate to
dry in air. Expose the plate to iodine vapour for 20 min. Examine in daylight. The principal spot in
the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless the
chromatogram obtained with reference solution (b) shows two clearly separated principal spots.
TESTS
Appearance of solution Dissolve 2 g in alcohol R and dilute to 20 ml with the same solvent. The
solution is clear (2.2.1) and not more intensely coloured than reference solution BY5 (Method II,
2.2.2).
Reference solution (a). Dissolve 10 mg of praziquantel impurity A CRS and 10 mg of praziquantel CRS
in the mobile phase and dilute to 25.0 ml with the mobile phase. Dilute 1.0 ml of this solution to
32-44
volumes of water R,
Inject 20 l of reference solution (a) and adjust the sensitivity of the detector so that the heights of
the two principal peaks in the chromatogram obtained are not less than 50 per cent of the full scale of
the recorder. The test is not valid unless the resolution between the peaks corresponding to
praziquantel impurity and praziquantel is at least three.
Inject separately 20 l of the test solution and 20 l of reference solution (b). In the chromatogram
obtained with the test solution, the sum of the areas of all the peaks, apart from the principal peak, is
(b) (0.5 per cent) and not more than one such peak has an area greater than 0.4 times the area of the
50C over diphosphorus pentoxide R at a pressure not exceeding 700 Pa for 2 h.
ASSAY
Dissolve 40.0 mg in alcohol R and dilute to 100.0 ml with the same solvent. Prepare a reference
solution in the same manner using 40.0 mg of praziquantel CRS. Measure the absorbances (2.2.25) of
the two solutions at the maximum at 265 nm.
Calculate the content of C19H24N2O2 from the absorbances measured and the concentrations of
the solutions.
STORAGE
IMPURITIES
A. (RS)-2-benzoyl-1,2,3,6,7,11b-hexahydro-4H-pyrazino[2,1-a]isoquinolin-4-one,
B. 2-cyclohexylcarbonyl-2,3,6,7-tetrahydro-4H-pyrazino[2,1-a]isoquinolin-4-one,
C. 2-[2-(N-formylcyclohexylcarbonylamino)acetyl]-1,2,3,4-tetrahydroisoquinolin-1-one.
__________________________________________________________________________________________________________________________________ Ph Eur
32-45
Prazosin Hydrochloride
O
O
N
MeO
N
,HCl
N
MeO
NH2
C19H21N5O4,HCl
419.9
19237-84-4
Prazosin Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Prazosin Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Prazosin hydrochloride contains not less than 98.5 per cent and not more than the equivalent of
101.0 per cent of 1-(4-amino-6,7-dimethoxyquinazolin-2-yl)-4-(furan-2-ylcarbonyl)piperazine hydrochloride, calculated with reference to the anhydrous substance.
CHARACTERS
A white or almost white powder, very slightly soluble in water, slightly soluble in alcohol and in
methanol, practically insoluble in acetone.
IDENTIFICATION
A. Dissolve 50.0 mg in a 0.1 per cent V/V solution of hydrochloric acid R in methanol R and dilute to
100.0 ml with the same acid solution. Dilute separately 1.0 ml and 5.0 ml of this solution to
100.0 ml with a 0.1 per cent V/V solution of hydrochloric acid R in methanol R (solution A and solution B, respectively). Examined between 220 nm and 280 nm (2.2.25), solution A shows an absorption maximum at 247 nm. The specific absorbance at the maximum is 1320 to 1400. Examined
between 280 nm and 400 nm, solution B shows two absorption maxima, at 330 nm and 343 nm.
The specific absorbances at the maxima are 260 to 280 and 240 to 265, respectively.
obtained with prazosin hydrochloride CRS. Examine the substances prepared as discs using potassium
chloride R. If the spectra obtained show differences, dissolve 50 mg of the substance to be examined
and 50 mg of prazosin hydrochloride CRS separately in a mixture of 10 ml of alcohol R and 10 ml of
water R, add 2 ml of dilute sodium hydroxide solution R and shake with two quantities, each of 25 ml, of
methylene chloride R. Evaporate the combined organic layers and dry the residue at 60C at a pressure
not exceeding 700 Pa. Record new spectra using the residues.
D. Dissolve about 2 mg in 2 ml of water R. The solution gives reaction (a) of chlorides (2.3.1).
TESTS
coating substance.
Test solution (a). Dissolve 0.10 g of the substance to be examined in a mixture of 1 volume of diethylamine R, 10 volumes of methanol R and 10 volumes of methylene chloride R and dilute to 10 ml with
Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with a mixture of 1 volume of diethylamine R, 10 volumes of methanol R and 10 volumes of methylene chloride R.
Reference solution (a). Dissolve 10 mg of prazosin hydrochloride CRS in a mixture of 1 volume of
32-46
diethylamine R, 10 volumes of methanol R and 10 volumes of methylene chloride R and dilute to 10 ml
Reference solution (b). Dilute 1 ml of test solution (b) to 50 ml with a mixture of 1 volume of diethylamine R, 10 volumes of methanol R and 10 volumes of methylene chloride R.
5 volumes of diethylamine R and 95 volumes of ethyl acetate R. Dry the plate in a current of warm air
with reference solution (b) (0.2 per cent).
Iron Not more than 100 ppm of Fe, determined by atomic absorption spectrometry (Method 1,
2.2.23).
Test solution. To 1.0 g add dropwise about 1.5 ml of nitric acid R. After fuming has subsided, heat on
a water-bath and then ignite by gradually raising the temperature from 150C to 1000C, maintaining the final temperature for 1 h. Cool, dissolve the residue in 20 ml of dilute hydrochloric acid R,
evaporate to about 5 ml and dilute to 25.0 ml with dilute hydrochloric acid R.
Reference solutions. Prepare the reference solutions using iron standard solution (8 ppm Fe) R, diluted as
necessary with water R.
Measure the absorbance at 248 nm using an iron hollow-cathode lamp as a source of radiation and
Nickel Not more than 50 ppm of Ni, determined by atomic C absorption spectrometry (Method I,
2.2.23).
Test solution. Use the test solution prepared in the test for iron.
Reference solutions. Prepare the reference solutions using nickel standard solution (10 ppm Ni) R, diluted
as necessary with water R.
Measure the absorbance at 232 nm using a nickel hollow-cathode lamp as a source of radiation and
of water. Use a mixture of equal volumes of methanol R and methylene chloride R as the solvent.
ASSAY
In order to avoid overheating in the reaction medium, mix thoroughly throughout and stop the titration
immediately after the end-point has been reached.
Dissolve 0.350 g in a mixture of 20 ml of anhydrous formic acid R and 30 ml of acetic anhydride R.
STORAGE
IMPURITIES
H3CO
Ar =
H3CO
NH2
A. Ar Cl: 2-chloro-6,7-dimethoxyquinazolin-4-amine,
O
O
N
N
B. 1,4-bis(furan-2-ylcarbonyl)piperazine,
NH
Ar
C. 6,7-dimethoxy-2-(piperazin-1-yl)quinazolin-4-amine,
32-47
O
N
HN
D. 1-(furan-2-ylcarbonyl)piperazine,
N
Ar
Ar
E. 2,2-(piperazine-1,4-diyl)bis(6,7-dimethoxyquinazolin-4-amine).
__________________________________________________________________________________________________________________________________ Ph Eur
33-1
Prednicarbate
O
O
Me
HO
OEt
Me
Et
H
O
H
O
C27H36O8
488.6
73771-04-7
Prednicarbate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1467].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Prednicarbate contains not less than 97.0 per cent and not more than the equivalent of 102.0 per
cent of 11,17,21-trihydroxypregna-1,4-diene-3,20-dione 17-ethylcarbonate 21-propionate, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, practically insoluble in water, freely soluble in alcohol
and in acetone, sparingly soluble in propylene glycol.
IDENTIFICATION
obtained with prednicarbate CRS. If the spectra obtained in the solid state show differences, dissolve
the substance to be examined and the reference substance separately in the minimum volume of
alcohol R, evaporate to dryness on a water-bath and record the spectra again using the residues.
Reference solution (a). Dissolve 10 mg of prednicarbate CRS in a mixture of 1 volume of methanol R
Reference solution (b). Dissolve 5 mg of prednisolone acetate CRS in 5 ml of reference solution (a).
reference solution (a). Spray the plate with alcoholic solution of sulphuric acid R. Heat at 120C for
10 min or until the spots appear. Allow to cool. Examine in daylight and in ultraviolet light at
365 nm. The principal spot in the chromatogram obtained with the test solution is similar in position, colour in daylight, flourescence in ultraviolet light at 365 nm and size to the principal spot in the
TESTS
substance.
two principal peaks in the chromatogram obtained are about 50 per cent of the full scale of the
recorder. When the chromatograms are recorded in the conditions described above, the retention
times are: prednicarbate about 17 min and impurity F about 19 min. The test is not valid unless the
33-2
resolution between the peaks corresponding to prednicarbate and impurity F is at least 3.0; if this
resolution is not achieved, adjust the composition of the mobile phase.
Inject separately 20 l of the test solution and 20 l of reference solution (b). Continue the chromatography for twice the retention time of the principal peak. In the chromatogram obtained with the
test solution: the area of any peak corresponding to impurity F is not greater than twice the area of
the principal peak in the chromatogram obtained with reference solution (b) (1 per cent); the area of
any peak, apart from the principal peak and a peak corresponding to impurity F, is not greater than
cent); the sum of the areas of all the peaks, apart from the principal peak in the chromatogram
obtained with reference solution (b) (2 per cent). Disregard any peak with an area less than 0.025
100C to 105C.
ASSAY
Reference solution (a). Dissolve 3 mg of prednicarbate impurity F CRS in the mobile phase, add 5.0 ml
of the test solution and dilute to 100.0 ml with the mobile phase. Dilute 1.0 ml to 10.0 ml with the
mobile phase.
Reference solution (c). Dissolve 30.0 mg of prednicarbate CRS in the mobile phase and dilute to
volumes of water R,
conditions the retention times are: prednicarbate about 17 min and impurity F about 19 min. The
test is not valid unless the resolution between the peaks corresponding to prednicarbate and impurity
F is not less than 3.0; if this resolution is not achieved, adjust the composition of the mobile phase.
Inject 20 l of reference solution (c). Adjust the sensitivity of the system so that the height of the
Inject 20 l of the test solution.
Calculate the percentage content of prednicarbate.
STORAGE
IMPURITIES
A. prednisolone,
O
Me
HO
OH
O
Me
OEt
H
O
H
B. prednisolone 17-ethylcarbonate,
O
O
Me
HO
O
OH
Me
H
H
C. prednisolone 21-propionate,
Et
33-3
O
O
Me
HO
Et
OH
Me
H
H
D. prednisolone 21-ethylcarbonate,
O
O
Me
HO
O
O
Me
Me
OEt
H
O
H
E. prednisolone 17-ethylcarbonate-21-acetate,
O
O
Me
HO
O
O
Me
Et
OEt
H
O
H
F. 1,2-dihydroprednicarbate.
__________________________________________________________________________________________________________ Ph Eur
33-4
Prednisolone
O
Me
HO
OH
OH
Me
H
H
O
C21H28O5
360.4
50-24-8
Prednisolone complies with the requirements of the 3rd edition of the European Pharmacopoeia [0353]. These
Preparations
Prednisolone Tablets
Enteric-coated Prednisolone Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Prednisolone contains not less than 97.0 per cent and not more than the equivalent of 103.0 per cent
of 11,17,21-trihydroxypregna-1,4-diene-3,20-dione, calculated with reference to the dried
substance.
CHARACTERS
A white or almost white, crystalline powder, hygroscopic, very slightly soluble in water, soluble in
alcohol and in methanol, sparingly soluble in acetone, slightly soluble in methylene chloride.
IDENTIFICATION
obtained with prednisolone CRS. If the spectra obtained in the solid state show differences, dissolve
the substance to be examined and the reference substance separately in the minimum volume of
acetone R and evaporate to dryness on a water-bath. Record new spectra using the residues.
Reference solution (a). Dissolve 20 mg of prednisolone CRS in a mixture of 1 volume of methanol R and
9 volumes of methylene chloride R and dilute to 20 ml with the same mixture of solvents.
Reference solution (b). Dissolve 10 mg of hydrocortisone CRS in reference solution (a) and dilute to
10 ml with reference solution (a).
volumes of methylene chloride R. Develop over a path of 15 cm. Carry out a second development over
a path of 15 cm using a mixture of 5 volumes of butanol R saturated with water R, 15 volumes of
toluene R and 80 volumes of ether R. Allow the plate to dry in air and examine in ultraviolet light at
254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position
and size to the principal spot in the chromatogram obtained with reference solution (a). Spray with
alcoholic solution of sulphuric acid R. Heat at 120C for 10 min or until the spots appear. Allow to cool.
Examine the chromatograms in daylight and in ultraviolet light at 365 nm. The principal spot in the
chromatogram obtained with the test solution is similar in position, colour in daylight, fluorescence in
ultraviolet light at 365 nm and size to the principal spot in the chromatogram obtained with reference
33-5
Test solution (b). Transfer 0.4 ml of the solution obtained during preparation of test solution (a) to a
glass tube 100 mm long and 20 mm in diameter and fitted with a ground-glass stopper or a polytetrafluoroethylene cap and evaporate the solvent with gentle heating under a stream of nitrogen R. Add
2 ml of a 15 per cent V/V solution of glacial acetic acid R and 50 mg of sodium bismuthate R. Stopper
the tube and shake the suspension for 1 h in a mechanical shaker protected from light. Add 2 ml of a
15 per cent V/V solution of glacial acetic acid R and filter into a 50 ml separating funnel, washing the
filter with two quantities, each of 5 ml, of water R. Shake the clear filtrate with 10 ml of methylene
chloride R. Wash the organic layer with 5 ml of 1M sodium hydroxide and two quantities, each of 5 ml,
of water R. Dry over anhydrous sodium sulphate R.
Reference solution (a). Dissolve 25 mg of prednisolone CRS in methanol R and dilute to 5 ml with the
same solvent. This solution is also used to prepare reference solution (b). Dilute 2 ml of the solution
to 10 ml with methylene chloride R.
Reference solution (b). Transfer 0.4 ml of the solution obtained during preparation of reference
solution (a) to a glass tube 100 mm long and 20 mm in diameter and fitted with a ground-glass
stopper or a polytetrafluoroethylene cap and evaporate the solvent with gentle heating under a stream
of nitrogen R. Add 2 ml of a 15 per cent V/V solution of glacial acetic acid R and 50 mg of sodium
bismuthate R. Stopper the tube and shake the suspension for 1 h in a mechanical shaker protected
from light. Add 2 ml of a 15 per cent V/V solution of glacial acetic acid R and filter into a 50 ml
Apply separately to the plate 5 l of test solution (a), 5 l of reference solution (a) and 10 l each of
test solution (b) and reference solution (b), applying the latter two in small quantities in order to
obtain small spots. Prepare the mobile phase by adding a mixture of 1.2 volumes of water R and 8
volumes of methanol R to a mixture of 15 volumes of ether R and 77 volumes of methylene chloride R.
Develop over a path of 15 cm. Carry out a second development over a path of 15 cm using a mixture
of 5 volumes of butanol R saturated with water R, 15 volumes of toluene R and 80 volumes of ether R.
Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in each of
the chromatograms obtained with the test solutions is similar in position and size to the principal spot
in the chromatogram obtained with the corresponding reference solution. Spray with alcoholic solution
of sulphuric acid R. Heat at 120C for 10 min or until the spots appear. Allow to cool. Examine the
chromatograms in daylight and in ultraviolet light at 365 nm. The principal spot in each of the two
chromatograms obtained with the test solutions is similar in position, colour in daylight, fluorescence
in ultraviolet light at 365 nm and size to the principal spot in the chromatogram obtained with the
corresponding reference solution. The principal spots in the chromatograms obtained with test
solution (b) and reference solution (b) have an Rf value distinctly higher than that of the principal
spots in the chromatograms obtained with test solution (a) and reference solution (a).
colour develops. When examined in ultraviolet light at 365 nm a reddish-brown fluorescence is seen.
After 5 min, add the solution to 10 ml of water R and mix. The colour fades and there is a yellow
fluorescence in ultraviolet light at 365 nm and a grey, flocculent precipitate is formed.
TESTS
substance.
Reference solution (a). Dissolve 2 mg of prednisolone CRS and 2 mg of hydrocortisone CRS in the mobile
a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with basedeactivated end-capped octadecylsilyl silica gel for chromatography R (5 m),
as mobile phase at a flow rate of 1 ml/min a mixture prepared as follows: in a 1000 ml
volumetric flask mix 220 ml of tetrahydrofuran R with 700 ml of water R and allow to equilibrate;
dilute to 1000 ml with water R and mix again,
33-6
conditions, the retention times are: prednisolone, about 14 min and hydrocortisone, about 15.5 min.
The test is not valid unless in the chromatogram obtained with reference solution (a) the resolution
between the peaks due to prednisolone and hydrocortisone is at least 2.2. If necessary, adjust the
concentration of tetrahydrofuran R in the mobile phase.
Inject separately 20 l of the solvent mixture of the test solution as a blank, 20 l of the test
solution and 20 l of reference solution (b). Continue the chromatography of the test solution for 4.5
times the retention time of the principal peak. In the chromatogram obtained with the test solution:
the area of any peak, apart from the principal peak, is not greater than the area of the principal peak
in the chromatogram obtained with reference solution (b) (1 per cent) and not more than one such
peak has an area greater than half the area of the principal peak in the chromatogram obtained with
reference solution (b) (2 per cent). Disregard any peak obtained with the blank run and any peak
100C to 105C.
ASSAY
solution to 100.0 ml with alcohol R. Measure the absorbance (2.2.25) at the maximum at 243.5 nm.
STORAGE
IMPURITIES
A. hydrocortisone.
__________________________________________________________________________________________________________ Ph Eur
33-7
Prednisolone Acetate
O
H
Me
HO
OAc
OH
Me
H
H
O
C23H30O6
402.5
52-21-1
Prednisolone Acetate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Prednisolone acetate contains not less than 97.0 per cent and not more than the equivalent of
103.0 per cent of 11,17-dihydroxy-3,20-dioxopregna-1,4-diene-21-yl acetate, calculated with
CHARACTERS
IDENTIFICATION
Second identification: C, D, E.
obtained with prednisolone acetate CRS. Examine the substances prepared as discs.
Reference solution (a). Dissolve 20 mg of prednisolone acetate CRS in a mixture of 1 volume of
solvents.
Reference solution (b). Dissolve 10 mg of prednisolone pivalate CRS in reference solution (a) and dilute
to 10 ml with the same solution.
Test solution (a). Dissolve 25 mg of the substance to be examined in methanol R with gentle heating
and dilute to 5 ml with the same solvent. This solution is also used to prepare test solution (b).
15 ml glass tube with a ground-glass stopper or a polytetrafluoroethylene cap. Add 10 ml of saturated
33-8
methanolic potassium hydrogen carbonate solution R and immediately pass a stream of nitrogen R briskly
for 2 h 30 min. Allow to cool.
Reference solution (a). Dissolve 25 mg of prednisolone acetate CRS in methanol R with gentle heating
and dilute to 5 ml with the same solvent. This solution is also used to prepare reference solution (b).
saturated methanolic potassium hydrogen carbonate solution R and immediately pass a stream of
nitrogen R briskly through the solution for 5 min. Stopper the tube. Heat in a water-bath at 45C,
protected from light, for 2 h 30 min. Allow to cool.
obtained with the corresponding reference solution. Spray the plate with alcoholic solution of sulphuric
acid R. Heat at 120C for 10 min or until the spots appear. Allow to cool. Examine in daylight and in
ultraviolet light at 365 nm. The principal spot in each of the chromatograms obtained with the test
solutions is similar in position, colour in daylight, fluorescence in ultraviolet light at 365 nm and size
to the principal spot in the chromatogram obtained with the corresponding reference solution. The
principal spot in each of the chromatograms obtained with test solution (b) and reference solution (b)
has an Rf value distinctly lower than that of the principal spots in each of the chromatograms
obtained with test solution (a) and reference solution (a).
Add the solution to 10 ml of water R and mix. The colour fades and there is greenish-yellow fluorescence in ultraviolet light at 365 nm.
E. About 10 mg gives the reaction of acetyl (2.3.1).
TESTS
substance.
Reference solution (a). Dissolve 2 mg of prednisolone acetate CRS and 2 mg of hydrocortisone acetate CRS
a stainless steel column 0.25 m long and 4.6 mm in internal diameter, packed with basedeactivated end-capped octadecylsilyl silica gel for chromatography R (5 m),
volumetric flask mix 350 ml of acetonitrile R with 600 ml of water R and allow to equilibrate;
adjust the volume to 1000 ml with water R and mix again,
conditions, the retention times are: prednisolone acetate, about 24 min and hydrocortisone acetate,
about 26 min. The test is not valid unless the resolution between the peaks corresponding to
prednisolone acetate and hydrocortisone acetate is at least 2.5; if necessary, adjust the concentration
of acetonitrile in the mobile phase.
Inject separately 20 l of the test solution and 20 l of reference solution (b). Continue the chromatogram for 2.5 times the retention time of the principal peak in the chromatogram obtained with the
test solution. In the chromatogram obtained with the test solution: the area of any peak, apart from
with reference solution (b) (1 per cent) and not more than one such peak has an area greater than
half the area of the principal peak in the chromatogram obtained with reference solution (b) (0.5 per
cent); the sum of the areas of all the peaks, apart from the principal peak, is not greater than twice the
area of the principal peak in the chromatogram obtained with reference solution (b) (2 per cent).
33-9
Disregard any peak due to the solvent and any peak with an area less than 0.05 times the area of the
100C to 105C.
ASSAY
STORAGE
IMPURITIES
A. hydrocortisone acetate,
B. prednisolone.
__________________________________________________________________________________________________________________________________ Ph Eur
33-10
Prednisolone Pivalate
O
H
OCOBut
Me
HO
OH
Me
H
H
O
C26H36O6
444.6
1107-99-9
Prednisolone Pivalate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Prednisolone pivalate contains not less than 97.0 per cent and not more than the equivalent of
103.0 per cent of 11,17,21-trihydroxypregna-1,4-diene-3,20-dione 21-pivalate, calculated with
CHARACTERS
A white or almost white, crystalline powder, practically insoluble in water, slightly soluble in alcohol,
IDENTIFICATION
A. Dissolve 10.0 mg in ethanol R and dilute to 100.0 ml with the same solvent. Place 2.0 ml of this
solution in a ground-glass-stoppered tube, add 10.0 ml of phenylhydrazine-sulphuric acid solution R,
mix and heat in a water-bath at 60C for 20 min. Cool immediately. The absorbance (2.2.25) of the
solution at the maximum at 415 nm is 0.20 to 0.30.
obtained with prednisolone pivalate CRS. If the spectra obtained in the solid state show differences,
volume of alcohol R, evaporate to dryness on a water-bath and record the spectra again using the
residues.
Reference solution (a). Dissolve 10 mg of prednisolone pivalate CRS in a mixture of 1 volume of
solvents.
Reference solution (b). Dissolve 10 mg of prednisolone acetate CRS in a mixture of 1 volume of
solvents. Dilute 5 ml of this solution to 10 ml with reference solution (a).
33-11
TESTS
substance.
Test solution. Dissolve 62.5 mg of the substance to be examined in 2 ml of a mixture of 1 volume of
water R and 4 volumes of tetrahydrofuran R and dilute to 25.0 ml with the mobile phase.
Reference solution (a). Dissolve 25 mg of prednisolone acetate CRS, 25 mg of cortisone acetate CRS and
25 mg of prednisolone pivalate CRS in 2 ml of a mixture of 1 volume of water R and 4 volumes of
tetrahydrofuran R and dilute to 25.0 ml with the mobile phase. Dilute 1.0 ml of this solution to
as mobile phase at a flow rate of 1 ml per minute a mixture prepared as follows: mix 19 ml of
butyl acetate R1 carefully with 37 ml of tetrahydrofuran R and 213 ml of ethylene glycol monomethyl
ether R and then with 231 ml of water R, leave to equilibrate for 1 h and filter through a 0.45 m
filter,
Adjust the sensitivity so that the height of the principal peak in the chromatogram obtained with
reference solution (b) is 70 per cent to 90 per cent of the full scale.
described above the retention times are: prednisolone acetate about 3.5 min, cortisone acetate about
4.5 min and prednisolone pivalate about 13 min. The test is not valid unless the resolution between
the peaks corresponding to prednisolone acetate and cortisone acetate is at least 2.5; if this resolution
is not achieved, adjust the concentration of water R in the mobile phase.
Inject separately 20 l of the test solution and 20 l of reference solution (b). Continue the chromatography for 1.5 times the retention time of the principal peak. In the chromatogram obtained with
the test solution: the area of any peak apart from the principal peak is not greater than the area of the
principal peak in the chromatogram obtained with reference solution (b) (2.0 per cent) and not more
than one such peak has an area greater than half the area of the principal peak in the chromatogram
obtained with reference solution (b) (1.0 per cent); the sum of the areas of all the peaks apart from
the principal peak is not greater than 1.25 times the area of the principal peak in the chromatogram
100C to 105C.
ASSAY
STORAGE
__________________________________________________________________________________________________________________________________ Ph Eur
33-12
Prednisolone Sodium Phosphate

corrected 1/01
ONa
O
H
Me
HO
O
OH
Me
ONa
H
H
O
C21H27Na2O8P
484.4
125-02-0
Prednisolone Sodium Phosphate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0735]. These requirements are reproduced after the heading Definition below.
Preparations
Prednisolone Enema
Prednisolone Sodium Phosphate Eye Drops
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Prednisolone sodium phosphate contains not less than 96.0 per cent and not more than the equivalent of 103.0 per cent of 11,17-dihydroxy-3,20-dioxpregna-1,4-dien-21-yl disodium phosphate,
CHARACTERS
A white or almost white, crystalline powder, hygroscopic, freely soluble in water, very slightly soluble
in alcohol.
IDENTIFICATION
A. Dissolve 10.0 mg in 5 ml of water R and dilute to 100.0 ml with ethanol R. Place 2.0 ml of this
solution in a ground-glass-stoppered tube, add 10.0 ml of phenylhydrazine-sulphuric acid solution R,
mix and heat in a water-bath at 60C for 20 min. Cool immediately. The absorbance (2.2.25) of the
solution at the maximum at 415 nm is 0.10 to 0.20.
obtained with prednisolone sodium phosphate CRS. If the spectra obtained in the solid state show
minimum volume of alcohol R, evaporate to dryness on a water-bath and record the spectra again
using the residues.
the same solvent.
Reference solution (a). Dissolve 10 mg of prednisolone sodium phosphate CRS in methanol R and dilute to
Reference solution (b). Dissolve 10 mg of dexamethasone sodium phosphate CRS in methanol R and dilute
to 10 ml with the same solvent. Dilute 5 ml of this solution to 10 ml with reference solution (a).
obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a). Spray the plate with alcoholic solution of sulphuric acid R.
Heat at 120C for 10 min or until the spots appear. Allow to cool. Examine in daylight and in
ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution is
similar in position, colour in daylight, fluorescence in ultraviolet light at 365 nm and size to the
33-13
the chromatogram obtained with reference solution (b) shows two spots which may however not be
completely separated.
E. To about 40 mg add 2 ml of sulphuric acid R and heat gently until white fumes are evolved. Add
nitric acid R dropwise, continue the heating until the solution is almost colourless and cool. Add 2 ml
of water R, heat until white fumes are again evolved, cool, add 10 ml of water R and neutralise to red
litmus paper R with dilute ammonia R1. The solution gives reaction (a) of sodium (2.3.1) and reaction
(b) of phosphates (2.3.1).
TESTS
substance.
Reference solution (a). Dissolve 25 mg of prednisolone sodium phosphate CRS and 25 mg of prednisolone
CRS in the mobile phase and dilute to 25.0 ml with the same solvent. Dilute 1.0 ml of this solution
as mobile phase at a flow rate of 1 ml/min a mixture prepared as follows: in a 250 ml conical
flask weigh 1.360 g of potassium dihydrogen phosphate R and 0.600 g of hexylamine R, mix and
allow to stand for 10 min and then dissolve in 185 ml of water R; add 65 ml of acetonitrile R, mix
and filter through a 0.45 m filter,
principal peak in the chromatogram obtained with reference solution (b) is 70 per cent to 90 per cent
of the full scale of the recorder.
described above the retention times are: prednisolone sodium phosphate, about 6.5 min and
prednisolone, about 8.5 min. The test is not valid unless the resolution between the peaks corresponding to prednisolone sodium phosphate and prednisolone is at least 4.5; if this resolution is not
achieved, increase the concentration of acetonitrile R or increase the concentration of water R in the
mobile phase.
Inject separately 20 l of the test solution and 20 l of reference solution (b). Continue the chromatography for three times the retention time of the principal peak. In the chromatogram obtained
with the test solution: the area of any peak apart from the principal peak is not greater than the area
of the principal peak in the chromatogram obtained with reference solution (b) (2 per cent) and not
more than one such peak has an area greater than half the area of the principal peak in the chromatogram obtained with reference solution (b) (1 per cent); the sum of the areas of all the peaks apart
from the principal peak is not greater than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (b) (3 per cent). Disregard any peak due to the solvent and
Inorganic phosphate Dissolve 50 mg in water R and dilute to 100 ml with the same solvent. To
10 ml of this solution add 5 ml of molybdovanadic reagent R, mix and allow to stand for 5 min. Any
yellow colour in the solution is not more intense than that in a standard prepared at the same time in
the same manner using 10 ml of phosphate standard solution (5 ppm PO4 ) R (1 per cent).
33-14
ASSAY
Dissolve 0.100 g in water R and dilute to 100.0 ml with the same solvent. Dilute 5.0 ml of the
solution to 250.0 ml with water R. Measure the absorbance (2.2.25) at the maximum at 247 nm.
Calculate the content of C21H27Na2O8P taking the specific absorbance to be 312.
STORAGE
__________________________________________________________________________________________________________________________________ Ph Eur
33-15
Prednisone
O
OH
Me
O
OH
Me
H
H
O
C21H26O5
358.4
53-03-2
Prednisone complies with the requirements of the 3rd edition of the European Pharmacopoeia [0354]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Prednisone contains not less than 97.0 per cent and not more than the equivalent of 103.0 per cent
of 17,21-dihydroxypregna-1,4-diene-3,11,20-trione, calculated with reference to the dried substance.
CHARACTERS
IDENTIFICATION
obtained with prednisone CRS. If the spectra obtained in the solid state show differences, dissolve the
substance to be examined and the reference substance separately in the minimum volume of
acetone R, evaporate to dryness on a water-bath and record new spectra using the residues.
Reference solution (a). Dissolve 20 mg of prednisone CRS in a mixture of 1 volume of methanol R and 9
volumes of methylene chloride R and dilute to 20 ml with the same mixture of solvents.
Reference solution (b). Dissolve 10 mg of betamethasone CRS in reference solution (a) and dilute to
solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a). Spray with alcoholic solution of sulphuric acid R. Heat at 120C for 10 min or until
the spots appear. Allow to cool. Examine the plate in daylight and in ultraviolet light at 365 nm. The
Test solution (b). Transfer 0.4 ml of the solution obtained during preparation of test solution (a) to a
glass tube 100 mm long and 20 mm in diameter and fitted with a ground-glass stopper or a poly-
33-16
tetrafluoroethylene cap and evaporate the solvent with gentle heating under a stream of nitrogen R.
Add 2 ml of a 15 per cent V/V solution of glacial acetic acid R and 50 mg of sodium bismuthate R.
Stopper the tube and shake the suspension in a mechanical shaker, protected from light, for 1 h. Add
2 ml of a 15 per cent V/V solution of glacial acetic acid R and filter into a 50 ml separating funnel,
washing the filter with two quantities, each of 5 ml, of water R. Shake the clear filtrate with 10 ml of
methylene chloride R. Wash the organic layer with 5 ml of 1M sodium hydroxide and two quantities,
each of 5 ml, of water R. Dry over anhydrous sodium sulphate R.
Reference solution (a). Dissolve 25 mg of prednisone CRS in methanol R and dilute to 5 ml with the
same solvent. This solution is also used to prepare reference solution (b). Dilute 2 ml of the solution
Reference solution (b). Transfer 0.4 ml of the solution obtained during preparation of reference solution (a) to a glass tube 100 mm long and 20 mm in diameter and fitted with a ground-glass stopper
or a polytetrafluoroethylene cap and evaporate the solvent with gentle heating under a stream of
nitrogen R. Add 2 ml of a 15 per cent V/V solution of glacial acetic acid R and 50 mg of sodium
bismuthate R. Stopper the tube and shake the suspension in a mechanical shaker, protected from
light, for 1 h. Add 2 ml of a 15 per cent V/V solution of glacial acetic acid R and filter into a 50 ml
Apply separately to the plate 5 l of test solution (a) and reference solution (a) and 50 l of test
solution (b) and reference solution (b), applying the latter two in small quantities in order to obtain
small spots. Prepare the mobile phase by adding a mixture of 1.2 volumes of water R and 8 volumes
of methanol R to a mixture of 15 volumes of ether R and 77 volumes of methylene chloride R. Develop
over a path of 15 cm. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The
principal spot in each of the chromatograms obtained with the test solutions is similar in position and
size to the principal spot in the chromatogram obtained with the corresponding reference solution.
Spray with alcoholic solution of sulphuric acid R. Heat at 120C for 10 min or until the spots appear.
Allow to cool. Examine the chromatograms in daylight and in ultraviolet light at 365 nm. The principal spot in each of the chromatograms obtained with the test solutions is similar in position, colour in
daylight, fluorescence in ultraviolet light at 365 nm and size to the principal spot in the chromatogram obtained with the corresponding reference solution. The principal spots in the chromatograms
obtained with test solution (b) and reference solution (b) have an R value distinctly higher than that
of the principal spots in the chromatograms obtained with test solution (a) and reference solution (a).
D. Add about 2 mg to 2 ml of sulphuric acid R and shake to dissolve. Within 5 min, a yellow colour
develops with a blue fluorescence in ultraviolet light at 365 nm. Add the solution to 10 ml of water R
and mix. The colour fades but the blue fluorescence in ultraviolet light does not disappear.
TESTS
substance.
Reference solution (a). Dissolve 2 mg of prednisone CRS and 2 mg of prednisolone CRS in methanol R
Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with methanol R.
as mobile phase at a flow rate of 2.5 ml per minute a linear gradient programme using the
following conditions:
Mobile phase A. In a 1000 ml volumetric flask mix 100 ml of acetonitrile R with 200 ml of
methanol R and 650 ml of water R and allow to equilibrate; adjust the volume to 1000 ml with
water R and mix again,
33-17
Time
(min)
Mobile
phase A
(per cent V/V)
Mobile
phase B
(per cent V/V)
Comment
0
25
100
100
0
0
40
40
60
41
100
46
100
47
100
52 = 0
100
isocratic
begin linear
gradient
end
chromatogram,
change to 100B
begin treatment
with B
end treatment,
return to 100A
begin equilibration
with A
end equilibration,
begin next
chromatogram

Equilibrate the column for at least 30 min with mobile phase B at a flow rate of 2.5 ml per minute
and then with the mobile phase A for 5 minutes. For subsequent chromatograms, use the conditions
described from 40.0 to 52.0 min. Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with 20 l of reference solution (b) is not less than 50 per
cent of the full scale of the recorder.
conditions, the retention times are: prednisone, about 19 minutes and prednisolone, about 23
minutes. The test is not valid unless the resolution between the peaks corresponding to prednisone
and prednisolone is at least 2.7; if necessary, adjust the concentration of acetonitrile in mobile phase
A.
solution (b). In the chromatogram obtained with the test solution: the area of any peak apart from
the principal peak is not greater than 0.25 times the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.75 per cent). Disregard any peak due to the blank run and
100C to 105C.
ASSAY
STORAGE
__________________________________________________________________________________________________________________________________ Ph Eur
33-18
Prilocaine
Me
H
N
Me
NHPr n
O
and enantiomer
C13H20N2O
220.3
721-50-6
Prilocaine complies with the requirements of the 3rd edition of the European Pharmacopoeia [1362]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Prilocaine contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of
(RS)-N-(2-methylphenyl)-2-(propylamino)propanamide, calculated with reference to the anhydrous
substance.
CHARACTERS
A white or almost white, crystalline powder, slightly soluble in water, very soluble in acetone and in
alcohol.
IDENTIFICATION
A. Melting point (2.2.14): 36C to 39C, determined without previous drying.
obtained with prilocaine CRS. Examine the substances by applying 50 l of a 30 g/l solution in ether R
to potassium bromide R discs and evaporating the solvent.
C. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel GF254 plate R.
Test solution. Dissolve 20.0 mg of the substance to be examined in alcohol R and dilute to 5 ml with
the same solvent.
Reference solution (a). Dissolve 20.0 mg of prilocaine CRS in alcohol R and dilute to 5 ml with the same
solvent.
Reference solution (b). Dissolve 20.0 mg of prilocaine CRS and 20.0 mg of lidocaine CRS in alcohol R
Apply to the plate 10 l of each solution. Develop over a path of 12 cm, using a mixture of 1 volume
TESTS
Solution S Dissolve 2.50 g in 15 ml of dilute hydrochloric acid R and dilute to 50.0 ml with water R.
+0.10.
Reference solution (a). Dissolve 2.5 mg of the substance to be examined and 3.0 mg of prilocaine
impurity E CRS in the mobile phase and dilute to 100.0 ml with the mobile phase. Dilute 1.0 ml to
33-19
volumes of a solution prepared as follows: dissolve 0.180 g of sodium dihydrogen phosphate
monohydrate R and 2.89 g of disodium hydrogen phosphate dihydrate R in 1000 ml of water R (pH
8.0),
recorder. The test is not valid unless the resolution between the peaks corresponding to impurity E
and prilocaine is at least 3.0. Inject 20 l of the test solution and 20 l of reference solution (b).
Continue the chromatography of the test solution for twice the retention time of prilocaine, which is
about 7 min. In the chromatogram obtained with the test solution: the area of any peak, apart from
the principal peak, is not greater than twice the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.2 per cent) and not more than one such peak has an area
five times the area of the principal peak in the chromatogram obtained with reference solution (b)
o-Toluidine Use freshly prepared solutions. Examine by liquid chromatography (2.2.29).
Reference solution. Dissolve 10.0 mg of o-toluidine hydrochloride R in the mobile phase and dilute to
Carry out the chromatography using the same system as described under Related substances. Inject
20 l of the reference solution. Adjust the sensitivity of the system so that the height of the peak in
the chromatogram obtained is at least 50 per cent of the full scale of the recorder. Inject 20 l of the
test solution. Continue the chromatography of the test solution for twice the retention time of
prilocaine. In the chromatogram obtained with the test solution, the area of any peak corresponding
to o-toluidine is not greater than the area of the principal peak in the chromatogram obtained with
of water.
ASSAY
IMPURITIES
Me
H
N
H Me
Cl
and enantiomer
A. (RS)-2-chloro-N-(2-methylphenyl)propanamide,
Me
NH2
B. 2-methylbenzenamine (o-toluidine),
Me
H
N
H Me
NHEt
and enantiomer
C. (RS)-2-(ethylamino)-N-(2-methylphenyl)propanamide,
33-20
H
N
Me
H Me
NHPr n
and enantiomer
D. (RS)-N-(3-methylphenyl)-2-(propylamino)propanamide,
H
N
H Me
NHPr n
and enantiomer
Me
E. (RS)-N-(4-methylphenyl)-2-(propylamino)propanamide,
H
N
H Me
NHPr n
and enantiomer
F. (RS)-N-phenyl-2-(propylamino)propanamide.
__________________________________________________________________________________________________________ Ph Eur
33-21
Prilocaine Hydrochloride
Me
H
N
Me
NHPr n
,HCl
O
and enantiomer
C13H20N2O,HCl
256.8
1786-81-8
Prilocaine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Prilocaine Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Prilocaine hydrochloride contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of (RS)-N-(2-methylphenyl)-2-(propylamino)propanamide hydrochloride, calculated
CHARACTERS
A white, crystalline powder or colourless crystals, freely soluble in water and in alcohol, very slightly
soluble in acetone.
IDENTIFICATION
obtained with prilocaine hydrochloride CRS. Examine the substances prepared as discs.
Test solution. Dissolve 20.0 mg of the substance to be examined in alcohol R and dilute to 5 ml with
the same solvent.
Reference solution (a). Dissolve 20.0 mg of prilocaine hydrochloride CRS in alcohol R and dilute to 5 ml
Reference solution (b). Dissolve 20.0 mg of prilocaine hydrochloride CRS and 20.0 mg of lidocaine
hydrochloride CRS in alcohol R and dilute to 5 ml with the same solvent.
Apply to the plate 10 l of each solution. Develop over a path of 12 cm, using a mixture of 1 volume
TESTS
+0.10.
Acidity or alkalinity Dilute 4 ml of solution S to 10 ml with carbon dioxide-free water R. Add 0. 1 ml
of bromocresol green solution R and 0.40 ml of 0.01M sodium hydroxide. The solution is blue. Add
0.80 ml of 0.01M hydrochloric acid. The solution is yellow.
Reference solution (a). Dissolve 3.0 mg of the substance to be examined and 3.0 mg of prilocaine
33-22
impurity E CRS in the mobile phase and dilute to 100.0 ml with the mobile phase. Dilute 1.0 ml of
this solution to 10.0 ml with the mobile phase.
volumes of a solution prepared as follows: dissolve 0.180 g of sodium dihydrogen phosphate
monohydrate R and 2.89 g of disodium hydrogen phosphate dihydrate R in 1000 ml of water R (pH
8.0),
recorder. The test is not valid unless the resolution between the peaks corresponding to impurity E
and prilocaine is at least 3.0. Inject 20 l of the test solution and 20 l of reference solution (b).
Continue the chromatography of the test solution for twice the retention time of prilocaine, which is
about 7 min. In the chromatogram obtained with the test solution: the area of any peak, apart from
obtained with reference solution (b) (0.2 per cent) and not more than one such peak has an area
five times the area of the principal peak in the chromatogram obtained with reference solution (b)
o-Toluidine Use freshly prepared solutions. Examine by liquid chromatography (2.2.29).
Reference solution. Dissolve 10.0 mg of o-toluidine hydrochloride R in the mobile phase and dilute to
Carry out the chromatography using the same system as described under Related substances. Inject
20 l of the reference solution. Adjust the sensitivity of the system so that the height of the peak in
the chromatogram obtained is at least 50 per cent of the full scale of the recorder. Inject 20 l of the
test solution. Continue the chromatography of the test solution for twice the retention time of
prilocaine. In the chromatogram obtained with the test solution, the area of any peak corresponding
to o-toluidine is not greater than the area of the principal peak in the chromatogram obtained with
100C to 105C.
ASSAY
1 ml of 0.1M sodium hydroxide is equivalent to 25.68 mg of C13H21ClN20O.
IMPURITIES
Me
H
N
H Me
Cl
and enantiomer
A. (RS)-2-chloro-N-(2-methylphenyl)propanamide,
Me
NH2
B. 2-methylbenzenamine (o-toluidine),
33-23
Me
H
N
H Me
NHEt
and enantiomer
C. (RS)-2-(ethylamino)-N-(2-methylphenyl)propanamide,
H
N
Me
H Me
NHPr n
and enantiomer
D. (RS)-N-(3-methylphenyl)-2-(propylamino)propanamide,
H
N
H Me
NHPr n
and enantiomer
Me
E. (RS)-N-(4-methylphenyl)-2-(propylamino)propanamide,
H
N
H Me
NHPr n
and enantiomer
F. (RS)-N-phenyl-2-(propylamino)propanamide.
__________________________________________________________________________________________________________ Ph Eur
33-24
Primaquine Phosphate
1/01
H
Me
NH2
HN
N
,2H3PO4
MeO
and enantiomer
C15H21N3O,2H3PO4
455.3
63-45-6
Primaquine Phosphate complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Primaquine Diphosphate [0635]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Primaquine diphosphate contains not less than 98.5 per cent and not more than the equivalent of
101.5 per cent of (RS)-8-(4-amino-1-methylbutylamino)-6-methoxyquinoline diphosphate, calculated with reference to the dried substance.
CHARACTERS
An orange, crystalline powder, soluble in water, practically insoluble in alcohol.
IDENTIFICATION
A. Dissolve 15 mg in 0.01M hydrochloric acid and dilute to 100.0 ml with the same acid. Examined
415 nm. The specific absorbances at the maxima are 45 to 52 and 27 to 35, respectively. Dilute
310 nm, the solution shows three absorption maxima, at 225 nm, 265 nm and 282 nm. The specific
absorbances at the maxima are 495 to 515, 335 to 350 and 330 to 345, respectively.
obtained with primaquine diphosphate CRS. Examine the substances as discs prepared as follows:
dissolve separately 0.1 g of the substance to be examined and 0.1 g of the reference substance in 5 ml
of water R, add 2 ml of dilute ammonia R2 and 5 ml of methylene chloride R and shake; dry the chloroform layer over 0.5 g of anhydrous sodium sulphate R; prepare a blank disc using about 0.3 g of
potassium bromide R; apply dropwise to the disc 0.1 ml of the chloroform layer, allowing the chloroform to evaporate between applications; dry the disc at 50C for 2 min.
Carry out all operations as rapidly as possible, protected from light. Prepare the test and reference solutions
immediately before use.
Test solution. Dissolve 0.20 g of the substance to be examined in 5 ml of water R and dilute to 10 ml
with methanol R. Dilute 1 ml of the solution to 10 ml with a mixture of equal volumes of methanol R
and water R.
Reference solution. Dissolve 20 mg of primaquine diphosphate CRS in 5 ml of water R and dilute to
10 ml with methanol R.
Carry out pre-washing of the plate with a mixture of 1 volume of concentrated ammonia R, 40 volumes
of methanol R and 60 volumes of methylene chloride R. Allow the plate to dry in air. Apply to the plate
5 l of each solution. Develop over a path of 15 cm using the mixture of solvents prescribed for prewashing. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot
in the chromatogram obtained with the test solution is similar in position and size to the principal
D. Dissolve 50 mg in 5 ml of water R. Add 2 ml of dilute sodium hydroxide solution R and shake with
two quantities, each of 5 ml, of methylene chloride R. The aqueous layer, acidified by addition of nitric
acid R, gives reaction (b) of phosphates (2.3.1).
33-25
TESTS
same solvent. To 1.0 ml of the solution add 0.2 ml of concentrated ammonia R and shake with 10.0 ml
of the mobile phase. Use the clear lower layer.
Reference solution (a). Dissolve 50 mg of primaquine diphosphate CRS in water R and dilute to 5.0 ml
with the same solvent. To 1.0 ml of the solution add 0.2 ml of concentrated ammonia R and shake
with 10.0 ml of the mobile phase. Use the clear lower layer.
Reference solution (c). Dilute 1.0 ml of the test solution to 10.0 ml with the mobile phase. Dilute
a column 0.2 m long and 4.6 mm in internal diameter packed with silica gel for chromatography R
(10 m),
as mobile phase at a flow rate of 3.0 ml/min a mixture of 0.1 volumes of concentrated ammonia R,
10 volumes of methanol R, 45 volumes of methylene chloride R and 45 volumes of hexane R,
a loop injector.
Inject 20 l of each solution and continue the chromatography for at least twice the retention time
of primaquine. The test is not valid unless in the chromatogram obtained with reference solution (a),
just before the principal peak there is a peak whose area is about 6 per cent of that of the principal
peak and the resolution between these peaks is not less than 2.0; in the chromatogram obtained with
reference solution (c) the signal-to-noise ratio of the principal peak is not less than five. In the
chromatogram obtained with the test solution, the sum of the areas of any peaks, apart from the
reference solution (b) (3.0 per cent). Disregard the peak due to the solvent and any peak whose area
is less than that of the principal peak in the chromatogram obtained with reference solution (c).
100C to 105C.
ASSAY
Dissolve 0.2000 g in 40 ml of anhydrous acetic acid R, heating gently. Allow to cool and titrate with
1 ml of 0.1M perchloric acid is equivalent to 22.77 mg of C15H27N3O9P2.
STORAGE
__________________________________________________________________________________________________________________________________ Ph Eur
33-26
Primidone
H
N
Et
NH
O
C12H14N2O2
218.3
125-33-7
Primidone complies with the requirements of the 3rd edition of the European Pharmacopoeia [0584]. These
Preparations
Primidone Oral Suspension
Primidone Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Primidone contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent of
5-ethyl-2,3-dihydro-5-phenyl-4,6(1H,5H)-pyrimidine dione, calculated with reference to the dried
substance.
CHARACTERS
A white or almost white, crystalline powder, very slightly soluble in water, slightly soluble in alcohol,
IDENTIFICATION
A. Use the solution prescribed for the assay. The solution, examined between 240 nm and 300 nm
(2.2.25), shows three absorption maxima, at 252 nm, at 257 nm and at 264 nm and two absorption
minima, at 254 nm and 261 nm. The ratio of the absorbance measured at the maximum at 257 nm
to that measured at the minimum at 261 nm is 2.00 to 2.20. The identification is valid if, in the test
for resolution (2.2.25), the ratio of the absorbances is not less than 2.0.
obtained with primidone CRS. Examine the substances as discs prepared using potassium bromide R.
C. Dissolve 0.1 g in 5 ml of a 5 g/l solution of chromotropic acid, sodium salt R in a mixture of 4
volumes of water R and 9 volumes of sulphuric acid R. A pinkish-blue colour develops on heating.
D. Mix 0.2 g with 0.2 g of anhydrous sodium carbonate R. Heat until the mixture melts. Ammonia is
evolved which is detectable by its odour and its alkaline reaction (2.2.4).
TESTS
Water-soluble substances absorbing ultraviolet light To 2.0 g add 50 ml of water R and heat in
a water-bath for 30 min, shaking from time to time. Cool the solution to 20C and filter. Wash the
filter with 10 ml of water R at 20C adding the washings to the main filtrate. Dilute the combined
filtrate and washings to 100.0 ml with water R. The absorbance (2.2.25) of the solution measured at
the maximum at 257 nm is not greater than 0.35.
100C to 105C for 2 h.
ASSAY
Dissolve 60.0 mg with heating in 70 ml of alcohol R, cool and dilute to 100.0 ml with the same
solvent. Prepare in the same manner a reference solution using 60.0 mg of primidone CRS. Measure
the absorbance (2.2.25) of the two solutions at the maximum at 257 nm.
the solutions.
__________________________________________________________________________________________________________________________________ Ph Eur
33-27
Primula Root
Primula Root complies with the requirements of the 3rd edition of the European Pharmacopoeia [1364].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Primula root consists of the whole or cut, dried rhizome and root of Primula veris L. or P. elatior (L.)
Hill.
CHARACTERS
Primula root has a bitter taste.
IDENTIFICATION
A. The coarsely torose, greyish-brown rhizome is straight or slightly curved, about 1 cm to 5 cm long
and about 2 mm to 4 mm thick. The rhizome crown often bears the remains of stems and leaves.
Attached to the rhizome are numerous brittle roots, about 1 mm thick and usually 6 cm to 8 cm
long. The root of P. elatior is light brown to reddish-brown, that of P. veris light yellow to yellowishwhite. The fracture is smooth.
B. Reduce to a powder (355). The powder is greyish-brown. Examine under a microscope, using
chloral hydrate solution R. The powder shows fragments of parenchyma from the root bark, medulla
and bark of the rhizome consisting of rounded cells with thickened and pitted walls; brownish
fragments from the surface tissue showing root hairs; vessels with reticulate thickening. Yellowishgreen groups of strongly pitted stone cells are characteristic of the presence of P. elatior. Examine
under a microscope using a 50 per cent V/V solution of glycerol R. The powder shows single or
compound starch grains of various size and shape.
C. Use the chromatograms obtained in the test for Vincetoxicum hirundinaria medicus root. Spray the
plate with anisaldehyde solution R. Heat at 100C to 105C for 5 min to 10 min. Examine in daylight.
The main zone (aescin) in the chromatogram obtained with the reference solution is bluish-violet and
is situated near the boundary between the lower and middle thirds. The chromatogram obtained with
the test solution shows one or two strong dark violet zones a little below the aescin zone in the
chromatogram obtained with the reference solution; further pale violet, yellowish or brownish-green
zones may be visible.
TESTS
Vincetoxicum hirundinaria medicus root Examine by thin-layer chromatography (2.2.27), using
a TLC silica gel F254 plate R.
Test solution. To 1.0 g of the powdered drug (500) add 10 ml of alcohol (70 per cent V/V) R and heat
under a reflux condenser for 15 min. Cool and filter.
Reference solution. Dissolve 10 mg of aescin R in 1.0 ml of alcohol (70 per cent V/V) R.
Apply to the plate as bands 20 l of each solution. Develop over a path of 12 cm using the upper
phase of a mixture of 10 volumes of glacial acetic acid R, 40 volumes of water R and 50 volumes of
butanol R. Allow the plate to dry in an oven at 100C to 105C. Examine in ultraviolet light at
254 nm. The chromatograms obtained with the reference solution and the test solution show a
quenching zone (aescin) near the boundary between the lower and the middle thirds. Mark this zone.
Examine in ultraviolet light at 365 nm. In the chromatogram obtained with the test solution no zones
of light-blue or greenish fluorescence occur below the main zone of aescin in the chromatogram
STORAGE
__________________________________________________________________________________________________________ Ph Eur
33-28
Probenecid
O
O
S
NPrn2
HOOC
C13H19NO4S
285.4
57-66-9
Probenecid complies with the requirements of the 3rd edition of the European Pharmacopoeia [0243]. These
Action and use Used in treatment of gout and to delay renal excretion of penicillins and
cephalosporins.
Preparation
Probenecid Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Probenecid contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of 4-(dipropylsulphamoyl)benzoic acid, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder or small crystals, practically insoluble in water, soluble in
acetone, sparingly soluble in ethanol, slightly soluble in ether.
IDENTIFICATION
B. Dissolve 20 mg in a mixture of 1 volume of 0.1M hydrochloric acid and 9 volumes of alcohol R and
dilute to 100.0 ml with the same mixture of solvents. Dilute 5.0 ml of the solution to 100.0 ml with a
mixture of 1 volume of 0.1M hydrochloric acid and 9 volumes of alcohol R. Examined between 220 nm
and 350 nm (2.2.25), the solution shows two absorption maxima, at 223 nm and 248 nm. The
specific absorbance at the maximum at 248 nm is 310 to 350.
obtained with probenecid CRS.
D. Dissolve 0.2 g in the smallest necessary quantity of dilute ammonia R2 (about 0.6 ml). Add 3 ml of
silver nitrate solution R2. A white precipitate is formed which dissolves in an excess of ammonia.
TESTS
Appearance of solution Dissolve 1.0 g in 1M sodium hydroxide and dilute to 10 ml with the same
solvent. The solution is clear (2.2.1) and not more intensely coloured than reference solution Y6
(Method II, 2.2.2).
Acidity To 2.0 g add 100 ml of water R and heat on a water-bath for 30 min. Make up to the
original volume with water R, allow to cool to room temperature and filter. To 50 ml of the filtrate
add 0.1 ml of phenolphthalein solution R. Not more than 0.5 ml of 0.1M sodium hydroxide is required to
coating substance.
same solvent.
10 volumes of glacial acetic acid R, 15 volumes of chloroform R, 20 volumes of di-isopropyl ether R and
55 volumes of toluene R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Any
100C to 105C.
33-29
ASSAY
Dissolve 0.250 g in 50 ml of alcohol R, shaking and heating slightly if necessary. Titrate with 0.1M
sodium hydroxide, determining the end-point potentiometrically (2.2.20).
1 ml of 0.1M sodium hydroxide is equivalent to 28.54 mg of C13H19NO4S.
__________________________________________________________________________________________________________________________________ Ph Eur
33-30
Procainamide Hydrochloride
O
NEt2
N
H
,HCl
H2N
C13H21N3O,HCl
271.8
614-39-1
Procainamide Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia [0567]. These requirements are reproduced after the heading Definition below.
Preparations
Procainamide Injection
Procainamide Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Procainamide hydrochloride contains not less than 98.0 per cent and not more than the equivalent of
101.0 per cent of 4-amino-N-[2-(diethylamino)ethyl]benzamide hydrochloride, calculated with
CHARACTERS
A white or very slightly yellow, crystalline powder, hygroscopic, very soluble in water, freely soluble in
alcohol, slightly soluble in acetone, practically insoluble in ether.
IDENTIFICATION
B. Dissolve 10.0 mg in 0.1M sodium hydroxide and dilute to 100.0 ml with the same solvent. Dilute
10.0 ml of the solution to 100.0 ml with 0.1M sodium hydroxide. Examined between 220 nm and
obtained with procainamide hydrochloride CRS.
D. Dilute 1 ml of solution S to 5 ml with water R. The solution gives reaction (a) of chlorides (2.3.1).
E. Dilute 1 ml of solution S (see Tests) to 2 ml with water R. 1 ml of this solution gives the reaction
of primary aromatic amines (2.3.1).
TESTS
solution B6 (2.2.2).
coating substance.
the same solvent.
Reference solution. Dilute 1 ml of the test solution to 200 ml with alcohol R.
15 volumes of glacial acetic acid R, 30 volumes of water R and 60 volumes of butanol R. Place the
plate in a stream of cold air until the plate appears dry. Examine in ultraviolet light at 254 nm. Any
100C to 105C.
33-31
ASSAY
Dissolve 0.2500 g in 50 ml of dilute hydrochloric acid R. Carry out the determination of primary
aromatic amino-nitrogen (2.5.8).
1 ml of 0.1M sodium nitrite is equivalent to 27.18 mg of C 13H22ClN3O.
STORAGE
__________________________________________________________________________________________________________________________________ Ph Eur
33-32
Procaine Benzylpenicillin / Procaine Penicillin

1/01
together when the substance is stated as an active ingredient in a formulated preparation (See Supplementary
O
COOH
H
N
S
H
H2N
NEt2
C13H20N2O2,C16H18N2O4S,H2O
Me
Me
588.7 6130-64-9
Procaine Benzylpenicillin complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparations
Procaine Benzylpenicillin Injection/Procaine Penicillin Injection
Fortified Procaine Benzylpenicillin Injection/Fortified Procaine Penicillin Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Procaine benzylpenicillin is the monohydrate of the (2S,5R,6R)-3,3-dimethyl-7-oxo-6[(phenylacetyl)amino]-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid compound with
2-(diethylamino)ethyl 4-aminobenzoate. It contains not less than 96.0 per cent and not more than
the equivalent of 102.0 per cent of procaine benzylpenicillin and not less than 39.0 per cent and not
more than 42.0 per cent of procaine (C13H20N2O2, 236.3), both calculated with reference to the
anhydrous substance. Dispersing or suspending agents (for example, lecithin and polysorbate 80)
may be added.
PRODUCTION
Benzylpenicillin procaine complies with the requirements of the monograph on Products of fermentation (1468).
CHARACTERS
A white, crystalline powder, slightly soluble in water, sparingly soluble in alcohol.
IDENTIFICATION
obtained with procaine benzylpenicillin CRS.
B. Examine by thin-layer chromatography (2.2.27), using a TLC silanised silica gel plate R.
Test solution. Dissolve 25 mg of the substance to be examined in 5 ml of acetone R.
Reference solution. Dissolve 25 mg of procaine benzylpenicillin CRS in 5 ml of acetone R.
of acetone R and 70 volumes of a 154 g/l solution of ammonium acetate R, the pH of which has been
adjusted to 7.0 with ammonia R. Allow the plate to dry in air and expose it to iodine vapour until the
spots appear. Examine in daylight. The two principal spots in the chromatogram obtained with the
test solution are similar in position, colour and size to the two principal spots in the chromatogram
obtained with the reference solution. The test is not valid unless the chromatogram obtained with the
reference solution shows two clearly separated spots.
by swirling; the solution is practically colourless. Place the test-tube on a water-bath for 1 min; a
D. Dissolve 0.1 g in 2 ml of dilute hydrochloric acid R and use the solution which may be turbid. The
solution gives the reaction of primary aromatic amines (2.3.1).
33-33
TESTS
pH (2.2.3). Dissolve 50 mg in carbon dioxide-free water R and dilute to 15 ml with the same solvent.
Shake until dissolution is complete. The pH of the solution is 5.0 to 7.5.
Specific optical rotation (2.2.7). Dissolve 0.250 g in a mixture of 2 volumes of water R and 3
volumes of acetone R and dilute to 25.0 ml with the same mixture of solvents. The specific optical
rotation is +165 to +180, calculated with reference to the anhydrous substance.
10 l of reference solution (c). Adjust the sensitivity of the system so that the height of the peak due
to benzylpenicillin is at least 50 per cent of the full scale of the recorder. Inject 10 l of test solution
(a) and continue the chromatography for 1.5 times the retention time of the benzylpenicillin peak. In
the chromatogram obtained with test solution (a): the area of any peak corresponding to 4-aminobenzoic acid is not greater than the area of the corresponding peak in the chromatogram obtained
with reference solution (c) (0.024 per cent); the area of any peak, apart from the two principal peaks
and any peak corresponding to 4-aminobenzoic acid, is not greater than the area of the peak corresponding to benzylpenicillin in the chromatogram obtained with reference solution (c) (1 per cent).
Bacterial endotoxins (2.6.14, Method E): less than 0.10 I.U. per milligram, if intended for use in
the manufacture of parenteral dosage forms without a further appropriate procedure for the removal
of bacterial endotoxins.
ASSAY
Test solution (b). Dissolve 70.0 mg of the substance to be examined in the mobile phase and dilute to
Reference solution (a). Dissolve 70.0 mg of procaine benzylpenicillin CRS in the mobile phase and dilute
Reference solution (b). Dissolve 4 mg of 4-aminobenzoic acid R in reference solution (a) and dilute to
Reference solution (c). Dissolve 16.8 mg of 4-aminobenzoic acid R in water R and dilute to 50.0 ml with
the same solvent. Dilute 1.0 ml of the solution to 10.0 ml with water R. To 1.0 ml of this solution,
add 1.0 ml of test solution (a) and dilute to 100.0 ml with the mobile phase.
as mobile phase at a flow rate of 1.75 ml/min a mixture prepared as follows: mix 250 ml of
acetonitrile R, 250 ml of water R and 500 ml of a solution containing 14 g/l of potassium
dihydrogen phosphate R and 6.5 g/l of tetrabutylammonium hydroxide solution (400 g/l) R adjusted
to pH 7.0 with 1M potassium hydroxide; adjust the mixture to pH 7.2 with dilute phosphoric acid R,
if necessary,
Inject 10 l of reference solution (b). When the chromatogram is recorded in the prescribed
conditions, the substances elute in the following order: 4-aminobenzoic acid, procaine,
benzylpenicillin. Adjust the sensitivity of the system so that the height of the peak corresponding to 4aminobenzoic acid is at least 50 per cent of the full scale of the recorder. The test is not valid unless,
the resolution between the first peak (4-aminobenzoic acid) and the second peak (procaine) is at least
2.0. If necessary, adjust the concentration of acetonitrile in the mobile phase. Inject reference
solution (a) six times. The test is not valid unless the relative standard deviation for the areas of the
two peaks is at most 1.0 per cent. Inject alternately test solution (b) and reference solution (a).
Calculate the percentage contents of procaine and procaine benzylpenicillin. Calculate the latter by
multiplying the percentage content of benzylpenicillin by 1.67.
STORAGE
Store in an airtight container. If the substance is sterile, store in a sterile, airtight, tamper-proof
container.
LABELLING
The label states:
where applicable, the name and quantity of any added dispersing or suspending agents,
33-34
IMPURITIES
COOH
H2N
A. 4-aminobenzoic acid,
H COOH
HN
H
N
Me
* S
Me
COOH
B. (4S)-2-[carboxy[(phenylacetyl)amino]methyl]-5,5-dimethylthiazolidine-4-carboxylic acid
(penicilloic acids of benzylpenicillin),
H COOH
HN
H
N
Me
* S
Me
and epimer at C*
C. (2RS,4S)-2-[[(phenylacetyl)amino]methyl]-5,5-dimethylthiazolidine-4-carboxylic acid
(penilloic acids of benzylpenicillin),
H COOH
N
HOOC
Me
S
Me
HH
D. (3S,7R,7aR)-5-benzyl-2,2-dimethyl-2,3,7,7a-tetrahydroimidazo[5,1-b]thiazole-3,7dicarboxylic acid (penillic acid of benzylpenicillin),

COOH
E. phenylacetic acid.
__________________________________________________________________________________________________________ Ph Eur
33-35
Procaine Hydrochloride
O
NEt2
,HCl
H2N
C13H20N2O2,HCl,
272.8
51-05-8
Procaine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Procaine hydrochloride contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of 2-diethylaminoethyl-4-aminobenzoate hydrochloride, calculated with reference to
CHARACTERS
A white, crystalline powder or colourless crystals, very soluble in water, soluble in alcohol, practically
insoluble in ether.
IDENTIFICATION
obtained with procaine hydrochloride CRS.
C. To about 5 mg add 0.5 ml of fuming nitric acid R. Evaporate to dryness on a water-bath, allow to
cool and dissolve the residue in 5 ml of acetone R. Add 1 ml of 0.1M alcoholic potassium hydroxide.
Only a brownish-red colour develops.
D. To 0.2 ml of solution S (see Tests) add 2 ml of water R and 0.5 ml of dilute sulphuric acid R and
shake. Add 1 ml of a 1 g/l solution of potassium permanganate R. The colour is immediately
discharged.
F. Dilute 1 ml of solution S to 100 ml with water R. 2 ml of this solution gives the reaction of primary
aromatic amines (2.3.1).
TESTS
pH (2.2.3). Dilute 4 ml of solution S to 10 ml with carbon dioxide-free water R. The pH of the solution is 5.0 to 6.5.
coating substance.
same solvent.
Reference solution. Dissolve 50 mg of 4-aminobenzoic acid R in water R and dilute to 100 ml with the
same solvent. Dilute 1 ml of the solution to 10 ml with water R.
volumes of glacial acetic acid R, 16 volumes of hexane R and 80 volumes of dibutyl ether R. Dry the
plate at 100C to 105C for 10 min and examine in ultraviolet light at 254 nm. Any spot in the
the spot in the chromatogram obtained with the reference solution (0.05 per cent). The principal
spot in the chromatogram obtained with the test solution remains on the starting point.
Heavy metals (2.4.8). Dissolve 1.0 g in water R and dilute to 25.0 ml with the same solvent. Carry
out the prefiltration. 10 ml of the prefiltrate complies with limit test E for heavy metals (5 ppm).
33-36
100C to 105C.
ASSAY
Dissolve 0.400 g in 50 ml of dilute hydrochloric acid R. Carry out the determination of primary
aromatic amino nitrogen (2.5.8).
1 ml of 0.1M sodium nitrite is equivalent to 27.28 mg of C 13H21ClN2O2.
STORAGE
__________________________________________________________________________________________________________________________________ Ph Eur
33-37
Prochlorperazine Maleate
NMe
COOH
COOH
,
2
Cl
C20H24ClN3S,2C4H4O4
606.2
84-02-6
Prochlorperazine Maleate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparations
Prochlorperazine Tablets
Prochlorperazine Buccal Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Prochlorperazine maleate contains not less than 98.0 per cent and not more than the equivalent of
101.0 per cent of 2-chloro-10-[3-(4-methylpiperazin-1-yl)propyl]phenothiazine dimaleate, calculated
CHARACTERS
A white or pale-yellow, crystalline powder, very slightly soluble in water and in alcohol, practically
insoluble in ether.
IDENTIFICATION
First identification: B, C, D.
A. Carry out the test protected from light. Dissolve 50 mg in 0.1M hydrochloric acid and dilute to
500.0 ml with the same acid. Examined immediately between 280 nm and 350 nm (2.2.25), the
solution shows an absorption maximum at 305 nm. Dilute 10.0 ml of the solution to 100.0 ml with
0.1M hydrochloric acid. Examined immediately between 230 nm and 280 nm, the solution shows an
absorption maximum at 255 nm. The specific absorbance at the maximum is 525 to 575.
obtained with prochlorperazine maleate CRS.
C. It complies with the identification of phenothiazines by thin-layer chromatography (2.3.3), with
the following modifications:
Test solution. Dissolve 20 mg of the substance to be examined in a mixture of equal volumes of chloroform R and methanol R and dilute to 20 ml with the same mixture of solvents.
Reference solution. Dissolve 20 mg of prochlorperazine maleate CRS in a mixture of equal volumes of
chloroform R and methanol R and dilute to 20 ml with the same mixture of solvents.
Apply separately to the plate 4 l of each solution.
D. Triturate 0.2 g with a mixture of 1 ml of strong sodium hydroxide solution R and 3 ml of water R.
Shake with three quantities, each of 5 ml, of ether R. To 0.1 ml of the aqueous layer add a solution of
10 mg of resorcinol R in 3 ml of sulphuric acid R. Heat in a water-bath for 15 min. No colour develops.
To the remainder of the aqueous layer add 2 ml of bromine solution R. Heat in a water-bath for
15 min and then heat to boiling. Cool. To 0.1 ml of the solution add a solution of 10 mg of
resorcinol R in 3 ml of sulphuric acid R. Heat in a water-bath for 15 min. A blue colour develops.
TESTS
pH (2.2.3). The pH of a freshly prepared saturated solution in carbon dioxide-free water R is 3.0 to
4.0.
(2.2.27), using silica gel GF254 R as the coating substance.
33-38
Test solution. Dissolve 0.2 g of the substance to be examined in a mixture of 5 volumes of diethylamine R and 95 volumes of methanol R and dilute to 10 ml with the same mixture of solvents. Prepare
Reference solution. Dilute 1 ml of the test solution to 200 ml with a mixture of 5 volumes of diethylamine R and 95 volumes of methanol R.
10 volumes of acetone R, 10 volumes of diethylamine R and 80 volumes of cyclohexane R. Allow the
plate to dry in air and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained
with the test solution, apart from the principal spot, is not more intense than the spot in the chromatogram obtained with the reference solution (0.5 per cent). Disregard any spots remaining at the
starting-points.
100C to 105C.
ASSAY
Dissolve 0.200 g of the powdered substance to be examined in 50 ml of anhydrous acetic acid R,
warming on a water-bath. Allow to cool to room temperature. Titrate with 0.1M perchloric acid determining the end-point potentiometrically (2.2.20).
1 ml of 0.1M perchloric acid is equivalent to 30.31 mg of C28H32ClN3O8S.
STORAGE
__________________________________________________________________________________________________________________________________ Ph Eur
33-39
Prochlorperazine Mesilate
NMe ,CH3SO3
Cl
C20H24ClN3S,2CH4SO3
566.2
5132-55-8
Definition Prochlorperazine Mesilate is 2-chloro-10-[3-(4-methylpiperazin-1yl)propyl]phenothiazine di(methanesulphonate). It contains not less than 98.0% and not more than
101.0% of C20H24ClN3S,2CH4SO3, calculated with reference to the dried substance.
Very soluble in water; sparingly soluble in ethanol (96%); slightly soluble in chloroform; practically
insoluble in ether.
Identification
prochlorperazine mesilate (RS 290).
B. Dissolve 5 mg in 2 ml of sulphuric acid and allow to stand for 5 minutes. A red colour is produced.
Related substances Complies with the test for related substances in phenothiazines, Appendix III A,
using mobile phase A and dissolving the substance being examined in methanol containing 0.5% v/v of
13.5M ammonia.
Assay Dissolve 0.8 g in 10 ml of water, add 5 ml of 1M sodium hydroxide and extract by shaking with
successive quantities of 50, 25, 25 and 25 ml of ether. Wash the combined ether extracts with 5 ml of
water, shake the washings with 5 ml of ether, add the ether to the combined ether extracts and
evaporate to dryness. Add 2 ml of absolute ethanol to the residue, evaporate to dryness and carry out
Method I for non-aqueous titration, Appendix VIII A, using 1-naphtholbenzein solution as indicator.
Each ml of 0.1M perchloric acid VS is equivalent to 28.31 mg of C20H24ClN3S,2CH4SO3.
Storage Prochlorperazine Mesilate should be protected from light.
Preparations
Prochlorperazine Injection
Prochlorperazine Oral Solution
When prochlorperazine mesylate is prescribed or demanded, Prochlorperazine Mesilate shall be
33-40
Procyclidine Hydrochloride
OH
N
C19H29NO,HCl
323.9
1508-76-5
Definition Procyclidine Hydrochloride is (RS)-1-cyclohexyl-1-phenyl-3-pyrrolidin-1-ylpropan-1-ol

Sparingly soluble in water; soluble in ethanol (96%); practically insoluble in acetone and in ether.
Identification
procyclidine hydrochloride (RS 292).
B. Dissolve 0.25 g in 10 ml of water, make alkaline with 5M ammonia and extract with three 10-ml
quantities of ether. Dry the combined extracts over anhydrous sodium sulphate, filter, remove the ether
and scratch the residue with a glass rod to induce solidification. The melting point of the residue is
about 85, Appendix V A.
Related substances
A. Carry out the method for thin-layer chromatography, Appendix III A, using silica gel GF254 as the
coating substance and a mixture of 100 volumes of ether and 1 volume of 13.5M ammonia as the
mobile phase. Apply separately to the plate 5 l of each of three solutions in chloroform containing (1)
2.0% w/v of the substance being examined, (2) 0.0040% w/v of 1-phenyl-3-pyrrolidinopropan-1-one
hydrochloride BPCRS and (3) 0.010% w/v of the substance being examined. After removal of the
plate, dry it at 105 for 15 minutes and examine under ultraviolet light (254 nm). Any spot corresponding to 1-phenyl-3-pyrrolidinopropan-1-one in the chromatogram obtained with solution (1) is
not more intense than the spot in the chromatogram obtained with solution (2). Spray the plate with
dilute potassium iodobismuthate solution. Any secondary spot in the chromatogram obtained with solution
(1) is not more intense than the spot in the chromatogram obtained with solution (3).
B. Carry out the method for gas chromatography, Appendix III B, using the following solutions. For
solution (1) add 5 ml of 1.25 M sodium hydroxide to 20 ml of a 0.015% w/v solution of the substance
being examined and mix. Extract with two 20-ml quantities of ether, add to the combined extracts
5 ml of a 0.06% w/v solution of triphenylethylene (internal standard) in ether, shake with anhydrous
sodium sulphate and filter; evaporate the filtrate and dissolve the residue in 1 ml of ether. Prepare
solution (2) in the same manner as solution (1) but using 20 ml of a 0.50% w/v solution of the
substance being examined and omitting the addition of the internal standard solution. Prepare solution (3) in the same manner as solution (1) but using 20 ml of a 0.50% w/v solution of the substance
being examined.
with acid-washed, silanised diatomaceous support (HP Chromosorb W is suitable) (80 to 100 mesh),
coated with 10% w/w of modified polyethylene glycol 20M (SP-1000 is suitable) and 2% w/w of
potassium hydroxide and maintained at 240, and using on-column injection.
The ratio of the sum of the areas of any secondary peaks to the area of the peak due to the internal
standard in the chromatogram obtained with solution (3) is not more than the ratio of the area of the
principal peak to the area of the internal standard peak in the chromatogram obtained with solution
(1).
1 g.
solution as indicator. Each ml of 0.1M perchloric acid VS is equivalent to 32.39 mg of C19H29NO,HCl.
Preparations
Procyclidine Injection
Procyclidine Tablets
33-41
Progesterone
O
Me
Me
H
Me
H
H
O
C21H30O2
314.5
57-83-0
Progesterone complies with the requirements of the 3rd edition of the European Pharmacopoeia [0429]. These
Preparation
Progesterone Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Progesterone contains not less than 97.0 per cent and not more than the equivalent of 103.0 per cent
of pregn-4-ene-3,20-dione, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder or colourless crystals, practically insoluble in water, freely
soluble in ethanol, sparingly soluble in acetone, in ether and in fatty oils.
IDENTIFICATION
obtained with progesterone CRS. If the spectra obtained in the solid state with the substance to be
examined and the reference substance show differences, record further spectra using 50 g/l solutions
in chloroform R.
C. Examine by thin-layer chromatography (2.2.27), using as coating substance a suitable silica gel
with a fluorescent indicator having an optical intensity at 254 nm.
Reference solution. Dissolve 10 mg of progesterone CRS in a mixture of 1 volume of methanol R and 9
volumes of chloroform R and dilute to 10 ml with the same mixture of solvents.
Apply separately to the plate 5 l of each solution. Develop over a path of 15 cm, using a mixture of
33 volumes of ethyl acetate R and 66 volumes of chloroform R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test
solution is similar in position, colour and size to the principal spot in the chromatogram obtained
with the reference solution. Spray with alcoholic solution of sulphuric acid R, heat at 120C for 15 min
and allow to cool. Examine in daylight and in ultraviolet light at 365 nm. The principal spot in the
chromatogram obtained with the test solution is similar in position, fluorescence in ultraviolet light at
365 nm and size to the principal spot in the chromatogram obtained with the reference solution.
TESTS
substance.
coating substance.
Reference solution. Dilute 1 ml of the test solution to 100 ml with a mixture of 1 volume of methanol R
Apply separately to the plate 5 l of each solution. Develop over a path of 15 cm, using a mixture of
33-42
33 volumes of ethyl acetate R and 66 volumes of chloroform R. Allow the plate to dry in air and spray
with a saturated solution of potassium dichromate R in a mixture of 30 volumes of water R and 70
volumes of sulphuric acid R. Heat at 130C for 30 min and allow to cool. Any spot in the chromatogram obtained with the test solution, apart from the principal spot, is not more intense than the spot
in the chromatogram obtained with the reference solution (1.0 per cent).
100C to 105C for 2 h.
ASSAY
STORAGE
__________________________________________________________________________________________________________________________________ Ph Eur
33-43
Proguanil Hydrochloride
H
N
H
N
NH
H
N
Pri
,HCl
NH
Cl
C11H16ClN5,HCl
290.2
637-32-1
Definition Proguanil Hydrochloride is 1-(4-chlorophenyl)-5-isopropylbiguanide hydrochloride. It

contains not less than 99.0% and not more than 101.0% of C11H16ClN5,HCl, calculated with reference to the dried substance.
Slightly soluble in water, more soluble in hot water; sparingly soluble in ethanol (96%); practically
insoluble in chloroform and in ether.
Identification
proguanil hydrochloride (RS 294).
B. To 15 ml of a saturated solution add 2 ml of 5M sodium hydroxide and extract with 20 ml of ether.
Wash the ether extract with water and evaporate to dryness at 105. The melting point of the residue is
Acidity or alkalinity To 35 ml of water maintained at 60 to 65 add 0.2 ml of methyl red mixed
solution, neutralise with 0.01M sodium hydroxide VS or 0.01M hydrochloric acid VS, add 0.4 g of the
substance being examined and stir until dissolved. The resulting solution is not acidic and requires
for neutralisation not more than 0.2 ml of 0.01M hydrochloric acid VS.
4-Chloroaniline Dissolve 0.10 g in 1 ml of 2M hydrochloric acid, add sufficient water to produce
20 ml, cool to 5, add 1 ml of 0.05M sodium nitrite, allow to stand at 5 for 5 minutes, add 2 ml of a
5% w/v solution of ammonium sulphamate and allow to stand for 10 minutes. Add 2 ml of a 0.1% w/v
solution of N-(1-naphthyl)ethylenediamine dihydrochloride, dilute to 50 ml with water and allow to
stand for 30 minutes. Any magenta colour produced is not more intense than that obtained by treating in the same manner and at the same time 20 ml of a solution containing 1.25 g per ml of
4-chloroaniline (250 ppm).
following solutions. For solution (1) dilute 1 volume of a 0.1% w/v solution of the substance being
examined in methanol to 10 volumes with the mobile phase. For solution (2) dilute 1 volume of
solution (1) to 100 volumes with the mobile phase. For solution (3) mix 1 volume of a 0.05% w/v
solution of 1-(2,5-dichlorophenyl)-5-isopropylbiguanide hydrochloride BPCRS in methanol with 9
volumes of solution (1).
5 mm) packed with octadecylsilyl silica gel for chromatography (5 m) (Nucleosil C18 is suitable) and
maintained at 40, (b) as the mobile phase with a flow rate of 1 ml per minute a solution prepared by
dissolving 1.89 g of sodium hexanesulphonate in a mixture of 500 ml of methanol, 500 ml of water and
5 ml of glacial acetic acid and (c) a detection wavelength of 254 nm.
The test is not valid unless the chromatogram obtained with solution (3) shows two clearly
separated peaks due to proguanil hydrochloride and 1-(2,5-dichlorophenyl)-5-isopropylbiguanide
hydrochloride.
The sum of the areas of any secondary peaks in the chromatogram obtained with solution (1) is not
greater than the area of the principal peak in the chromatogram obtained with solution (2) (1%).
1 g.
C11H16ClN5,HCl.
Storage Proguanil Hydrochloride should be protected from light.
Preparation
Proguanil Tablets
33-44
IMPURITIES
Cl
Pr i HN
H
N
H
N
H
N
NH
NH
Cl
A. 1-(2,5-dichlorophenyl)-5-isopropylbiguanide hydrochloride
Cl
Pr i HN
H
N
H
N
H
N
NH
NH
B. 1-(2-chlorophenyl)-5-isopropylbiguanide hydrochloride
Pr i HN
H
N
H
N
H
N
NH
NH
C. proguanil aniline analogue

Cl
H
N
Cl
H
N
NH
D. bis compound
NH2
Cl
E. 4-chloroaniline
H
N
NH
Cl
33-45
Proline
H
N
H
COOH
C5H9NO2
115.1
147-85-3
Proline complies with the requirements of the 3rd edition of the European Pharmacopoeia [0785]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Proline contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of
(S)-pyrrolidine-2-carboxylic acid, calculated with reference to the dried substance.
PRODUCTION
When Proline is produced by a process involving fermentation steps, it complies with the requirements of the monograph on Products of fermentation (1468).
CHARACTERS
White or almost white, crystalline powder or colourless crystals, very soluble in water, freely soluble
in alcohol, practically insoluble in ether.
IDENTIFICATION
obtained with proline CRS. Examine the substances prepared as discs.
TESTS
substance.
gel plate R.
Test solution (a). Dissolve 0.10 g of the substance to be examined in 0.1M hydrochloric acid and dilute
to 10 ml with the same acid.
Reference solution (a). Dissolve 10 mg of proline CRS in 0.1M hydrochloric acid and dilute to 50 ml with
the same acid.
Reference solution (c). Dissolve 10 mg of proline CRS and 10 mg of threonine CRS in 0.1M hydrochloric
acid and dilute to 25 ml with the same acid.
Apply separately to the plate 5 l of each solution. Allow the plate to dry in air. Develop over a path
of 15 cm using a mixture of 20 volumes of glacial acetic acid R, 20 volumes of water R and 60 volumes
of butanol R. Allow the plate to dry in air, spray with ninhydrin solution R and heat at 100C to 105C
33-46
To the inner wall of the upper watch-glass stick a piece of red litmus paper R 5 mm square and wetted
Briefly triturate with a glass rod. Immediately close the cell by putting the two watch-glasses together.
(100 ppm NH4 ) R, 0.5 ml of water R and 0.30 g of heavy magnesium oxide R (200 ppm).
the solution complies with limit test A for heavy metals (10 ppm). Prepare the standard using the lead
100C to 105C.
ASSAY
0.1M perchloric acid using 0.1 ml of naphtholbenzein solution R as indicator, until the colour changes
from brownish-yellow to green.
STORAGE
__________________________________________________________________________________________________________________________________ Ph Eur
33-47
Promazine Hydrochloride
NMe2
,HCl
C17H20N2S,HCl
320.9
53-60-1
Promazine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparations
Promazine Injection
Promazine Oral Suspension
Promazine Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Promazine hydrochloride contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of 3-(10H-phenothiazin-10-yl)-N,N-dimethylpropan-1-amine hydrochloride, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, slightly hygroscopic, very soluble in water, in alcohol and
IDENTIFICATION
obtained with promazine hydrochloride CRS.
B. It complies with the identification test for phenothiazines by thin-layer chromatography (2.3.3).
Use promazine hydrochloride CRS to prepare the reference solution.
C. Dissolve about 5 mg in 2 ml of sulphuric acid R and allow to stand for 5 min. An orange colour is
produced.
D. It gives reaction (b) of chlorides (2.3.1).
TESTS
The pH of the freshly prepared solution is 4.2 to 5.2.
Related substances Carry out the test protected from bright light. Prepare the solutions immediately before
use.
Examine by thin layer chromatography (2.2.27), using a TLC silica gel F254 plate R.
Test solution. Dissolve 0.10 g of the substance to be examined in a mixture of 5 volumes of diethylamine R and 95 volumes of methanol R and dilute to 10 ml with the same mixture of solvents.
Reference solution (a). Dilute 1 ml of the test solution to 200 ml with a mixture of 5 volumes of
diethylamine R and 95 volumes of methanol R.
Reference solution (b). Dissolve 10 mg of chlorprothixene hydrochloride CRS in a mixture of 5 volumes of
diethylamine R and 95 volumes of methanol R, add 1 ml of the test solution and dilute to 10 ml with
volumes of acetone R, 10 volumes of diethylamine R and 80 volumes of cyclohexane R. Allow the plate
to dry in air and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with
the test solution, apart fom the principal spot, is not more intense than the spot in the chromatogram
obtained with reference solution (a) (0.5 per cent).
33-48
Disregard any spot at the starting point. The test is not valid unless the chromatogram obtained
with reference solution (b) shows two clearly separated principal spots.
100C to 105C.
ASSAY
1 ml of 0.1M sodium hydroxide is equivalent to 32.09 mg of C17H21ClN2S.
STORAGE
IMPURITIES
NMe2
N
S
O
A. 3-(10H-phenothiazin-10-yl)-N,N-dimethylpropan-1-amine S-oxide
(promazine sulphoxide).
__________________________________________________________________________________________________________ Ph Eur
33-49
Promethazine Hydrochloride
NMe2
N
H
Me
, HCl
and enantiomer
C17H20N2S,HCl
320.9
58-33-3
Promethazine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia [0524]. These requirements are reproduced after the heading Definition below.
Action and use Histamine H1-receptor antagonist; anti-emetic.
Preparations
Promethazine Injection
Promethazine Oral Solution
Promethazine Hydrochloride Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Promethazine hydrochloride contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of (2RS)-N,N-dimethyl-1-(10H-phenothiazin-10-yl)propan-2-amine hydrochloride,
CHARACTERS
A white or faintly yellowish, crystalline powder, very soluble in water, freely soluble in alcohol and in
methylene chloride.
IDENTIFICATION
obtained with promethazine hydrochloride CRS.
C. Dissolve 0.1 g in 3 ml of water R. Add dropwise 1 ml of nitric acid R. A precipitate is formed which
rapidly dissolves to give a red solution, becoming orange and then yellow. Heat to boiling. The
solution becomes orange and an orange-red precipitate is formed.
TESTS
The pH of the solution measured immediately after preparation is 4.0 to 5.0.
Related substances Carry out the test protected from bright light. Prepare the solutions immediately before
use. Examine by thin-layer chromatography (2.2.27), using a suitable silica gel as the coating
substance.
Test solution. Dissolve 0.20 g of the substance to be examined in a mixture of 5 volumes of diethylamine R and 95 volumes of methanol R and dilute to 10 ml with the same mixture of solvents.
Reference solution (a). Dissolve 20 mg of isopromethazine hydrochloride CRS in a mixture of 5 volumes
of diethylamine R and 95 volumes of methanol R and dilute to 100 ml with the same mixture of
solvents.
Reference solution (b). Dilute 0.5 ml of the test solution to 100 ml with a mixture of 5 volumes of
Reference solution (c). Dilute 0.2 ml of the test solution to 100 ml with a mixture of 5 volumes of
Apply separately to the plate 10 l of the test solution and 10 l of each reference solution. Develop
33-50
in an unsaturated tank over a path of 12 cm using a mixture of 5 volumes of diethylamine R, 10
volumes of acetone R and 85 volumes of cyclohexane R. Allow the plate to dry in air and examine in
ultraviolet light at 254 nm. Disregard any spot at the starting point.
In the chromatogram obtained with the test solution: any spot corresponding to isopromethazine
hydrochloride is not more intense that the spot in the chromatogram obtained with reference solution
(a) (1 per cent); any spot, apart from the principal spot and the spot corresponding to isopromethazine hydrochloride, is not more intense than the spot in the chromatogram obtained with
reference solution (b) (0.5 per cent) and at most three such spots are more intense than the spot in
the chromatogram obtained with reference solution (c) (0.2 per cent).
Heavy metals (2.4.8). Dissolve 1.0 g in 5 ml of water R, add 5 ml of acetone R and 5 ml of buffer
solution pH 3.5 R. Carry out the prefiltration. The prefiltrate complies with limit test E for heavy
metals (10 ppm). Prepare the standard using 5 ml of lead standard solution (2 ppm Pb) R.
100C to 105C.
ASSAY
1 ml of 0.1M sodium hydroxide is equivalent to 32.09 mg of C17H21ClN2S.
STORAGE
IMPURITIES
H
N
S
A. phenothiazine,
Me
N(Me)2
N
S
and enantiomer
B. (2RS)-N,N-dimethyl-2-(10H-phenothiazin-10-yl)propan-1-amine (isopromethazine),
NHMe
and enantiomer
H Me
C. (2RS)-N-methyl-1-(10H-phenothiazin-10-yl)propan-2-amine,
N
S
N(Me)2
H Me
and enantiomer
D. (2RS)-N,N-dimethyl-1-(10H-phenothiazin-10-yl)propan-2-amine S-oxide.
__________________________________________________________________________________________________________ Ph Eur
33-51
Promethazine Teoclate
NMe2
N
H
Me
H
N
MeN
Cl
O
Me
and enantiomer
C17H20N2S,C7H7ClN4O2
499.0
17693-51-5
Definition Promethazine Teoclate is the (RS)-dimethyl(2-phenothiazin-10-ylpropyl)amine salt of

8-chlorotheophylline. It contains not less than 98.0% and not more than 101.0% of
C17H20N2S,C7H7ClN4O2, calculated with reference to the dried substance.
Very slightly soluble in water; freely soluble in chloroform; sparingly soluble in ethanol (96%);
Identification
A. Shake 0.15 g with 2.5 ml of water, add 1 ml of 5M ammonia and extract with 30 ml of ether. Wash
the ether extract with 10 ml of water, dry with anhydrous sodium sulphate and evaporate the ether to
dryness. Dissolve the residue in 1 ml of chloroform IR. The infrared absorption spectrum of the resulting
solution, Appendix II A, is concordant with the reference spectrum of promethazine (RS 297).
B. Dissolve 5 mg in 2 ml of sulphuric acid and allow to stand for 5 minutes. A red colour is produced.
C. Shake 0.4 g with 10 ml of water, add 4 ml of 5M ammonia, shake with two 30-ml quantities of
ether and add 4 ml of hydrochloric acid to the aqueous solution. Filter the white precipitate, wash with
water and dry at 105. Dissolve 10 mg of the residue in 1 ml of hydrochloric acid, add 0.1 g of
potassium chlorate and evaporate to dryness. A reddish residue remains which becomes purple on
exposure to the vapour of ammonia.
Chloride Shake 0.3 g with 30 ml of water for 2 minutes and filter. 15 ml of the filtrate complies with
the limit test for chlorides, Appendix VII, but using 2 ml of nitric acid in place of the 1 ml of 2M nitric
acid (350 ppm).
silica gel F254 precoated plate (Merck silica gel 60 F254 plates are suitable) and a mixture of 5 volumes
of diethylamine, 10 volumes of acetone and 85 volumes of cyclohexane as the mobile phase. Pour the
mobile phase into an unlined tank, immediately place the prepared plate in the tank, close the tank
and allow the solvent front to ascend 12 cm above the line of application. Apply separately to the
plate 10 l of each of the following solutions in a mixture of 5 volumes of diethylamine and 95
volumes of methanol. Solution (1) contains 2% w/v of the substance being examined. Solution (2)
contains 0.02% w/v of isopromethazine hydrochloride EPCRS. For solution (3) dilute 1 volume of
solution (1) to 200 volumes. For solution (4) dilute 1 volume of solution (1) to 500 volumes. Allow
the plate to dry in air and examine under ultraviolet light (254 nm). In the chromatogram obtained
with solution (1) any spot corresponding to isopromethazine is not more intense than the spot in the
chromatogram obtained with solution (2) (1%), any other secondary spot is not more intense than the
spot in the chromatogram obtained with solution (3) (0.5%) and not more than three such spots are
more intense than the spot in the chromatogram obtained with solution (4) (0.2%). Disregard any
spot remaining on the line of application.
1 g.
Assay Dissolve 1 g in 200 ml of acetone and carry out Method I for non-aqueous titration, Appendix
VIII A, using 3 ml of a saturated solution of methyl orange in acetone as indicator. Each ml of 0.1M
perchloric acid VS is equivalent to 49.90 mg of C17H20N2S,C7H7ClN4O2.
Storage Promethazine Teoclate should be kept in a well-closed container and protected from light.
Action and use Histamine H1-receptor antagonist; anti-emetic.
Preparation
Promethazine Teoclate Tablets
When promethazine theoclate is prescribed or demanded, Promethazine Teoclate shall be dispensed
or supplied.
33-52
Propacetamol Hydrochloride
NHAc
O
Et2N
,HCl
O
C14H20N2O3,HCl
300.8
66532-86-3
Propacetamol Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia [1366]. These requirements are reproduced after the heading Definition below.
Action and use Analgesic and antipyretic.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Propacetamol hydrochloride contains not less than 98.0 per cent and not more than the equivalent of
102.0 per cent of 4-(acetylamino)phenyl diethylamino(acetate) hydrochloride, calculated with
CHARACTERS
A white or almost white, crystalline powder, freely soluble in water, slightly soluble in ethanol,
practically insoluble in acetone.
IDENTIFICATION
spectrum of propacetamol hydrochloride.
TESTS
Solution S Prepare the solution immediately before use. Dissolve 1.75 g in water R and dilute to 10.0 ml
Absorbance The absorbance (2.2.25) of solution S measured at 390 nm is not greater than 0.05.
Solution A. Dissolve 2.16 g of sodium octanesulphonate R in 900 ml of water R and dilute to 1000 ml
with the same solvent. Adjust to pH 3.0 with acetic acid R.
Test solution. Suspend 1.00 g of the substance to be examined in 10.0 ml of acetonitrile R. Shake for
10 min. Allow to stand. Take 3.0 ml of the supernatant solution and dilute to 10.0 ml with solution
A. Inject this solution immediately after preparation.
Reference solution (a). Dissolve 50 mg of paracetamol R in acetonitrile R and dilute to 50.0 ml with the
same solvent. Dilute 1.0 ml of the solution to 50.0 ml with acetonitrile R. Dilute 3.0 ml of the latter
solution to 10.0 ml with solution A.
Reference solution (b). Dissolve 10 mg of paracetamol R and 0.100 g of 4-aminophenol R in acetonitrile R
and dilute to 50.0 ml with the same solvent. Dilute 1.0 ml of the solution to 50.0 ml with acetonitrile R. Dilute 3.0 ml of the latter solution to 10.0 ml with solution A.
volumes of solution A,
Inject 20 l of reference solution (b). The chromatogram obtained shows a peak corresponding to
paracetamol (first peak) and a peak corresponding to 4-aminophenol (second peak) with a retention
time relative to paracetamol of about 1.6. Adjust the sensitivity of the system so that the height of the
two principal peaks is at least 20 per cent of the full scale of the recorder.
Inject 20 l of the test solution and 20 l of reference solution (a). Continue the chromatography of
the test solution for twice the retention time of the principal peak. In the chromatogram obtained
with the test solution: the area of any peak corresponding to paracetamol is not greater than that of
the peak due to paracetamol in the chromatogram obtained with reference solution (a) (200 ppm);
the area of any peak, apart from the principal peak and the peak due to paracetamol, is not greater
33-53
(a) (0.1 per cent taking into account the response factor of paracetamol of 1.6); the sum of the areas
of any peak, apart from the principal peak, is not greater than 6.4 times the area of the paracetamol
peak in the chromatogram obtained with reference solution (a) (0.2 per cent taking into account the
relative response factor of paracetamol of 1.6). Disregard any peak with an area less than 0.01 times
the area of the principal peak in the chromatogram obtained with reference solution (a).
4-Aminophenol Examine by thin-layer chromatography (2.2.27), using a TLC silica gel F254 plate R.
Test solution. Suspend 4.00 g of the substance to be examined in 8 ml of acetonitrile R. Shake for
30 min and filter. Dilute to 10 ml with acetonitrile R.
Reference solution (a). Dissolve 25 mg of 4-aminophenol R in acetonitrile R and dilute to 50 ml with the
same solvent. Dilute 10 ml of the solution to 50 ml with acetonitrile R.
Reference solution (b). Dilute 5 ml of reference solution (a) to 50 ml with acetonitrile R.
Reference solution (c). Dilute 0.2 ml of reference solution (a) to 5 ml with the test solution.
Apply to the plate 50 l of the test solution and 50 l each of reference solutions (b) and (c). Develop
over a path of 15 cm using a mixture of 3 volumes of anhydrous formic acid R, 4 volumes of water R,
30 volumes of methanol R and 64 volumes of methylene chloride R. Allow the plate to dry in air and
examine in ultraviolet light at 254 nm. Spray with a 10 g/l solution of dimethylaminobenzaldehyde R in
alcohol R. Reference solution (c) shows two spots: one visible in ultraviolet light and corresponding to
propacetamol hydrochloride and the other one yellow, visible after spraying and corresponding to
4-aminophenol. An additional spot may appear in ultraviolet light and corresponds to paracetamol.
In the chromatogram obtained with the test solution, any yellow spot corresponding to 4aminophenol and not visible in ultraviolet light is not more intense than the spot in the chromatogram obtained with reference solution (b) (25 ppm). The test is not valid unless the chromatogram
obtained with reference solution (c) shows two clearly separated spots.
Methanol Examine by gas chromatography (2.2.28), using propanol R as the internal standard.
Internal standard solution. Dilute 2.0 ml of propanol R to 20.0 ml with water R. Dilute 1.0 ml of the
solution to 25.0 ml with water R. Dilute 1.0 ml of this solution to 25.0 ml with water R.
Test solution. Dissolve 2.00 g of the substance to be examined in water R, add 2.0 ml of the internal
Reference solution. Dilute 0.8 ml of methanol R to 50.0 ml with water R. Dilute 1.0 ml of the solution
to 25.0 ml with water R. To 2.0 ml of the solution, add 2.0 of the internal standard solution and
a glass column 2 m long and 2 mm in internal diameter packed with carbon molecular sieve
impregnated with 0.2 per cent of macrogol 1500,
Time
(min)
Column 01.5
1.55.5
Temperature
(C)
Rate
(C per min)
60
6080
5.515.5 80
Injection
port
170
Detector
220
Comment
isothermal
linear
gradient
isothermal

Calculate the ratio of the area of the peak due to methanol to the area of the peak due to propanol
for both chromatograms. The ratio for the test solution is not greater than that for the reference
solution (500 ppm).
standard solution 1 ppm (Pb) R.
100C to 105C for 3 h.
ASSAY
33-54
STORAGE
IMPURITIES
A. paracetamol,
NH2
HO
B. 4-aminophenol.
__________________________________________________________________________________________________________ Ph Eur
33-55
Propantheline Bromide
O
O
H
+
NPri2
Me
Br
O
C23H30BrNO3
448.4
50-34-0
Propantheline Bromide complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Propantheline Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Propantheline bromide contains not less than 98.5 per cent and not more than the equivalent of
101.0 per cent of (methyl)bis(1-methylethyl)[2-[(9H-xanthen-9-ylcarbonyl)oxy]ethyl]ammonium
bromide, calculated with reference to the dried substance.
CHARACTERS
A white or yellowish-white powder, slightly hygroscopic, very soluble in water, in alcohol and in
methylene chloride.
IDENTIFICATION
A. Dissolve 60 mg in methanol R and dilute to 100.0 ml with the same solvent. Dilute 10.0 ml of the
shows two absorption maxima, at 246 nm and 282 nm. The specific absorbances at the maxima are
115 to 125 and 57 to 63, respectively.
B. Dissolve 0.2 g in 15 ml of water R and add 1 ml of strong sodium hydroxide solution R. Boil for
2 min and cool slightly. Add 7.5 ml of dilute hydrochloric acid R and filter. Wash the residue with
water R and recrystallise from alcohol (50 per cent V/V) R. Dry at 100C to 105C for 1 h. Dissolve
about 10 mg of the residue in 5 ml of sulphuric acid R. The solution has an intense yellow colour and
shows an intense yellowish-green fluorescence when examined in ultraviolet light at 365 nm.
C. Dissolve 50 mg in 0.1 ml of water R in a 25 ml flask and add 1 ml of a saturated solution of
potassium permanganate R. Attach a fractionating column and a condenser, with the end of the
delivery tube immersed in 1 ml of water R in a test-tube placed in a bath of iced water. Distil fairly
vigorously and continue heating for 1 min after a dry residue has been obtained in the flask. Prepare a
blank by introducing into an identical test-tube a volume of water R equal to that of the distillate.
Place the tubes in a bath of iced water. To each tube, add 0.5 ml of a 20 per cent V/V solution of
morpholine R and 0.5 ml of a freshly prepared 50 g/l solution of sodium nitroprusside R. Mix and allow
to stand at 0C for 5 min and then at room temperature for 3 min. No blue colour develops in either
tube. Add 1 g of ammonium sulphate R, mix and allow to stand for 15 min. A stable, intense pink
colour develops in the test solution. A brownish-yellow colour develops in the blank.
D. It gives reaction (a) of bromides (2.3.1).
TESTS
Appearance of solution. Dissolve 0.6 g in water R and dilute to 20 ml with the same solvent. The
solution is clear (2.2.1).
Related substance. Examine by liquid chromatography (2.2.29).
Test solution (a). Dissolve 6 mg of the substance to be examined in a mixture of 40 volumes of acetonitrile R and 60 volumes of water R and dilute to 50 ml with the same mixture of solvents.
Test solution (b). Dissolve 6 mg of the substance to be examined in 30 ml of a mixture of 40 volumes
of acetonitrile R and 60 volumes of water R. Add 5 ml of reference solution (b) and dilute to 50 ml
with a mixture of 40 volumes of acetonitrile R and 60 volumes of water R.
Test solution (c). Dissolve 6 mg of xanthydrol R1 and 6 mg of the substance to be examined in a
mixture of 40 volumes of acetonitrile R and 60 volumes of water R and dilute to 50 ml with the same
33-56
Reference solution (a). Dissolve 6 mg of xanthydrol R1 in a mixture of 40 volumes of acetonitrile R and
60 volumes of water R and dilute to 50 ml with the same mixture of solvents.
Reference solution (b). Dilute 5 ml of reference solution (a) to 50 ml with a mixture of 40 volumes of
acetonitrile R and 60 volumes of water R.
as mobile phase at a flow rate of 1 ml per minute a mixture of equal volumes of acetonitrile R and
a solution containing 28 g/l of sodium perchlorate R and 11 g/l of phosphoric acid R, adjusted to pH
3.8 with strong sodium hydroxide solution R and then with 0.1M sodium hydroxide,
a loop injector.
Inject 20 l of test solution (c). The test is not valid unless the resolution between the peaks corresponding to propantheline and xanthydrol is at least 8.0. Inject 20 l of test solution (a), 20 l of
reference solution (a) and 20 l of test solution (b). Continue the chromatography for twice the
retention time of the propantheline peak. In the chromatogram obtained with test solution (a), there
is no peak corresponding to the principal peak in the chromatogram obtained with reference solution
(a). In the chromatogram obtained with test solution (b), the area of any peak, apart from the principal peak and the xanthydrol peak, is not greater than the area of the xanthydrol peak (1.0 per cent)
and not more than one such peak has an area greater than or equal to half the area of the xanthydrol
peak (0.5 per cent). Disregard any peak with a retention time relative to that of the propantheline
peak, less than 0.2 (bromide).
at 100C to 105C.
ASSAY
1 ml of 0.1M perchloric acid corresponds to 44.84 mg of C23H30BrNO3.
STORAGE
__________________________________________________________________________________________________________________________________ Ph Eur
33-57
Propofol
1/01
OH
Pr i
C12H18O
Pr i
178.3
2078-54-8
Propofol complies with the requirements of the 3rd edition of the European Pharmacopoeia [1558]. These
Action and use Anaesthetic.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Propofol contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent of
2,6-bis(1-methylethyl)phenol.
CHARACTERS
A colourless or very light yellow, clear liquid, very slightly soluble in water, miscible with hexane and
with methanol.
IDENTIFICATION
Examine by infrared absorption spectrophotometry (2.2.24) as a thin film between potassium
bromide plates, comparing with the spectrum obtained with the propofol CRS.
TESTS
Refractive index (2.2.6) The refractive index is not less than 1.5125 and not more than 1.5145.
Impurity J Examine by liquid chromatography (2.2.29).
Prepare the solutions immediately before use and protect from light.
Test solution. Dissolve 0.5 g of the substance to be examined in hexane R and dilute to 10.0 ml with
the same solvent.
Reference solution. Dissolve 5 l of propofol impurity J CRS (corresponding to 5 mg) in hexane R and
dilute to 50.0 ml with the same solvent. Dilute 5.0 ml of this solution to 100.0 ml with hexane R.
The chromatographic procedure used in the test for Related substances may be used, by monitoring
the eluate of the column at 254 nm in place of 275 nm.
Inject 20 l of the test solution and 20 l of the reference solution and continue the chromatography for six times the retention time of the propofol peak. In the chromatogram obtained with the
test solution the area of any peak due to impurity J is not greater than five times the area of the peak
due to impurity J in the chromatogram obtained with the reference solution (0.05 per cent).
Inject 10 l of reference solution (a) and continue the chromatography for 15 min. The resolution
of the two principal peaks observed (impurity J, approximate retention time 2.5 min and propofol,
approximate retention time 3 min) is at least 4.0.
Inject 10 l of reference solution (b) and locate the peaks due to impurity G (relative retention
about 0.5) and impurity E (relative retention about 5), both with respect to propofol.
Inject 10 l of reference solution (c) and adjust the sensitivity of the system so that the height of the
peak due to propofol is at least 50 per cent of the full scale of the recorder.
Inject separately 10 l of test solution (a) and reference solution (c) and record the chromatogram
of test solution (a) for six times the retention time of the peak due to propofol. In the chromatogram
obtained with test solution (a): the area of any peak due to impurity G is not greater than 0.4 times
the area of the peak due to propofol in the chromatogram obtained with reference solution (c)
(0.2 per cent taking into account a response factor of 0.2); and the area of any peak due to impurity
E is not greater than 0.4 times the area of the peak due to propofol in the chromatogram obtained
with reference solution (c) (0.01 per cent taking into account a response factor of 4.0). The area of
any other peak apart from that due to propofol and the two impurities is not greater than 0.5 times
the area of the peak due to propofol in the chromatogram obtained with reference solution (c)
(0.05 per cent). The sum of all impurities including impurity G and impurity F is not greater than
0.3 per cent. Disregard any peak with an area less than 0.25 times the area of the peak due to
propofol in the chromatogram obtained with reference solution (c) (0.025 per cent).
33-58
ASSAY
Test solution (a). Dissolve 1.00 g of the substance to be examined in hexane R and dilute to 10.0 ml
Test solution (b). Dissolve 0.240 g of the substance to be examined in hexane R and dilute to 100.0 ml
Reference solution (a). Dissolve 5 l of the substance to be examined and 15 l of propofol impurity J
CRS in hexane R and dilute to 50.0 ml with the same solvent.
Reference solution (b). Dilute 1.0 ml of propofol for system suitability CRS to 10.0 ml with hexane R.
Reference solution (c). Dilute 1.0 ml of test solution (a) to 100.0 ml with hexane R. Dilute 1.0 ml of
this solution to 10.0 ml with hexane R.
Reference solution (d). Dissolve 0.240 g of propofol CRS in hexane R and dilute to 100.0 ml with the
same solvent.
a stainless steel column 0.20 m long and 5 mm in internal diameter packed with silica gel for
as mobile phase at a flow rate of 2.0 ml/min a mixture of 1.0 volumes of ethanol R, 7.5 volumes
of acetonitrile R and 990 volumes of hexane R,
Inject 10 l of test solution (b) and 10 l of reference solution (d). Continue the chromatography
for five times the retention time of the peak due to propofol.
STORAGE
Store protected from light under an inert atmosphere of gas.
IMPURITIES
OH
Pr i
Pr i
A. 2,4-bis(1-methylethyl)phenol,
OH
CH2
Pr i
CH3
B. 2-(1-methylethenyl)-6-(1-methylethyl)phenol,
OH
Pr i
C. 2-(1-methylethyl)phenol,
OH
Pr i
Pr i
D. 2,5-bis(1-methylethyl)phenol,
OH
Pr i
Pr i
Pr i
Pr i
OH
E. 3,3,5,5-tetrakis(1-methylethyl)biphenyl-4,4-diol,
OH
Pr i
F. 3-(1-methylethyl)phenol,
33-59
OPr i
Pr i
Pr i
G. 2-(1-methylethoxy)-1,3-bis(1-methylethyl)benzene,
OH
Pr i
H. 4-(1-methylethyl)phenol,
O
I. oxydibenzene,
O
Pr i
Pr i
J. 2,6-bis(1-methylethyl)-1,4-benzoquinone.
__________________________________________________________________________________________________________ Ph Eur
33-60
Propranolol Hydrochloride
NHPri
O
H
OH
,HCl
and enantiomer
C16H21NO2,HCl
295.8
318-98-9
Propranolol Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparations
Prolonged-release Propranolol Capsules
Propranolol Injection
Propranolol Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Propranolol hydrochloride contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of (2RS)-1-[(1-methylethyl)amino]-3-(naphthalen-1-yloxy)propan-2-ol hydrochloride,
CHARACTERS
A white or almost white powder, soluble in water and in alcohol.
IDENTIFICATION
obtained with propranolol hydrochloride CRS.
Reference solution. Dissolve 10 mg of propranolol hydrochloride CRS in 1 ml of methanol R.
1 volume of concentrated ammonia R1 and 99 volumes of methanol R. Dry the plate at 100C to 105C
and spray with anisaldehyde solution R. Heat the plate at 100C to 105C until the colour of the spots
reaches maximum intensity (10 min to 15 min). The principal spot in the chromatogram obtained
TESTS
The solution is clear (2.2.1) and not more intensely coloured than intensity 6 of the range of reference solutions of the most appropriate colour (Method II, 2.2.2).
Acidity or alkalinity Dissolve 0.20 g in carbon dioxide-free water R and dilute to 20 ml with the same
solvent. Add 0.2 ml of methyl red solution R and 0.2 ml of 0.01M hydrochloric acid. The solution is red.
Add 0.4 ml of 0.01M sodium hydroxide. The solution is yellow.
Reference solution (a). Dissolve 10.0 mg of propranolol hydrochloride for performance test CRS in the
mobile phase and dilute to 10.0 ml with the same mobile phase.
33-61
as mobile phase at a flow rate of 1.8 ml/min a mixture prepared as follows: mix 1.6 g of sodium
lauryl sulphate R and 0.31 g of tetrabutylammonium dihydrogen phosphate R in a mixture of 1 ml of
sulphuric acid R, 450 ml of water R and 550 ml of acetonitrile R; adjust to pH 3.3 using dilute
sodium hydroxide solution R,
Equilibrate the column for at least 30 min.
conditions, the appearance and acceptance criteria specified in the leaflet which accompanies
propranolol for performance test CRS are met.
Inject 20 l of the test solution. Continue the chromatography for five times the retention time of
the principal peak. In the chromatogram obtained with the test solution, the area of any peak apart
from the principal peak, is not greater than half the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.1 per cent) and the sum of the area of all peaks, apart from
methanol R and dilute to 20 ml with the same mixture of solvents. 12 ml of the solution complies with
limit test B for heavy metals (20 ppm).
Prepare the standard using lead standard solution (1 ppm Pb) prepared by diluting lead standard
solution (100 ppm Pb) R with a mixture of 15 volumes of water R and 85 volumes of methanol R.
100C to 105C.
ASSAY
Dissolve 0.250 g in 25 ml of alcohol R. Titrate with 0.1M sodium hydroxide, determining the end-point
IMPURITIES
O
OH
H OH
and enantiomer
A. 3-(naphthalen-1-yloxy)propane-1,2-diol-(diol derivative),
N
O
OH
Pr i
O
OH
B. 1,1-[(1-methylethyl)imino]bis[3-(naphthalen-1-yloxy)-propan-2-ol] (tertiary amine

derivative),
O
O
OH
O
OH
C. 1,1-oxybis[3-(naphthalen-1-yloxy)propan-2-ol] (bis-ether derivative).

__________________________________________________________________________________________________________ Ph Eur
33-62
Propyl Gallate
COOPrn
HO
HO
OH
C10H12O5
212.2
121-79-9
Propyl Gallate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1039].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Propyl gallate contains not less than 97.0 per cent and not more than the equivalent of 102.0 per cent
of propyl 3,4,5-trihydroxybenzoate, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, very slightly soluble in water, freely soluble in alcohol
and in ether. It dissolves in dilute solutions of alkali hydroxides.
IDENTIFICATION
obtained with propyl gallate CRS.
C. Examine the chromatograms obtained in the test for gallic acid. The principal spot in the chromatogram obtained with test solution (b) is similar in position, colour and size to the principal spot in
D. Dissolve about 10 mg in 10 ml of water R by heating to about 70C. Cool and add 1 ml of bismuth
subnitrate solution R. A bright yellow precipitate is formed.
TESTS
solution is clear (2.2.1) and not more intensely coloured than reference solution BY5 (Method II,
2.2.2).
Gallic acid Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating
substance.
Reference solution (a). Dissolve 10 mg of propyl gallate CRS in acetone R and dilute to 10 ml with the
same solvent.
Reference solution (b). Dissolve 20 mg of gallic acid R in acetone R and dilute to 20 ml with the same
solvent. Dilute 1 ml to 10 ml with acetone R.
Reference solution (c). Dilute 0.5 ml of test solution (b) to 5 ml with reference solution (b).
volumes of anhydrous formic acid R, 40 volumes of ethyl formate R and 50 volumes of toluene R. Allow
the plate to dry in air for 10 min and spray with a mixture of 1 volume of ferric chloride solution R1 and
9 volumes of alcohol R. Any spot corresponding to gallic acid in the chromatogram obtained with test
solution (a) is not more intense than the spot in the chromatogram obtained with reference solution
(b) (0.5 per cent). The test is not valid unless the chromatogram obtained with reference solution (c)
Total chlorine Mix 0.5 g with 2 g of calcium carbonate R1. Dry and ignite at 700C. Take up the
residue with 20 ml of dilute nitric acid R and dilute to 30 ml with water R. 15 ml of the solution,
without further addition of dilute nitric acid R, complies with the limit test for chlorides (2.4.4)
(200 ppm).
33-63
Chlorides (2.4.4). To 1.65 g add 50 ml of water R. Shake for 5 min. Filter. 15 ml of the filtrate
complies with the limit test for chlorides (100 ppm).
Zinc Not more than 25 ppm of Zn, determined by atomic absorption spectrometry (Method II,
2.2.23).
Test solution. To 2.5 ml of the solution obtained in the test for heavy metals, add 2.5 ml of water R.
Reference solutions. Prepare the reference solutions using zinc standard solution (10 ppm Zn) R, diluted
Measure the absorbance at 213.9 nm using a zinc hollow-cathode lamp as the source of radiation
100C to 105C.
ASSAY
Dissolve 0.400 g in 150 ml of water R heated to about 70C. Heat to boiling and add, with constant
stirring, 50.0 ml of bismuth subnitrate solution R. Cool and transfer the mixture quantitatively into a
250.0 ml volumetric flask and dilute to volume with a 0.5 per cent V/V solution of nitric acid R.
Filter, reject the first 20 ml of the filtrate and carry out the assay of bismuth by complexometry
(2.5.11) using 100.0 ml of the filtrate (n1 ml). Carry out a blank titration (n2 ml). The difference (n2
n1) ml between the volumes of 0.1M sodium edetate used is equivalent to the mass of C10H12O5
contained in the volume taken.
1 ml of 0.1M sodium edetate is equivalent to 21.22 mg of C10H12O5.
STORAGE
IMPURITIES
HO
COOH
HO
OH
A. gallic acid (3,4,5-trihydroxybenzoic acid).

__________________________________________________________________________________________________________ Ph Eur
33-64
Propyl Hydroxybenzoate
Propylparaben
COOPrn
HO
C10H12O3
180.2
94-13-3
Propyl Hydroxybenzoate complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Propyl Parahydroxybenzoate [0431]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Propyl parahydroxybenzoate contains not less than 99.0 per cent and not more than the equivalent of
100.5 per cent of propyl 4-hydroxybenzoate.
CHARACTERS
A white, crystalline powder, very slightly soluble in water, freely soluble in alcohol and in methanol.
IDENTIFICATION
obtained with propyl parahydroxybenzoate CRS.
D. To about 10 mg in a test-tube add 1 ml of sodium carbonate solution R, boil for 30 s and cool
(solution a). To a further 10 mg in a similar test-tube add 1 ml of sodium carbonate solution R; the
substance partly dissolves (solution b). Add at the same time to solution (a) and solution (b) 5 ml of
aminopyrazolone solution R and 1 ml of potassium ferricyanide solution R and mix. Solution (b) is yellow
to orange-brown. Solution (a) is orange to red, the colour being clearly more intense than any similar
colour which may be obtained with solution (b).
TESTS
Acidity To 2 ml of solution S add 3 ml of alcohol R, 5 ml of carbon dioxide-free water R and 0.1 ml of
bromocresol green solution R. Not more than 0.1 ml of 0.1M sodium hydroxide is required to change the
Reference solution (a). Dilute 0.5 ml of test solution (a) to 100 ml with acetone R.
Reference solution (b). Dissolve 10 mg of propyl parahydroxybenzoate CRS in acetone R and dilute to
Reference solution (c). Dissolve 10 mg of ethyl parahydroxybenzoate CRS in 1 ml of test solution (a) and
dilute to 10 ml with acetone R.
glacial acetic acid R, 30 volumes of water R and 70 volumes of methanol R. Allow the plate to dry in air
with reference solution (c) shows two clearly separated principal spots.
33-65
ASSAY
Place 2.000 g in a ground-glass-stoppered flask and add 40.0 ml of 1M sodium hydroxide. Heat gently
under a reflux condenser for 1 h. Cool to room temperature and rinse the condenser with water R.
Titrate the excess sodium hydroxide with 0.5M sulphuric acid continuing the titration until the second
point of inflexion and determining the end-point potentiometrically (2.2.20). Carry out a blank
titration.
IMPURITIES
COOR
HO
B. R = CH3: methyl 4-hydroxybenzoate,
C. R = CH2-CH3: ethyl 4-hydroxybenzoate,
D. R = CH2-CH2-CH2-CH3: butyl 4-hydroxybenzoate.
__________________________________________________________________________________________________________ Ph Eur
33-66
Propylene Glycol
H
OH
OH
H3C
and enantiomer
C3H8O2
76.10
57-55-6
Propylene Glycol complies with the requirements of the 3rd edition of the European Pharmacopoeia [0430].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Propylene glycol is (RS)-propane-1,2-diol.
CHARACTERS
A viscous, clear, colourless, hygroscopic liquid, miscible with water and with alcohol.
IDENTIFICATION
B. It complies with the test for refractive index (see Tests).
C. Boiling point (2.2.12): 184C to 189C.
D. To 0.5 ml add 5 ml of pyridine R and 2 g of finely ground nitrobenzoyl chloride R. Boil for 1 min
and pour into 15 ml of cold water R with shaking. Filter, wash the precipitate with 20 ml of a
saturated solution of sodium hydrogen carbonate R and then with water R and dry. Dissolve in boiling
alcohol (80 per cent V/V) R and filter the hot solution. On cooling, crystals are formed which, after
drying at 100C to 105C, melt (2.2.14) at 123C to 128C.
TESTS
Appearance It is clear (2.2.1) and colourless (Method II, 2.2.2).
Acidity To 10 ml add 40 ml of water R and 0.1 ml of bromothymol blue solution R1. The solution is
greenish-yellow. Not more than 0.05 ml of 0.1M sodium hydroxide is required to change the colour of
the indicator to blue.
Oxidising substances To 10 ml add 5 ml of water R, 2 ml of potassium iodide solution R and 2 ml of
dilute sulphuric acid R and allow to stand in a ground-glass-stoppered flask protected from light for
15 min. Titrate with 0.05M sodium thiosulphate, using 1 ml of starch solution R as indicator. Not more
than 0.2 ml of 0.05M sodium thiosulphate is required.
Reducing substances To 1 ml add 1 ml of dilute ammonia R1 and heat in a water-bath at 60C for
5 min. The solution is not yellow. Immediately add 0.15 ml of 0.1M silver nitrate and allow to stand
for 5 min. The solution does not change its appearance.
Heavy metals (2.4.8). Mix 3 ml with 12 ml of water R. 12 ml of the solution complies with limit test
A for heavy metals (5 ppm m/V). Prepare the standard using lead standard solution (1 ppm Pb) R.
of water.
Sulphated ash (2.4.14). Heat 50 g until it burns and ignite. Allow to cool. Moisten the residue with
sulphuric acid R and ignite; repeat the operations. The residue weighs not more than 5 mg (0.01 per
cent).
STORAGE
__________________________________________________________________________________________________________________________________ Ph Eur
33-67
Propylene Glycol Monopalmitostearate

Propylene Glycol Monostearate
Propylene Glycol Monopalmitostearate complies with the requirements of the 3rd edition of the European
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Propylene glycol monopalmitostearate is a mixture of propylene glycol mono- and diesters and of
stearic and palmitic acids. It contains not less than 50.0 per cent of monoesters produced from the
condensation of propylene glycol and stearic acid 50 of vegetable or animal origin.
PRODUCTION
CHARACTERS
A white or almost white, waxy solid, practically insoluble in water, soluble in acetone and in hot
alcohol.
IDENTIFICATION
A. It complies with the test for melting point (see Tests).
B. It complies for the test for composition of fatty acids (see Tests).
C. It complies with the assay (monoesters content).
TESTS
Melting point (2.2.15): 33C to 40C.
sum of contents of palmitic acid and stearic acid: not less than 90.0 per cent.
Free propylene glycol Not more than 5.0 per cent, determined as prescribed under Assay.
ASSAY
Determine the free propylene glycol content and monoesters content by size-exclusion chromatography (2.2.30).
Test solution. Into a 15 ml flask, weigh about 0.2 g (m), to the nearest 0.1 mg. Add 5.0 ml of tetrahydrofuran R and shake to dissolve. Heat gently, if necessary. Reweigh the flask and calculate the total
mass of solvent and substance (M).
Reference solutions. Into four 15 ml flasks, respectively weigh, to the nearest 0.1 mg, about 2.5 mg,
5.0 mg, 10.0 mg and 20.0 mg of propylene glycol R. Add 5.0 ml of tetrahydrofuran R and shake to
dissolve. Weigh the flasks again and calculate the concentration of propylene glycol in milligrams per
gram for each reference solution.
a gel-permeation column 0.6 m long and 7 mm in internal diameter packed with styrenedivinylbenzene copolymer R (particle diameter 5 m and pore size 10 nm),
as mobile phase at a flow rate of 1 ml/min tetrahydrofuran R,
a differential refractive index detector.
Inject 40 l of each solution. When the chromatograms are recorded in the prescribed conditions,
the retention times relative to propylene glycol are about 0.84 for the monoesters and about 0.78 for
the diesters. From the calibration curve obtained with the reference solutions determine the concentration (C) in milligrams per gram of propylene glycol in the test solution.
Calculate the percentage content of free propylene glycol in the substance to be examined using the
following expression:
33-68
CM
m 100
From the peak area of the monoesters (A) and the diesters (B), calculate the percentage content of
monoesters using the following expression:
A
(100 D )
A+ B
D = the percentage content of free propylene glycol plus the percentage content of free fatty
acids.
Calculate the percentage content of free fatty acids using the expression:
I A 270
561.1
IA = acid value.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
33-69
Propylthiouracil
Pr n
H
N
S
NH
O
C7H10N2OS
170.2
51-52-5
Propylthiouracil complies with the requirements of the 3rd edition of the European Pharmacopoeia [0525].
Action and use Antithyroid.
Preparation
Propylthiouracil Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Propylthiouracil contains not less than 98.0 per cent and not more than the equivalent of 100.5 per
cent of 2,3-dihydro-6-propyl-2-thioxopyrimidin-4(1H)-one, calculated with reference to the dried
substance.
CHARACTERS
White or almost white, crystalline powder or crystals, very slightly soluble in water, sparingly soluble
in alcohol, very slightly soluble in ether. It dissolves in solutions of alkali hydroxides.
IDENTIFICATION
obtained with propylthiouracil CRS. Examine as discs prepared using 1 mg of substance and 0.3 g of
potassium bromide R.
C. Examine the chromatograms obtained in the test for impurity A and related substances in ultraviolet light at 254 nm before exposure of the plate to iodine vapour. The principal spot in the
D. To about 20 mg add 8 ml of bromine water R and shake for a few minutes. Boil until the mixture is
decolourised, allow to cool and filter. To the filtrate add 2 ml of barium chloride solution R1. A white
precipitate is formed whose colour does not become violet on the addition of 5 ml of dilute sodium
hydroxide solution R.
TESTS
Impurity A and related substances Examine by thin-layer chromatography (2.2.27), using a TLC
silica gel GF254 plate R.
Reference solution (a). Dissolve 10 mg of propylthiouracil CRS in methanol R and dilute to 10 ml with
the same solvent.
Reference solution (b). Dissolve 50 mg of thiourea R in methanol R and dilute to 100 ml with the same
solvent. Dilute 1 ml of this solution to 100 ml with methanol R.
Reference solution (c). Dilute 1 ml of test solution (a) to 100 ml with methanol R.
0.1 volumes of glacial acetic acid R, 6 volumes of 2-propanol R and 50 volumes of chloroform R. Allow
the plate to dry in air. Examine in ultraviolet light at 254 nm. Expose the plate to iodine vapour for
10 min. In the chromatogram obtained with test solution (a), any spot corresponding to impurity A is
not more intense than the spot in the chromatogram obtained with reference solution (b) (0.05 per
cent) and any spot apart from the principal spot and any spot corresponding to impurity A is not
33-70
100C to 105C.
ASSAY
To 0.300 g add 30 ml of water R and 30.0 ml of 0.1M sodium hydroxide. Boil and shake until solution
is complete. Add 50 ml of 0.1M silver nitrate while stirring, boil gently for 5 min and cool. Titrate with
0.1M sodium hydroxide, determining the end-point potentiometrically (2.2.20). The volume of 0.1M
sodium hydroxide used is equal to the sum of the volume added initially and the volume used in the
final titration.
1 ml of 0.1M sodium hydroxide is equivalent to 8.511 mg of C7H10N2OS.
STORAGE
IMPURITIES
S
H2N
NH2
A. thiourea.
__________________________________________________________________________________________________________ Ph Eur
34-1
Propyphenazone
Me
N
Me
N
Pri
C14H18N2O
Ph
O
230.3
479-92-5
Propyphenazone complies with the requirements of the 3rd edition of the European Pharmacopoeia [0636].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Propyphenazone contains not less than 99.0 per cent and not more than the equivalent of 101.0 per
cent of 1,2-dihydro-1,5-dimethyl-4-(1-methylethyl)-2-phenyl-3H-pyrazol-3-one, calculated with
CHARACTERS
A white or slightly yellowish, crystalline powder, slightly soluble in water, freely soluble in alcohol and
in methylene chloride, soluble in ether.
IDENTIFICATION
obtained with propyphenazone CRS. Examine the substances prepared as discs.
chromatogram obtained with test solution (b) corresponds in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a).
D. To 1 ml of solution S (see Tests) add 0.1 ml of ferric chloride solution R1. A brownish-red colour
appears which becomes yellow on addition of 1 ml of dilute hydrochloric acid R.
TESTS
Solution S Dissolve 2 g in a mixture of equal volumes of alcohol R and carbon dioxide-free water R and
dilute to 50 ml with the same mixture of solvents.
colourless. Add 0.2 ml of 0.01M sodium hydroxide; the solution becomes pink. Add 0.4 ml of 0.01M
hydrochloric acid; the solution becomes colourless. Add 0.2 ml of methyl red solution R. The solution
becomes orange or red.
coating substance.
Reference solution (a). Dissolve 80 mg of propyphenazone CRS in methanol R and dilute to 5 ml with
the same solvent.
10 volumes of butanol R, 45 volumes of cyclohexane R and 45 volumes of ethyl acetate R. Dry the plate
in a current of hot air for 15 min and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with test solution (a), apart from the principal spot, is not more intense than the
spot in the chromatogram obtained with reference solution (b) (0.2 per cent). Spray with a mixture
of equal volumes of potassium ferricyanide solution R and ferric chloride solution R1. Any spot in the
chromatogram obtained with test solution (a), apart from the principal spot, is not more intense than
34-2
60C for 4 h.
ASSAY
Dissolve 0.2000 g in 10 ml of anhydrous acetic acid R and add 75 ml of ethylene chloride R. Titrate with
STORAGE
__________________________________________________________________________________________________________________________________ Ph Eur
34-3
Protamine Hydrochloride
Protamine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Protamine hydrochloride consists of the hydrochlorides of basic peptides extracted from the sperm or
roe of fish, usually species of Salmonidae and Clupeidae. It binds with heparin in solution, inhibiting
its anticoagulant activity; in the conditions of the assay this binding gives rise to a precipitate. Calculated with reference to the dried substance, 1 mg of protamine hydrochloride precipitates not less
than 100 International Units of heparin.
PRODUCTION
Protamine hydrochloride is prepared in conditions designed to minimise the risk of microbial
contamination.
The method of manufacture is validated to demonstrate that the product if tested would comply with
the following test:
Abnormal toxicity (2.6.9). It complies with the test for abnormal toxicity. Inject into each mouse
0.5 mg dissolved in 0.5 ml of water for injections R.
CHARACTERS
A white or almost white powder, hygroscopic, soluble in water, practically insoluble in alcohol and in
ether.
IDENTIFICATION
A. Dissolve 1.000 g in 0.1M hydrochloric acid and dilute to 100.0 ml with the same acid. The specific
optical rotation (2.2.7) is 40 to 60, calculated with reference to the dried substance.
B. In the conditions of the Assay, protamine hydrochloride forms a precipitate.
C. To 0.5 ml of solution S (see Tests) add 4.5 ml of water R, 1.0 ml of a 100 g/l solution of sodium
hydroxide R and 1.0 ml of a 0.2 g/l solution of -naphthol R and mix. Cool the mixture to 5C. Add
0.5 ml of sodium hypobromite solution R. An intense red colour is produced.
D. Heat 2 ml of solution S in a water-bath at 60C and add 0.1 ml of mercuric sulphate solution R.
Mix. No precipitate is formed. Cool the mixture in iced water. A precipitate is formed.
TESTS
Appearance of solution To 2.5 ml of solution S add 7.5 ml of water R. The solution is not more
opalescent than reference suspension II (2.2.1) and not more than intensely coloured than reference
solution BY6 or Y6 (Method II, 2.2.2).
Absorbance (2.2.25). Dilute 2.5 ml of solution S to 5.0 ml with water R. At wavelengths from
260 nm to 280 nm, the absorbance of the solution is not greater than 0.1.
Chloride 12.3 per cent to 19.0 per cent, calculated with reference to the dried substance. Dissolve
0.400 g in 50 ml of water R. Add 5 ml of dilute nitric acid R, 25.0 ml of 0.1M silver nitrate and 2 ml of
dibutyl phthalate R and shake. Titrate with 0.1M ammonium thiocyanate using 2 ml of ferric ammonium
sulphate solution R2 as indicator; shake vigorously when approaching the end-point.
1 ml of 0.1M silver nitrate is equivalent to 3.545 mg of chloride (Cl).
Sulphate Not more than 4.0 per cent, calculated with reference to the dried substance. Dissolve
0.500 g in 200 ml of distilled water R, add 5.0 ml of dilute hydrochloric acid R and heat to boiling. Add
dropwise 10 ml of a hot 100 g/l solution of barium chloride R while stirring with a glass rod, cover the
beaker with a watch glass and allow to stand on a water-bath for 2 h to obtain a coarse granular
precipitate. Add 0.1 ml of a 100 g/l solution of barium chloride R to the clear supernatant liquid. If a
turbidity develops, repeat the precipitation procedure. Transfer the precipitate quantitatively to a
previously ignited and tared porcelain crucible and wash with hot distilled water R until the addition of
silver nitrate solution R1 to the washings produces no opalescence. Ignite the precipitate at 600C for
1 h. Allow to cool in a desiccator and weigh.
1 mg of residue is equivalent to 0.412 mg of sulphate (SO4).
Barium Not more than 10 ppm of Ba, determined by atomic absorption spectrometry (Method I,
2.2.23).
34-4
Test solution. Dissolve 1.0 g of the substance to be examined in distilled water R, add 1 ml of a 250 g/l
solution of caesium chloride R and 0.2 ml of hydrochloric acid R and dilute to 20.0 ml with distilled
water R.
Reference solution. To 1.0 ml of barium standard solution (50 ppm Ba) R add 5 ml of a 250 g/l solution
of caesium chloride R and 1 ml of hydrochloric acid R and dilute to 100.0 ml with distilled water R.
Measure the absorbance at 553.3 nm using a barium hollow-cathode lamp as source of radiation and
an air-acetylene-nitrous oxide flame of suitable composition.
Iron (2.4.9). Dissolve 1.0 g with heating in water R and dilute to 10 ml with the same solvent. The
solution complies with the limit test for iron (10 ppm).
Mercury Not more than 10 ppm of Hg. Introduce 2.0 g of the substance to be examined into a
ground-glass-stoppered 250 ml conical flask and add 20 ml of a mixture of equal volumes of nitric
acid R and sulphuric acid R. Boil under a reflux condenser for 1 h, cool and cautiously dilute with
water R. Boil until nitrous fumes are no longer seen. Cool the solution, cautiously dilute to 200.0 ml
with water R, mix and filter. Transfer 50.0 ml of the filtrate to a separating funnel. Shake with
successive small portions of chloroform R until the chloroform layer remains colourless. Discard the
chloroform layers. To the aqueous layer add 25 ml of dilute sulphuric acid R, 115 ml of water R and
10 ml of a 200 g/l solution of hydroxylamine hydrochloride R. Titrate with dithizone solution R2; after
each addition, shake the mixture twenty times and towards the end of the titration allow to separate
and discard the chloroform layer. Titrate until a bluish-green colour is obtained. Calculate the
content of mercury using the equivalent in micrograms of mercury per millilitre of titrant determined
in the standardisation of the dithizone solution R2.
Nitrogen 23.0 per cent to 27.0 per cent, calculated with reference to the dried substance. Carry out
the determination of nitrogen by sulphuric acid digestion (2.5.9), using 10.0 mg; heat for 3 h to 4 h.
100C to 105C for 3 h.
Inject per kilogram of the rabbits mass 1.0 ml of a solution containing 10 mg of the substance to be
examined per millilitre.
ASSAY
Test solution (a). Dissolve 15.0 mg of the substance to be examined in water R and dilute to 100.0 ml
Test solution (b). Dilute 2.0 ml of test solution (a) to 3.0 ml with water R.
Test solution (c). Dilute 1.0 ml of test solution (a) to 3.0 ml with water R.
Use as titrant a 1 in 6 dilution of heparin sodium BRP (for example, 1.7 ml diluted to 10.0 ml with
water R). Titrate each of the test solutions in duplicate as follows: introduce an accurately measured
volume of the solution to be titrated, for example, 1.5 ml, into the cell of a suitable colorimeter, set
the apparatus for measurement at a suitable wavelength (none is critical) in the visible range, add the
titrant in small volumes until there is a sharp increase in the absorbance and note the volume of
titrant added.
Carry out three independent assays. For each individual titration, calculate the number of International Units of heparin in the volume of titrant added at the end-point per milligram of the substance
to be examined. Calculate the potency of the substance as the average of the eighteen values. Test the
linearity of the response by the usual statistical methods. Calculate the three standard deviations for
the results obtained with each of the three test solutions. Calculate the three standard deviations for
the results obtained with each of the three independent assays. The assay is not valid unless each of
the six standard deviations is less than 5 per cent of the average result.
STORAGE
Store in an airtight, tamper-proof container. If the substance is sterile, store in a sterile, airtight,
tamper-proof container.
LABELLING
The label states:
__________________________________________________________________________________________________________ Ph Eur
34-5
Protamine Sulphate
9009-65-8
Protamine Sulphate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Heparin antidote.
Preparation
Protamine Sulphate Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Protamine sulphate consists of the sulphates of basic peptides extracted from the sperm or roe of fish,
usually species of Salmonidae and Clupeidae. It binds with heparin in solution, inhibiting its
anticoagulant activity; in the conditions of the assay this binding gives rise to a precipitate. Calculated
with reference to the dried substance, 1 mg of protamine sulphate precipitates not less than 100
International Units of heparin.
PRODUCTION
Protamine sulphate is prepared in conditions designed to minimise the risk of microbial contamination.
The method of manufacture is validated to demonstrate that the product, if tested, would comply
with the following test:
Abnormal toxicity (2.6.9). It complies with the test for abnormal toxicity. Inject into each mouse
0.5 mg dissolved in 0.5 ml of water for injections R.
CHARACTERS
A white or almost white powder, hygroscopic, sparingly soluble in water, practically insoluble in
IDENTIFICATION
A. Dissolve 1.000 g in 0.1M hydrochloric acid and dilute to 100.0 ml with the same acid. The specific
optical rotation (2.2.7) is 65 to 85, calculated with reference to the dried substance.
B. In the conditions of the Assay, protamine sulphate forms a precipitate.
C. To 0.5 ml of solution S (see Tests) add 4.5 ml of water R, 1.0 ml of a 100 g/l solution of sodium
hydroxide R and 1.0 ml of a 0.2 g/l solution of -naphthol R and mix. Cool the mixture to 5C. Add
0.5 ml of sodium hypobromite solution R. An intense red colour is produced.
D. Heat 2 ml of solution S in a water-bath at 60C and add 0.1 ml of mercuric sulphate solution R.
Mix. No precipitate is formed. Cool the mixture in iced water. A precipitate is formed.
TESTS
Appearance of solution To 2.5 ml of solution S add 7.5 ml of water R. The solution is not more
opalescent than reference suspension II (2.2.1) and not more intensely coloured than reference
solution BY6 or Y6 (Method II, 2.2.2).
Absorbance (2.2.25). Dilute 2.5 ml of solution S to 5.0 ml with water R. At wavelengths from
260 nm to 280 nm, the absorbance of the solution is not greater than 0.1.
Iron (2.4.9). Dissolve 1.0 g with heating in water R and dilute to 10 ml with the same solvent. The
Mercury Not more than 10 ppm of Hg. Introduce 2.0 g of the substance to be examined into a
250 ml ground-glass-stoppered conical flask and add 20 ml of a mixture of equal volumes of nitric
acid R and sulphuric acid R. Boil under a reflux condenser for 1 h, cool and cautiously dilute with
water R. Boil until nitrous fumes are no longer seen. Cool the solution, cautiously dilute to 200.0 ml
with water R, mix and filter. Transfer 50.0 ml of the filtrate to a separating funnel. Shake with
successive small portions of chloroform R until the chloroform layer remains colourless. Discard the
chloroform layers. To the aqueous layer add 25 ml of dilute sulphuric acid R, 115 ml of water R and
10 ml of a 200 g/l solution of hydroxylamine hydrochloride R. Titrate with dithizone solution R2; after
each addition, shake the mixture twenty times and towards the end of the titration allow to separate
and discard the chloroform layer. Titrate until a bluish-green colour is obtained. Calculate the
content of mercury using the equivalent in micrograms of mercury per millilitre of titrant, determined
in the standardisation of the dithizone solution R2.
34-6
Sulphate 16 per cent to 24 per cent of sulphate (SO4), calculated with reference to the dried
substance. Dissolve 0.150 g in 15 ml of distilled water R in a beaker. Add 5 ml of dilute hydrochloric
acid R. Heat to boiling and slowly add to the boiling solution 10 ml of a 100 g/l solution of barium
chloride R. Cover the beaker and heat on a water-bath for 1 h. Filter. Wash the precipitate several
times with small quantities of hot water R. Dry and ignite the residue at 600C to constant mass.
1.0 g of residue is equivalent to 0.4117 g of sulphate (SO4) in the substance to be examined.
Nitrogen 21.0 per cent to 26.0 per cent, calculated with reference to the dried substance. Carry out
the determination of nitrogen by sulphuric acid digestion (2.5.9), using 10.0 mg; heat for 3 h to 4 h.
100C to 105C for 3 h.
Inject per kilogram of the rabbits mass 1.0 ml of a solution containing 10 mg of the substance to be
examined per millilitre.
ASSAY
Test solution (a). Dissolve 15.0 mg of the substance to be examined in water R and dilute to 100.0 ml
Test solution (b). Dilute 2.0 ml of test solution (a) to 3.0 ml with water R.
Test solution (c). Dilute 1.0 ml of test solution (a) to 3.0 ml with water R.
Use as titrant a 1 to 6 dilution of heparin sodium BRP (for example, 1.7 ml diluted to 10.0 ml with
water R). Titrate each of the test solutions in duplicate as follows: introduce an accurately measured
volume of the solution to be titrated, for example 1.5 ml, into the cell of a suitable colorimeter and
set the apparatus for measurement at a suitable wavelength (none is critical) in the visible range. Add
the titrant in small volumes until there is a sharp increase in the absorbance and note the volume of
titrant added.
Carry out three independent assays. For each individual titration, calculate the number of International Units of heparin in the volume of titrant added at the end-point per milligram of the substance
to be examined. Calculate the potency of the substance as the average of the eighteen values. Test the
linearity of the response by the usual statistical methods. Calculate the three standard deviations for
the results obtained with each of the three test solutions. Calculate the three standard deviations for
the results obtained with each of the three independent assays. The assay is not valid unless each of
the six standard deviations is less than 5 per cent of the average result.
STORAGE
Store in an airtight, tamper-proof container. If the substance is sterile, store in a sterile, airtight,
tamper-proof container.
LABELLING
The label states:
__________________________________________________________________________________________________________ Ph Eur
34-7
Protirelin
1/01
oxoPro-His-Pro-NH2
C16H22N6O4
362.4
24305-27-9
Protirelin complies with the requirements of the 3rd edition of the European Pharmacopoeia [1144]. These
Action and use Thyrotrophin-releasing hormone.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Protirelin is a synthetic tripeptide in which the sequence of amino acids is the same as that in the
natural hypothalamic neurohormone, that stimulates the release and synthesis of thyrotropin. It
contains not less than 97.0 per cent and not more than the equivalent of 102.0 per cent of 5-oxo-Lprolyl-L-histidyl-L-prolinamide, calculated with reference to the anhydrous, acetic acid-free
substance.
CHARACTERS
A white or yellowish-white powder, hygroscopic, very soluble in water, freely soluble in methanol.
IDENTIFICATION
obtained with protirelin CRS.
B. Examine the chromatograms obtained in the assay. The retention time and size of the principal
peak in the chromatogram obtained with the test solution are approximately the same as those of the
principal peak in the chromatogram obtained with the reference solution.
TESTS
Appearance of solution A 10 g/l solution is clear (2.2.1) and not more intensely coloured than
Specific optical rotation (2.2.7). Dissolve 10 mg in 1.0 ml of water R. The specific optical rotation
is 62 to 70, calculated with reference to the anhydrous, acetic acid-free substance.
Test solution. Dissolve 5.0 mg of the substance to be examined in mobile phase A and dilute to 5.0 ml
with the same mobile phase.
Reference solution (a). Dissolve the contents of a vial of D-His-protirelin CRS in an appropriate volume
of mobile phase A to obtain a concentration of 1 mg/ml. Mix equal volumes of this solution and the
test solution.
Inject 10 l of reference solution (a). With the aid of the reference chromatogram provided with
D-His-protirelin CRS, identify the peaks due to D-His-protirelin and protirelin. The test is not valid
unless the resolution between the peak due to protirelin and the peak due to D-His-protirelin in the
chromatogram obtained with reference solution (a) is not less than 2.5 and the symmetry factor for
the protirelin peak is between 0.9 and 1.2. If necessary, adjust the initial composition of the mobile
phase or the gradient profile.
principal peak in the chromatogram obtained with reference solution (b) is at least 50 per cent of the
Inject 10 l of the test solution and continue the chromatography for about 40 min. In the chromatogram obtained with the test solution: the area of any peak, apart from the principal peak, is not
(2 per cent); the sum of the areas of all the peaks, apart from the principal peak, is not greater than
1.5 times the area of the principal peak in the chromatogram obtained with reference solution (b)
(3 per cent). Disregard any peaks due to the solvent and any peak with an area less than 0.05 times
cent).
Acetic acid (2.5.34). Not more than 2.0 per cent.
34-8
ASSAY
Test solution. Dissolve 5.0 mg of the substance to be examined in mobile phase A and dilute to 5.0 ml
with the same mobile phase.
Reference solution. Dissolve the contents of a vial of protirelin CRS in mobile phase A. Dilute an
appropriate volume of this solution in the same mobile phase to obtain a final concentration of
1.0 mg/ml.
silica gel for chromatography R (5 m) with a porosity of 12 nm,
Mobile phase A. A mixture of 100 ml of acetonitrile for chromatography R, 1900 ml of water R and
2.0 g of sodium octanesulphonate R, containing 2.5 ml/l of tetraethylammonium hydroxide solution R;
adjust the pH of the solution to 3.5 using phosphoric acid R,
Mobile phase B. A mixture of 300 ml of acetonitrile for chromatography R, 1700 ml of water R and
2.0 g of sodium octanesulphonate R, containing 2.5 ml/l of tetraethylammonium hydroxide solution R;
adjust the pH of the solution to 3.5 using phosphoric acid R,
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
0 30
74 41
41 74
74
26 59
59 26
26
30 35
35 50

Equilibrate the column with a mixture of 74 volumes of mobile phase A and 26 volumes of mobile
phase B.
Inject 10 l of the test solution and 10 l of the reference solution and continue the chromatography for about 40 min.
Calculate the content of protirelin (C16H22N6O4) from the peak areas in the chromatograms
obtained with the test solution and the reference solution and the declared content of C16H22N6O4 in
protirelin CRS.
STORAGE
Store in an airtight container, protected from light at a temperature of 2C to 8C. If the substance is
sterile, store in a sterile, airtight, tamper-proof container.
LABELLING
The label states:
the mass of peptide in the container,
where applicable, that the substance is free from bacterial endotoxins,
where applicable, that the substance is sterile.
__________________________________________________________________________________________________________ Ph Eur
34-9
Protriptyline Hydrochloride
,HCl
NHMe
C19H21N,HCl
299.8
1225-55-4
Definition Protriptyline Hydrochloride is 3-(5H-dibenzo[a,d]cyclohept-5-yl)propyl(methyl)amine

hydrochloride. It contains not less than 99.0% and not more than 101.0% of C19H21N,HCl, calculated with reference to the dried substance.
Characteristics A white to yellowish white powder; odourless or almost odourless.
Freely soluble in water, in chloroform and in ethanol (96%); practically insoluble in ether.
Identification
A. Dissolve 0.1 g in 10 ml of water, make alkaline with 1M sodium hydroxide, extract with 5 ml of
chloroform, dry with anhydrous sodium sulphate and evaporate the solvent using a current of nitrogen.
The infrared absorption spectrum of the oily residue, Appendix II A, is concordant with the reference
spectrum of protriptyline (RS 301).
solution as indicator. Each ml of 0.1M perchloric acid VS is equivalent to 29.98 mg of C19H21N,HCl.
Storage Protriptyline Hydrochloride should be kept in a well-closed container.
Preparation
Protriptyline Tablets
34-10
Proxymetacaine Hydrochloride
O
H2N
NEt2
,HCl
PrnO
C16H26N2O3,HCl
330.9
5875-06-9
Definition Proxymetacaine Hydrochloride is 2-diethylaminoethyl 3-amino-4-propoxybenzoate

hydrochloride. It contains not less than 98.0% and not more than 102.0% of C16H26N2O3,HCl,
Soluble in water and in chloroform; very soluble in absolute ethanol; practically insoluble in ether.
Identification
A. The light absorption, Appendix II B, in the range 220 to 350 nm of a 0.002% w/v solution exhibits
three maxima, at 231, 268 and 310 nm. The absorbances at the maxima at 268 nm and at 310 nm are
about 0.58 and about 0.32, respectively.
proxymetacaine hydrochloride (RS 303).
C. A 5% w/v solution yields the reaction characteristic of primary aromatic amines and the reactions
characteristic of chlorides, Appendix VI.
Related substances
A. Carry out the method for thin-layer chromatography, Appendix III A, using silica gel GF254 as the
coating substance and a mixture of 75 volumes of toluene, 30 volumes of ethyl acetate and 5 volumes
of diethylamine as the mobile phase. Apply separately to the plate 10 l of each of three solutions of
the substance being examined in methanol containing (1) 2.0% w/v, (2) 0.020% w/v and (3) 0.010%
w/v. After removal of the plate, heat it at 105 for 10 minutes, allow to cool and examine under
ultraviolet light (254 nm). Any secondary spot in the chromatogram obtained with solution (1) is not
more intense than the spot in the chromatogram obtained with solution (2) (1%) and not more than
one such spot is more intense than the spot in the chromatogram obtained with solution (3) (0.5%).
Disregard any spot remaining on the line of application.
B. Carry out the method for thin-layer chromatography, Appendix III A, using silica gel GF254 as the
coating substance and a mixture of 80 volumes of 1,4-dioxan, 20 volumes of cyclohexane and 4
volumes of glacial acetic acid as the mobile phase. Apply separately to the plate 10 l of each of two
solutions in methanol containing (1) 2.0% w/v of the substance being examined and (2) 0.0050% w/v
of 3-amino-4-propoxybenzoic acid BPCRS. After removal of the plate, allow it to dry in air and examine
under ultraviolet light (254 nm). Any secondary spot in the chromatogram obtained with solution (1) is
not more intense than the spot in the chromatogram obtained with solution (2) (0.25%). The principal spot remains on the line of application.
Assay Carry out Method I for non-aqueous titration, Appendix VIII A, using 0.25 g, 20 ml of
mercury(II) acetate solution and 1-naphtholbenzein solution as indicator. Each ml of 0.1M perchloric acid
VS is equivalent to 16.54 mg of C16H26N2O3,HCl.
Storage Proxymetacaine Hydrochloride should be kept in a well-closed container and protected
from light.
Preparation
Proxymetacaine Eye Drops
34-11
Proxyphylline
H
O
CH3
N
MeN
O
OH
N
Me
and enantiomer
C10H14N4O3
238.2
603-00-9
Proxyphylline complies with the requirements of the 3rd edition of the European Pharmacopoeia [0526].
Action and use Xanthine bronchodilator.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Proxyphylline contains not less than 98.5 per cent and not more than the equivalent of 101.0 per
cent of 7-[(2RS)-2-hydroxypropyl]-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione, calculated with
CHARACTERS
A white, crystalline powder, very soluble in water, soluble in alcohol.
IDENTIFICATION
obtained with proxyphylline CRS. Examine the substances as discs prepared using 0.5 mg to 1 mg of
the substance to be examined in 0.3 g of potassium bromide R.
C. Dissolve 1 g in 5 ml of acetic anhydride R and boil under a reflux condenser for 15 min. Allow to
cool and add 100 ml of a mixture of 20 volumes of ether R and 80 volumes of light petroleum R. Cool
in iced water for at least 20 min, shaking from time to time. Filter, wash the precipitate with a
mixture of 20 volumes of ether R and 80 volumes of light petroleum R, recrystallise from alcohol R and
dry in vacuo. The crystals melt (2.2.14) at 87C to 92C.
D. It gives the reaction of xanthines (2.3.1).
TESTS
Acidity or alkalinity To 10 ml of solution S add 0.25 ml of bromothymol blue solution R1. The
solution is yellow or green. Not more than 0.4 ml of 0.01M sodium hydroxide is required to change the
coating substance.
Test solution. Dissolve 0.3 g of the substance to be examined in a mixture of 20 volumes of water R
and 30 volumes of methanol R and dilute to 10 ml with the same mixture of solvents. Prepare
Reference solution (b). Dilute 0.2 ml of the test solution to 100 ml with methanol R.
Reference solution (c). Dissolve 10 mg of theophylline R in methanol R, add 0.3 ml of the test solution
and dilute to 10 ml with methanol R.
1 volume of concentrated ammonia R, 10 volumes of ethanol R and 90 volumes of chloroform R. Allow
34-12
chromatogram obtained with reference solution (a) (1 per cent) and at most one such spot is more
separated spots.
100C to 105C.
ASSAY
In order to avoid overheating in the reaction medium, mix thoroughly throughout and stop the titration
immediately after the end-point has been reached.
Dissolve 0.200 g in 3.0 ml of anhydrous formic acid R and add 50.0 ml of acetic anhydride R. Titrate
with 0.1M perchloric acid determining the end-point potentiometrically (2.2.20).
STORAGE
__________________________________________________________________________________________________________ Ph Eur
34-13
Pseudoephedrine Hydrochloride
H
OH
CH3
,HCl
H
C10H15NO,HCl
NHMe
201.7
345-78-8
Pseudoephedrine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia [1367]. These requirements are reproduced after the heading Definition below.
Preparation
Pseudoephedrine Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Pseudoephedrine hydrochloride contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of (1S,2S)-2-(methylamino)-1-phenylpropan-1-ol hydrochloride, calculated
CHARACTERS
A white, crystalline powder or colourless crystals, freely soluble in water and in alcohol, sparingly
IDENTIFICATION
obtained with pseudoephedrine hydrochloride CRS. Examine the substances prepared as discs.
substance.
the same solvent.
Reference solution (a). Dissolve 20 mg of pseudoephedrine hydrochloride CRS in methanol R and dilute to
Reference solution (b). Dissolve 10 mg of ephedrine hydrochloride CRS in reference solution (a) and
of methylene chloride R, 15 volumes of concentrated ammonia R and 80 volumes of 2-propanol R. Allow
the plate to dry in air and spray with ninhydrin solution R. Heat at 110C for 5 min. The principal
D. Solution S (see Tests) gives reaction (a) of chlorides (2.3.1).
TESTS
Acidity or alkalinity Dilute 2 ml of solution S to 10 ml with carbon dioxide-free water R. Add 0.1 ml
of methyl red solution R and 0.1 ml of 0.01M sodium hydroxide; the solution is yellow. Add 0.2 ml of
0.01M hydrochloric acid; the solution is red.
Specific optical rotation (2.2.7). +61.0 to +62.5, determined on solution S and calculated with
Reference solution (a). Dissolve 20.0 mg of ephedrine hydrochloride CRS in the mobile phase and dilute
to 20.0 ml with the mobile phase. Dilute 1.0 ml of the solution to 50.0 ml with the mobile phase.
34-14
Reference solution (c). Dissolve 10 mg of ephedrine hydrochloride CRS in 5 ml of the test solution and
a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with phenylsilyl
as mobile phase at a flow rate of 1 ml/min a mixture of 6 volumes of methanol R and 94 volumes
of a 11.6 g/l solution of ammonium acetate R adjusted to pH 4.0 with glacial acetic acid R,
two peaks in the chromatogram obtained are at least 50 per cent of the full scale of the recorder. The
test is not valid unless the resolution between the peaks corresponding to ephedrine and pseudoephedrine is at least 2.0. If necessary reduce the content of methanol in the mobile phase. Inject 20 l
of the test solution, 20 l of reference solution (a) and 20 l of reference solution (b). Continue the
chromatography of the test solution for 1.5 times the retention time of pseudoephedrine. In the
chromatogram obtained with the test solution: the area of any peak due to ephedrine is not greater
than the area of the principal peak in the chromatogram obtained with reference solution (a) (1 per
cent); the area of any peak, apart from the principal peak and any peak due to ephedrine, is not
(0.5 per cent) and the sum of the areas of such peaks is not greater than twice the area of the principal peak in the chromatogram obtained with reference solution (b) (1 per cent). Disregard any peak
with an area less than 0.1 times that of the principal peak in the chromatogram obtained with
100C to 105C.
ASSAY
1 ml of 0.1M sodium hydroxide is equivalent to 20.17 mg of C10H16ClNO.
STORAGE
IMPURITIES
A. ephedrine.
__________________________________________________________________________________________________________ Ph Eur
34-15
Psyllium Seed
Psyllium Seed complies with the requirements of the 3rd edition of the European Pharmacopoeia [0858].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Psyllium seed consists of the ripe, whole, dry seeds of Plantago afra L. (Plantago psyllium L.) or
Plantago indica L. (Plantago arenaria Waldstein and Kitaibel).
CHARACTERS
Psyllium seed has a sweet taste.
It has the macroscopic characters described under Identification.
IDENTIFICATION
P. afra seeds are light brown to very dark brown but never black, smooth and shiny having an
elliptical oblong shape. They are 2 mm to 3 mm long and 0.8 mm to 1.0 mm wide, one end being
wider than the other. Towards the middle of the dorsal surface there is a fairly marked transverse
constriction of light colour. On the ventral surface, there is a linear lighter-coloured groove in the
middle of which is a clear spot corresponding to the hilum and bounded by swollen edges.
P. indica seeds are almost identical to the seeds of P. afra, but a little less shiny; they are 2 mm to
3 mm long and have a maximum diameter of 1.5 mm.
TESTS
Swelling index (2.8.4). Not less than 10.
Foreign matter (2.8.2). Not more than 1.0 per cent, determined on 10.0 g of the drug, including
greenish unripe seeds. Psyllium seed does not contain seeds having a dark central spot on the groove
(Plantago lanceolata L. and P. major L.) or seeds with brownish-grey or pinkish outer coats (P. ovata
Forssk. and P. sempervirens Crantz).
Loss on drying (2.2.32). Not more than 14.0 per cent, determined on 1.000 g of drug by drying in
an oven at 100C to 105C for 2 h.
STORAGE
Store protected from light and moisture.
__________________________________________________________________________________________________________ Ph Eur
34-16
Pyrazinamide
N
CONH2
N
C5H5N3O
123.1
98-96-4
Pyrazinamide complies with the requirements of the 3rd edition of the European Pharmacopoeia [0859].
Action and use Antituberculous.
Preparation
Pyrazinamide Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Pyrazinamide contains not less than 99.0 per cent and not more than the equivalent of 100.5 per cent
of pyrazine-2-carboxamide, calculated with reference to the anhydrous substance.
CHARACTERS
A white, crystalline powder, sparingly soluble in water, slightly soluble in alcohol and in methylene
chloride, very slightly soluble in ether.
IDENTIFICATION
B. Dissolve 50.0 mg in water R and dilute to 100.0 ml with the same solvent (solution (a)). Dilute
1.0 ml of solution (a) to 10.0 ml with water R. Examined between 290 nm and 350 nm (2.2.25), the
solution shows an absorption maximum at 310 nm. Dilute 2.0 ml of solution (a) to 100.0 ml with
water R. Examined between 230 nm and 290 nm, the solution shows an absorption maximum at
obtained with pyrazinamide CRS. Examine the substances prepared as discs. If the spectra obtained
alcohol R, evaporate to dryness and record new spectra using the residues.
D. Dissolve 0.1 g in 5 ml of water R. Add 1 ml of ferrous sulphate solution R2. The solution becomes
orange. Add 1 ml of dilute sodium hydroxide solution R. The solution becomes dark blue.
TESTS
Acidity or alkalinity To 25 ml of solution S add 0.05 ml of phenolphthalein solution R1 and 0.2 ml
of 0.01M sodium hydroxide. The solution is red. Add 1.0 ml of 0.01M hydrochloric acid. The solution is
colourless. Add 0.15 ml of methyl red solution R. The solution is red.
coating substance.
methanol R and 9 volumes of methylene chloride R. Dilute 1 ml of the solution to 10 ml with a mixture
of 1 volume of methanol R and 9 volumes of methylene chloride R.
Reference solution (b). Dissolve 10 mg of nicotinic acid CRS in a mixture of 1 volume of methanol R and
9 volumes of methylene chloride R, add 1 ml of the test solution and dilute to 10 ml with the same
plate to dry in air and examine immediately in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with the test solution, apart from the principal spot, is not more intense than the spot
34-17
in the chromatogram obtained with reference solution (a) (0.2 per cent). The test is not valid unless
the chromatogram obtained with reference solution (b) shows two clearly separated principal spots.
of water.
ASSAY
STORAGE
IMPURITIES
N
COOH
A. pyrazine-2-carboxylic acid.
__________________________________________________________________________________________________________ Ph Eur
34-18
Pyridostigmine Bromide
Me
N+
Br
OCONMe2
C9H13BrN2O2
261.1
101-26-8
Pyridostigmine Bromide complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Pyridostigmine Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Pyridostigmine bromide contains not less than 98.5 per cent and not more than the equivalent of
101.0 per cent of 3-(dimethylcarbamoyloxy)-1-methylpyridinium bromide, calculated with reference
CHARACTERS
A white or almost white, crystalline, deliquescent powder, very soluble in water and in alcohol,
IDENTIFICATION
obtained with pyridostigmine bromide CRS.
B. It gives reaction (a) of bromides (2.3.1).
TESTS
Acidity or alkalinity To 40 ml of solution S add a few drops of methyl red solution R. To 20 ml of
this solution add 0.2 ml of 0.02M sodium hydroxide. The solution is yellow. To the other 20 ml add
0.2 ml of 0.02M hydrochloric acid. The solution is red.
Test solution. Dissolve 50 mg of the substance to be examined in the mobile phase at about 40C.
Allow to cool and dilute to 50.0 ml with the same solvent.
Reference solution (a). Dissolve 4 mg of pyridostigmine impurity A CRS and 4 mg of pyridostigmine
bromide CRS in the mobile phase and dilute to 100.0 ml with the same solvent. Dilute 5.0 ml to
a stainless steel column 0.25 m long and 4.0 mm in internal diameter packed with base
deactivated octadecylsilyl silica gel for chromatography R (5 m to 10 m),
70 volumes of a 4.33 g/l solution of sodium dodecyl sulphate R, previously adjusted to pH 2.0 with
phosphoric acid R,
Inject 20 l of reference solution (a). The test is not valid unless in the chromatogram obtained, the
resolution between the peaks due to pyridostigmine and pyridostigmine impurity A is at least 1.5.
Inject separately 20 l of the test solution, 20 l of reference solution (b) and 20 l of reference
solution (c). Continue the chromatography for twice the retention time of pyridostigmine. In the
chromatogram obtained with the test solution: the area of any peak apart from the principal peak is
34-19
solution (b) (0.4 per cent), at most one such peak has an area greater than the area of the principal
peak in the chromatogram obtained with reference solution (b) (0.2 per cent) and at most one further
peak has an area greater than half of the area of the principal peak in the chromatogram obtained
with reference solution (b) (0.1 per cent); the sum of the area of all peaks apart from the principal
peak is not greater than 2.5 times the area of the principal peak in the chromatogram obtained with
reference solution (b) (0.5 per cent). Disregard any peak due to the solvent and any peak with an
area less than the area of the principal peak in the chromatogram obtained with reference solution
(c).
100C to 105C.
ASSAY
Dissolve 0.230 g in 10 ml of anhydrous acetic acid R. Add 40 ml of acetic anhydride R. Titrate with
1 ml of 0.1M perchloric acid is equivalent to 26.11 mg of C9H13BrN2O2.
STORAGE
airtight, tamper-proof container, protected from light.
LABELLING
The label states, where applicable, that the substance is sterile.
IMPURITIES
N
O
O
NMe2
A. pyridin-3-yl dimethylcarbamate,
Me
N+
OH
B. 3-hydroxy-1-methylpyridinium.
__________________________________________________________________________________________________________ Ph Eur
34-20
Pyridoxine Hydrochloride
1/01
Me
N
,HCl
HO
CH2OH
CH2OH
C8H11NO3,HCl
205.6
58-56-0
Pyridoxine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Used in treatment of sideroblastic anaemias.
Preparations
Pyridoxine Tablets
When vitamin B6 is prescribed or demanded, Pyridoxine Hydrochloride shall be dispensed or
supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Pyridoxine hydrochloride contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of (5-hydroxy-6-methylpyridine-3,4-diyl)dimethanol hydrochloride, calculated with
CHARACTERS
A white or almost white, crystalline powder, freely soluble in water, slightly soluble in alcohol.
IDENTIFICATION
A. Dilute 1.0 ml of solution S (see Tests) to 50.0 ml with 0.1M hydrochloric acid (solution (a)). Dilute
1.0 ml of solution (a) to 100.0 ml with 0.1M hydrochloric acid. Examined between 250 nm and
350 nm (2.2.25), the solution shows an absorption maximum at 288 nm to 296 nm. The specific
absorbance at the maximum is 425 to 445. Dilute 1.0 ml of solution (a) to 100.0 ml with a mixture
of equal volumes of 0.025M potassium dihydrogen phosphate solution and 0.025M disodium hydrogen
phosphate solution (2.2.3). Examined between 220 nm and 350 nm, the solution shows two absorption
maxima, at 248 nm to 256 nm and at 320 nm to 327 nm. The specific absorbances at the maxima
are 175 to 195 and 345 to 365, respectively.
obtained with pyridoxine hydrochloride CRS.
D. Solution S gives reaction (a) of chlorides (2.3.1).
TESTS
the same solvent.
Reference solution (a). Dissolve 0.10 g of pyridoxine hydrochloride CRS in water R and dilute to 10 ml
34-21
Reference solution (b). Dilute 2.5 ml of test solution (a) to 100 ml with water R. Dilute 1 ml of this
Apply to the plate 2 l of each solution. Develop in an unsaturated tank over a path of 15 cm using a
mixture of 9 volumes of concentrated ammonia R, 13 volumes of methylene chloride R, 13 volumes of
tetrahydrofuran R and 65 volumes of acetone R. Allow the plate to dry in air and spray with a 50 g/l
solution of sodium carbonate R in a mixture of 30 volumes of alcohol R and 70 volumes of water R. Dry
the plate in a current of air, spray with a 1 g/l solution of dichloroquinonechlorimide R in alcohol R and
examine the chromatograms immediately. Any spot in the chromatogram obtained with test solution
with reference solution (b) (0.25 per cent). Disregard any spots remaining on the starting line.
100C to 105C.
ASSAY
STORAGE
__________________________________________________________________________________________________________ Ph Eur
34-22
Pyrimethamine
Cl
Et
N
H2N
C12H13ClN4
NH2
248.7
58-14-0
Pyrimethamine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0288].
Preparation
Pyrimethamine Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Pyrimethamine contains not less than 99.0 per cent and not more than the equivalent of 101.0 per
cent of 2,4-diamino-5-(4-chlorophenyl)-6-ethylpyrimidine, calculated with reference to the dried
substance.
CHARACTERS
An almost white, crystalline powder or colourless crystals, practically insoluble in water, slightly
soluble in alcohol, very slightly soluble in ether.
IDENTIFICATION
B. Dissolve 0.14 g in ethanol R and dilute to 100.0 ml with the same solvent. Dilute 10.0 ml of this
solution to 100.0 ml with 0.1M hydrochloric acid. Dilute 10.0 ml of this solution to 100.0 ml with
0.1M hydrochloric acid. Examined between 250 nm and 300 nm (2.2.25), the solution shows an
absorption maximum at 272 nm and an absorption minimum at 261 nm. The specific absorbance at
obtained with pyrimethamine CRS.
D. Examine the chromatograms obtained in the test for related substances in ultraviolet light at
TESTS
Solution S Shake 1.0 g with 50 ml of distilled water R for 2 min and filter.
Appearance of solution Prepare the solution immediately before use.
Dissolve 0.25 g in a mixture of 1 volume of methanol R and 3 volumes of methylene chloride R and
dilute to 10 ml with the same mixture of solvents. The solution is clear (2.2.1) and not more
intensely coloured than reference solution BY6 (Method II, 2.2.2).
Acidity or alkalinity To 10 ml of solution S add 0.05 ml of phenolphthalein solution R1. The solution is colourless. Not more than 0.2 ml of 0.01M sodium hydroxide is required to change the colour of
the indicator to pink. Add 0.4 ml of 0.01M hydrochloric acid and 0.05 ml of methyl red solution R. The
solution is red or orange.
coating substance.
methanol R and 9 volumes of chloroform R and dilute to 25 ml with the same mixture of solvents.
34-23
Reference solution (a). Dissolve 0.1 g of pyrimethamine CRS in a mixture of 1 volume of methanol R
Reference solution (b). Dilute 2.5 ml of test solution (a) to 100 ml with a mixture of 1 volume of
methanol R and 9 volumes of chloroform R. Dilute 1 ml of the solution to 10 ml with a mixture of 1
volume of methanol R and 9 volumes of chloroform R.
4 volumes of chloroform R, 8 volumes of propanol R, 12 volumes of glacial acetic acid R and 76
volumes of toluene R. Allow the plate to dry in air. Examine in ultraviolet light at 254 nm. Any spot in
than the spot in the chromatogram obtained with reference solution (b) (0.25 per cent).
Sulphates (2.4.13). 15 ml of solution S complies with the limit test for sulphates (80 ppm). Prepare
the standard using a mixture of 2.5 ml of sulphate standard solution (10 ppm SO4 ) R and 12.5 ml of
distilled water R.
100C to 105C for 4 h.
ASSAY
Dissolve 0.200 g in 25 ml of anhydrous acetic acid R, heating gently. Cool. Titrate with 0.1M perchloric
acid determining the end-point potentiometrically (2.2.20).
STORAGE
__________________________________________________________________________________________________________ Ph Eur
34-24
Pyroxylin
Cellulose Nitrate
Definition Pyroxylin is a nitrated cellulose obtained by the action of a mixture of nitric and sulphuric
acids on wood pulp or cotton linters that have been freed from fatty matter. It must be damped with
not less than 25% by weight of Isopropyl Alcohol or of Industrial Methylated Spirit.
CAUTION Compliance is required with the provisions of relevant legislation relating to the storage of, use of
and containers used for Pyroxylin.
In the following tests, particular care should be exercised when drying pyroxylin. The material so obtained is
explosive and sensitive to ignition by impact or friction and it should be handled as carefully as possible.
Characteristics White or almost white, cuboid granules or fibrous material, the latter resembling
absorbent cotton but harsher to the touch and more powdery. Both the granules and the fibrous
material appear moist and smell strongly of the damping fluid. Highly flammable.
Soluble in glacial acetic acid and in acetone.
Identification Add water to a solution in acetone. A white, viscid mass is readily precipitated.
Clarity and colour of solution Dissolves at 20 in a mixture of 1 volume of ethanol (90%) and 3
volumes of ether to produce an almost clear and colourless to pale straw-coloured solution.
Kinematic viscosity 1160 to 2900 mm2s-1 when determined in the following manner. To 20 g,
previously dried to constant weight by heating on a water bath at 80 and allowing to cool in a
desiccator over silica gel, add 200 ml of a mixture of 19 volumes of acetone and 1 volume of water.
Shake vigorously to prevent the formation of aggregates of partly solvated pyroxylin and rotate
suitably to obtain a homogeneous solution. Measure the viscosity of the solution within 48 hours
using a falling sphere viscometer complying with British Standard 188: 1977 (Methods for the determination of viscosity of liquids). Fill the fall tube with the solution being examined to about 10 mm
above the 220-mm mark, place vertically in the bath and allow to stand for air bubbles to clear and
for temperature equilibrium to be attained. Clean the sphere, immerse it in a portion of the liquid
being examined maintained at a temperature of 19.9 to 20.1 and when it is at this temperature
introduce it, without wiping, into the delivery tube. Observe the time for the lowest part of the sphere
to pass through the planes of the tops of the 175-mm mark and the 25-mm mark, using a telescope
or other suitable device to avoid errors due to parallax. The average of three readings concordant to
within 0.5% is taken as the time of fall. Calculate the kinematic viscosity () in square millimetres per
second (mm2s-1) from the expression:
=
d 2 g( )
0.867
0.18v
where d = the diameter of the sphere in cm,

= density of the sphere in g cm-3
= density of the liquid being tested in g cm-3
v = velocity of fall in cm s-1,
g = local acceleration due to gravity in cm s-2.
Nitrogen 11.7 to 12.2%, calculated with reference to the material dried to constant weight by heating on a water bath at 80 and allowing to cool in a desiccator over silica gel, when determined by the
following method. Transfer 0.4 g of the dried substance to a 750-ml round-bottomed flask using
60 ml of water, add 20 ml of hydrogen peroxide solution (20 vol) and then add slowly, with shaking,
50 ml of a 30% w/w solution of potassium hydroxide and 5 ml of ethanol (96%). Agitate slowly for 3
hours or until the substance being examined has dissolved. Add 3 g of Devardas alloy and immediately connect the flask to the spray trap of an ammonia-distillation apparatus, the receiver of which
contains 50 ml of 0.1M hydrochloric acid VS and 0.15 ml of methyl red solution. Allow the reaction to
proceed until no further evolution of gas occurs and then heat the contents of the flask to boiling and
distil carefully until 30 ml remains in the round-bottomed flask. Rinse the delivery tube into the
receiver with water and titrate the combined distillate and rinsings with 0.1M sodium hydroxide VS.
Repeat the operation without the substance being examined. The difference between the titrations
represents the amount of acid required to neutralise the ammonia formed. Each ml of 0.1M hydrochloric acid VS is equivalent to 1.401 mg of nitrogen.
Storage Pyroxylin should be kept in a well-closed container, loosely packed, protected from light and
stored at a temperature not exceeding 15, remote from fire. The container should be suitably
designed to disrupt should the internal pressure reach or exceed 1400 kPa. The amount of damping
fluid must not be allowed to fall below 25% w/w; should this happen, the material should be either
rewetted or used immediately for the preparation of Collodion.
Preparation
Flexible Collodion
34-25
Quillaia
Quillaia Bark
Definition Quillaia is the dried inner part of the bark of Quillaja saponaria Molina and of other
species of Quillaja.
Characteristics Odourless or almost odourless; dust strongly sternutatory.
Macroscopical Pieces flat, up to about 1 metre long, 10 to 20 cm broad and 3 to 10 mm, usually
6 mm, thick. Outer surface brownish white or pale reddish brown, longitudinally striated or coarsely
reticulated, with occasional blackish brown patches of adherent outer bark; inner surface yellowish
white, smooth and very hard; fracture splintery and laminated, the broken surface showing numerous
large prisms of calcium oxalate as glistening points. Smoothed transversely cut surface appearing
chequered, with delicate radial lines representing medullary rays and tangential lines formed by
alternating tangential bands of fibrous and non-fibrous phloem.
Microscopical Outer bark, when present, consisting of reddish brown cork cells alternating with bands
of brown parenchyma containing numerous groups of phloem fibres and large prisms of calcium
oxalate. Inner bark consisting of alternating bands of tortuous fibres, irregularly enlarged at intervals,
about 500 to 1000 m long and 20 to 50 m wide and of sieve tissue mixed with parenchyma.
Medullary rays mostly three to four, but sometimes up to six cells wide, with occasional pitted,
subrectangular sclereids adjacent to the bundles of phloem fibres. Starch granules 5 to 20 m, usually
about 10 m, in diameter and prisms of calcium oxalate usually 50 to 170 m long and up to 30 m
wide present in the parenchymatous cells.
Extractive soluble in ethanol (45%) Not less than 22.0%, Appendix XI B.
Acid-insoluble ash Not more than 1.0%, Appendix XI K.
Foreign matter Not more than 2.0%, Appendix XI D.
Action and use Emulsifying agent.
Preparation
Quillaia Liquid Extract
When Powdered Quillaia is prescribed or demanded, material complying with the appropriate
requirements above shall be dispensed or supplied.
34-26
Quinidine Bisulphate
OMe
H H
H
CH2
N
H
OH
,H2SO4
N
C20H24N2O2,H2SO4
422.5
747-45-5
Definition Quinidine Bisulphate is (8R,9S)-6-methoxycinchonan-9-ol hydrogen sulphate. It

contains not less than 98.5% and not more than 101.5% of alkaloid hydrogen sulphates, calculated
as C20H24N2O2,H2SO4 with reference to the anhydrous substance.
Characteristics Colourless crystals; odourless or almost odourless.
Freely soluble in water and in ethanol (96%); practically insoluble in ether.
Identification
A. Carry out the method for thin-layer chromatography, Appendix III A, using silica gel G as the coating substance and a mixture of 15 volumes of diethylamine, 36 volumes of ether and 60 volumes of
toluene as the mobile phase. Apply separately to the plate 4 l of each of three solutions in methanol
containing (1) 1.0% w/v of the substance being examined, (2) 1.0% w/v of quinidine sulphate EPCRS
and (3) 1.0% w/v each of quinidine sulphate EPCRS and quinine sulphate EPCRS. After removal of the
plate, dry it in a current of air for 15 minutes and repeat the development. Dry the plate at 105 for
30 minutes, allow it to cool and spray with iodoplatinate reagent. The principal spot in the chromatogram obtained with solution (1) is similar in position, colour and size to that in the chromatogram
obtained with solution (2). The test is not valid unless the chromatogram obtained with solution (3)
B. Complies with the test for Acidity.
Specific optical rotation In a 2% w/v solution in 0.1M hydrochloric acid, +246 to +258, Appendix
V F, determined using a 2-dm layer and calculated with reference to the anhydrous substance.
Other cinchona alkaloids Carry out the method for liquid chromatography, Appendix III D, using
the following solutions. For solution (1) dissolve 20 mg of the substance being examined, with gentle
heating if necessary, in 5 ml of the mobile phase and dilute to 10 ml with the mobile phase. Prepare
solutions (2) and (3) in the same manner using quinine sulphate EPCRS and quinidine sulphate EPCRS
respectively in place of the substance being examined. Solution (4) is a mixture of equal volumes of
solutions (2) and (3). For solution (5) dilute 1 volume of solution (2) to 10 volumes with the mobile
phase and dilute 1 volume of the resulting solution to 50 volumes with the mobile phase. Solution (6)
contains 0.10% w/v of thiourea in the mobile phase.
The chromatographic procedure may be carried out using (a) a stainless steel column (15 to
25 cm 4.6 mm) packed with stationary phase C (5 m or 10 m) (Hypersil ODS 5 m is suitable),
(b) as the mobile phase with a flow rate of 1.5 ml per minute a solution prepared by dissolving 6.8 g
of potassium dihydrogen orthophosphate and 3.0 g of hexylamine in 700 ml of water, adjusting the pH to
2.8 with 1M orthophosphoric acid, adding 60 ml of acetonitrile and diluting to 1000 ml with water and
(c) a detection wavelength of 250 nm for recording the chromatogram obtained with solution (6) and
316 nm for the other solutions.
Inject separately 10 l of each of solutions (3) and (6). If necessary adjust the concentration of
acetonitrile in the mobile phase so that in the chromatogram obtained with solution (3) the capacity
factor of the peak due to quinidine is 3.5 to 4.5, VO being calculated from the peak due to thiourea in
the chromatogram obtained with solution (6). Inject 10 l of each of solutions (2), (3), (4) and (5).
The chromatogram obtained with solution (2) shows a principal peak due to quinine and a peak due
to dihydroquinine with a retention time relative to quinine of about 1.4. The chromatogram obtained
with solution (3) shows a principal peak due to quinidine and a peak due to dihydroquinidine, with a
retention time relative to quinine of about 1.2. The chromatogram obtained with solution (4) shows
four peaks due to quinine, dihydroquinine, quinidine and dihydroquinidine which are identified by
comparison of their retention times with those of the corresponding peaks in the chromatograms
obtained with solutions (2) and (3).
The test is not valid unless (a) in the chromatogram obtained with solution (4) the resolution factor
between the peaks due to quinine and quinidine is at least 1.5 and the resolution factor between the
peaks due to dihydroquinidine and quinine is at least 1.0 and (b) the signal-to-noise ratio of the principal peak in the chromatogram obtained with solution (5) is at least 5.
34-27
Inject 10 l of solution (1) and allow the chromatography to proceed for 2.5 times the retention
time of the principal peak. Calculate the percentage content of related substances by normalisation,
disregarding any peaks the areas of which are less than that of the peak in the chromatogram obtained
with solution (5) (0.2%). The content of dihydroquinidine is not greater than 15%, the content of
any related substance eluting before quinidine is not greater than 5% and the content of any other
related substance is not greater than 2.5%.
Titratable cation 75.3 to 79.6%, calculated with reference to the anhydrous substance, when
determined by the following method. To the combined aqueous solutions reserved in the Assay add
0.1 ml of phenolphthalein solution R1 and titrate with 0.1M hydrochloric acid VS. Each ml of 0.1M
sodium hydroxide VS is equivalent to 16.32 mg of [C20H26N2O2]2+.
Assay Dissolve 0.45 g in 15 ml of water, add 25 ml of 0.1M sodium hydroxide VS and extract with
three 25-ml quantities of chloroform. Wash each chloroform extract successively with the same 20 ml
of water, combine the aqueous solution and reserve for the test for Titratable cation. Dry the chloroform extracts with anhydrous sodium sulphate, evaporate to dryness at a pressure of 2 kPa and dissolve
the residue in 50 ml of anhydrous acetic acid. Carry out Method I for non-aqueous titration, Appendix
VIII A, using crystal violet solution as indicator. Each ml of 0.1M perchloric acid VS is equivalent to
21.13 mg of C20H24N2O2,H2SO4.
Storage Quinidine Bisulphate should be kept in a well-closed container and protected from light.
34-28
Quinidine Sulphate
OMe
HO
,H 2SO4
CH2
N
H
N
H
2
(C20H24N2O2)2H2SO4,2H2O
783
6591-63-5
Quinidine Sulphate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0017].
Preparation
Quinidine Sulphate Tablets.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Quinidine sulphate contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of alkaloid monosulphates, calculated as bis[(S)-(6-methoxyquinolin-4-yl)[(2R,4S,5R)-5-ethenyl-1-azabicyclo[2.2.2]oct-2-yl]methanol] sulphate, with reference to the dried
substance.
CHARACTERS
A white or almost white, crystalline powder or silky, colourless needles, slightly soluble in water,
soluble in boiling water and in alcohol, practically insoluble in acetone.
IDENTIFICATION
A. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel G plate R.
the same solvent.
Reference solution. Dissolve 0.10 g of quinidine sulphate CRS in methanol R and dilute to 10 ml with the
same solvent.
of diethylamine R, 24 volumes of ether R and 40 volumes of toluene R. Dry the plate in a current of air
for 15 min and repeat the development. Dry the plate at 105C for 30 min, allow to cool and spray
with iodoplatinate reagent R. The principal spot in the chromatogram obtained with the test solution is
similar in position, colour and size to the principal spot in the chromatogram obtained with the
reference solution.
B. Dissolve about 5 mg in 5 ml of water R. Add 0.2 ml of bromine water R and 1 ml of dilute
ammonia R2. A green colour develops.
C. Dissolve 0.1 g in 3 ml of dilute sulphuric acid R and dilute to 100 ml with water R. When examined
in ultraviolet light at 366 nm, an intense blue fluorescence appears which disappears almost
completely on addition of 1 ml of hydrochloric acid R.
D. Dissolve about 50 mg in 5 ml of hot water R, cool, add 1 ml of silver nitrate solution R1 and stir
with a glass rod. After a few minutes, a white precipitate is formed that dissolves on the addition of
dilute nitric acid R.
F. It complies with the test for pH (see Tests).
TESTS
Solution S Dissolve 0.500 g in 0.1M hydrochloric acid and dilute to 25.0 ml with the same acid.
34-29
Other cinchona alkaloids Examine by liquid chromatography (2.2.29).
Test solution. Dissolve 20 mg of the substance to be examined, with gentle heating if necessary, in
5 ml of the mobile phase and dilute to 10 ml with the mobile phase.
Reference solution (a). Dissolve 20 mg of quinine sulphate CRS, with gentle heating if necessary, in 5 ml
of the mobile phase and dilute to 10 ml with the mobile phase.
Reference solution (b). Dissolve 20 mg of quinidine sulphate CRS, with gentle heating if necessary, in
Reference solution (c). To 1 ml of reference solution (a) add 1 ml of reference solution (b).
Reference solution (d). Dilute 1.0 ml of reference solution (a) to 10.0 ml with the mobile phase. Dilute
Reference solution (e). Dissolve 10 mg of thiourea R in the mobile phase and dilute to 10 ml with the
mobile phase.
a stainless steel column 0.15 m to 0.25 m long and 4.6 mm in internal diameter packed with
octadecylsilyl silica gel for chromatography R (5 m or 10 m),
potassium dihydrogen phosphate R and 3.0 g of hexylamine R in 700 ml of water R, adjust to pH
2.8 with dilute phosphoric acid R, add 60 ml of acetonitrile R and dilute to 1000 ml with water R,
as detector a spectrophotometer set at 250 nm for recording the chromatogram obtained with
reference solution (e) and at 316 nm for the other solutions.
Inject 10 l of reference solution (b) and 10 l of reference solution (e). If necessary, adjust the
concentration of acetonitrile in the mobile phase so that, in the chromatogram obtained with reference solution (b), the mass distribution factor of the peak corresponding to quinidine is 3.5 to 4.5, tR
being calculated from the peak corresponding to thiourea in the chromatogram obtained with
reference solution (e).
Inject 10 l each of reference solutions (a), (b), (c) and (d). The chromatogram obtained with
reference solution (a) shows a principal peak corresponding to quinine and a peak corresponding to
dihydroquinine, with a retention time relative to quinine of about 1.4. The chromatogram obtained
with reference solution (b) shows a principal peak corresponding to quinidine and a peak corresponding to dihydroquinidine, with a retention time relative to quinidine of about 1.2. The chromatogram
obtained with reference solution (c) shows four peaks corresponding to quinidine, quinine,
dihydroquinidine and dihydroquinine which are identified by comparison of their retention times
with those of the corresponding peaks in the chromatograms obtained with reference solutions (a)
and (b).
The test is not valid unless: in the chromatogram obtained with reference solution (c), the resolution between the peaks corresponding to quinine and quinidine is at least 3.0 and the resolution
between the peaks corresponding to dihydroquinidine and quinine is at least 2.0; and the chromatogram obtained with reference solution (d) shows a principal peak with a signal-to-noise ratio of at
least four.
Inject 10 l of the test solution and record the chromatogram for 2.5 times the retention time of
the principal peak. Calculate the percentage content of related substances from the areas of the peaks
in the chromatogram obtained with the test solution by the normalisation procedure, disregarding
any peaks with an area less than that of the peak in the chromatogram obtained with reference
solution (d). The content of dihydroquinidine is not greater than 15 per cent; the content of any
related substance eluted before quinidine is not greater than 5 per cent; and the content of any other
related substance is not greater than 2.5 per cent.
Boron Avoid where possible the use of glassware.
Test solution. Dissolve 1.00 g in a mixture of 0.5 ml of hydrochloric acid R and 4.0 ml of water R.
Reference solution. Dissolve 0.572 g of boric acid R in water R and dilute to 1000.0 ml with the same
solvent. Dilute 5.0 ml to 100.0 ml with water R. To 1.0 ml of this solution add 3.0 ml of water R and
0.5 ml of hydrochloric acid R.
Blank solution. Add 0.5 ml of hydrochloric acid R to 4.0 ml of water R.
Add 3.0 ml of a 100 g/l solution of 2-ethylhexane-1,3-diol R in methylene chloride R to the test solution,
to the reference solution and to the blank solution and shake for 1 min. Allow to stand for 6 min. To
1.0 ml of the lower layer, add 2.0 ml of a 3.75 g/l solution of curcumin R in anhydrous acetic acid R
and 0.3 ml of sulphuric acid R. Mix and after 20 min add 25.0 ml of alcohol R. Mix. The colour of the
blank solution is yellow. Any red colour in the test solution is not more intense than that in the
reference solution (5 ppm of B).
130C.
ASSAY
Dissolve 0.200 g in 20 ml of acetic anhydride R. Titrate with 0.1M perchloric acid, using 0.15 ml of
naphtholbenzein solution R as indicator.
34-30
STORAGE
IMPURITIES
A. quinine,
HO H
H
CH2
N
N
B. (S)-(quinolin-4-yl)[(2R,4S,5R)-5-ethenyl-1-azabicyclo-[2.2.2]oct-2-yl]methanol
(cinchonine),
OMe
HO H
H Et
N
C. (S)-(6-methoxyquinolin-4-yl)[(2R,4S,5R)-5-ethyl-1-azabicyclo[2.2.2]oct-2-yl]methanol
(dihydroquinidine).
__________________________________________________________________________________________________________ Ph Eur
34-31
Quinine Bisulphate
OMe
HO
H H
CH2
N
N
C20H24N2O2,H2SO4,7H2O
,H2SO4
H
548.6
549-56-4
Definition Quinine Bisulphate is (8S,9R)-6-methoxycinchonan-9-ol hydrogen sulphate

heptahydrate. It contains not less than 98.5% and not more than 101.5% of alkaloid hydrogen
sulphates, calculated as C20H24N2O2,H2SO4 with reference to the anhydrous substance.
Characteristics Colourless crystals or a white, crystalline powder; efflorescent in dry air.
Freely soluble in water; sparingly soluble in ethanol (96%).
Identification
containing (1) 1.0% w/v of the substance being examined, (2) 1.0% w/v of quinine sulphate EPCRS
Specific optical rotation In a 3% w/v solution in 0.1M hydrochloric acid, 208 to 216, calculated with reference to the anhydrous substance, Appendix V F.
34-32
with solution (5) (0.2%). The content of dihydroquinine is not greater than 10%, the content of any
related substances eluting before quinine is not greater than 5% and the content of any other related
substances is not greater than 2.5%.
Titratable cation 75.3 to 79.6%, calculated with reference to the anhydrous substance, when
determined by the following method. Add to the combined aqueous solutions reserved in the Assay
0.1 ml of phenolphthalein solution R1 and titrate with 0.1M hydrochloric acid VS. Each ml of 0.1M
sodium hydroxide VS is equivalent to 16.32 mg of [C20H26N2O2]2+.
Assay Dissolve 0.45 g in 15 ml of water. Add 25 ml of 0.1M sodium hydroxide VS and extract with
three 25-ml quantities of chloroform. Wash the combined chloroform extracts with 20 ml of water,
combine the aqueous solutions and reserve for the test for Titratable cation. Dry the chloroform
extracts with anhydrous sodium sulphate, evaporate to dryness at a pressure of 2 kPa and dissolve the
residue in 50 ml of anhydrous acetic acid. Carry out method I for non-aqueous titration, Appendix
VIII A, using crystal violet solution as indicator. Each ml of 0.1M perchloric acid VS is equivalent to
21.13 mg of C20H24N2O2,H2SO4.
Storage Quinine Bisulphate should be kept in a well-closed container and protected from light.
Preparation
Quinine Bisulphate Tablets
34-33
Quinine Dihydrochloride
OMe
HO
H H
CH2
N
N
C20H24N2O2,2HCl
,2HCl
H
397.3
60-93-5
Definition Quinine Dihydrochloride is (8S,9R)-6-methoxycinchonan-9-ol dihydrochloride. It

contains not less than 99.0% and not more than 101.0% of alkaloid dihydrochlorides, calculated as
C20H24N2O2,2HCl, with reference to the dried substance.
Very soluble in water; freely soluble in ethanol (96%).
Identification
containing (1) 1.0% w/v of the substance being examined, (2) 1.0% w/v of quinine sulphate EPCRS
Specific optical rotation In a 3% w/v solution in 0.1M hydrochloric acid, 223 to 229 calculated
with reference to the dried substance, Appendix V F.
Barium To 15 ml of a 2.0% w/v solution add 1 ml of 1M sulphuric acid. The solution remains clear
for at least 15 minutes.
Sulphate 0.125 g complies with the limit test for sulphates, Appendix VII (0.12%).
Dihydroquinine dihydrochloride Not more than 10.0%, calculated with reference to the dried
substance, when determined by the following method. Dissolve 0.2 g in 20 ml of water and add 0.5 g
of potassium bromide and 15 ml of 2M hydrochloric acid. Titrate slowly with 0.0167M potassium bromate
VS, using methyl red solution as indicator, until a yellow colour is produced. Add a solution of 0.5 g of
potassium iodide in 200 ml of water and stopper the flask immediately. Allow to stand in the dark for 5
minutes and titrate with 0.1M sodium thiosulphate VS using starch solution, added towards the end of
the titration, as indicator. Repeat the operation without the substance being examined. Each ml of
0.0167M potassium bromate VS is equivalent to 19.87 mg of C20H24N2O2,2HCl. Calculate the
content of dihydroquinine dihydrochloride by subtracting the result from the assay result.
34-34
with solution (5). The content of dihydroquinine is not greater than 10%, the content of any related
substance eluting before quinine is not more than 5% and the content of any other related substance
is not more than 2.5%.
1 g.
Titratable cation 79.7 to 84.2%, calculated with reference to the dried substance, when determined
by the following method. Dissolve 0.4 g in 10 ml of water, add 40 ml of methanol and titrate with
0.1M sodium hydroxide VS using phenolphthalein solution R1 as indicator. Each ml of 0.1M sodium
hydroxide VS is equivalent to 16.32 mg of [C20H26N2O2]2+.
Assay Dissolve 0.3 g in a mixture of 50 ml of anhydrous acetic acid and 20 ml of acetic anhydride, add
10 ml of mercury(II) acetate solution and carry out method I for non-aqueous titration, Appendix VIII A,
using crystal violet solution as indicator. Each ml of 0.1M perchloric acid VS is equivalent to 19.87 mg
of C20H24N2O2,2HCl.
Storage Quinine Dihydrochloride should be kept in a well-closed container and protected from light.
34-35
Quinine Hydrochloride
OMe
HO
H H
CH2
N
N
C20H24N2O2,HCl,2H2O
,HCl
H
396.9
6119-47-7
Quinine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Quinine hydrochloride contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of alkaloid monohydrochlorides, calculated as (R)-(6-methoxyquinolin-4-yl)[(2S,4S,5R)-5-ethenyl-1-azabicyclo[2.2.2]oct-2-yl]methanol hydrochloride, with reference to the
dried substance.
CHARACTERS
Fine, silky needles, often in clusters, colourless, soluble in water, freely soluble in alcohol.
IDENTIFICATION
the same solvent.
Reference solution. Dissolve 0.10 g of quinine sulphate CRS in methanol R and dilute to 10 ml with the
same solvent.
10 volumes of diethylamine R, 24 volumes of ether R and 40 volumes of toluene R. Dry the plate in a
current of air for 15 min and repeat the development. Dry the plate at 105C for 30 min, allow to
cool and spray with iodoplatinate reagent R. The principal spot in the chromatogram obtained with the
test solution is similar in position, colour and size to the principal spot in the chromatogram obtained
B. Dissolve about 10 mg in water R and dilute to 10 ml with the same solvent. To 5 ml of the
solution add 0.2 ml of bromine water R and 1 ml of dilute ammonia R2. A green colour develops.
completely on the addition of 1 ml of hydrochloric acid R.
D. It gives the reactions of chlorides (2.3.1).
E. It complies with the test for pH (see Tests).
TESTS
pH (2.2.3). Dilute 10 ml of solution S to 20 ml with carbon dioxide-free water R. The pH of the
dried substance.
34-36
mobile phase.
Inject 10 l of each reference solutions (a), (b), (c) and (d). The chromatogram obtained with
reference solution (a) shows a principal peak corresponding to quinine and a peak corresponding to
with reference solution (b) shows a principal peak corresponding to quinidine and a peak corresponding to dihydroquinidine with a retention time relative to quinidine of about 1.2. The chromatogram
obtained with reference solution (c) shows four peaks corresponding to quinidine, quinine,
dihydroquinidine and dihydroquinine, which are identified by comparison of their retention times
and (b).
The chromatographic system is not satisfactory unless in the chromatogram obtained with reference solution (c), the resolution between the peaks corresponding to quinine and quinidine is at least
3.0 and the resolution between the peaks corresponding to dihydroquinidine and quinine is at least
2.0 and the chromatogram obtained with reference solution (d) shows a principal peak with a signalto-noise ratio of at least four.
Inject 10 l of the test solution. Record the chromatograms for 2.5 times the retention time of the
principal peak. Calculate the percentage content of related substances from the areas of the peaks in
the chromatogram obtained with the test solution by the normalisation procedure, ignoring any peaks
with an area less than that of the peak in the chromatogram obtained with reference solution (d). The
content of dihydroquinine is not greater than 10 per cent; the content of any related substance eluted
before quinine is not greater than 5 per cent; and the content of any other related substance is not
greater than 2.5 per cent.
Sulphates (2.4.13). 15 ml of solution S complies with the limit test for sulphate (500 ppm).
Barium To 15 ml of solution S add 1 ml of dilute sulphuric acid R. After at least 15 min, any
opalescence in the solution is not more intense than that in a mixture of 15 ml of solution S and 1 ml
of distilled water R.
at 100C to 105C.
ASSAY
Dissolve 0.250 g in 50 ml of alcohol R and add 5.0 ml of 0.01M hydrochloric acid. Titrate with 0.1M
sodium hydroxide, determining the end-point potentiometrically (2.2.20). Read the volume added
between the two inflexion points.
1 ml of 0.1M sodium hydroxide is equivalent to 36.09 mg of C20H25ClN2O2
STORAGE
IMPURITIES
A. quinidine,
34-37
HO H
H
CH2
N
N
B. (R)-(quinolin-4-yl)[(2S,4S,5R)-5-ethenyl-1-azabicyclo-[2.2.2]oct-2-yl]methanol
(cinchonidine),
OMe
HO H
H Et
N
C. (R)-(6-methoxyquinolin-4-yl)[(2S,4S,5R)-5-ethyl-1-azabicyclo[2.2.2]oct-2-yl]methanol
(dihydroquinine).
__________________________________________________________________________________________________________ Ph Eur
34-38
Quinine Sulphate
OMe
H
H
CH2
N
H
,H2SO4
H
OH
N
2
(C20H24N2O2)2,H2SO4,2H2O
783
6119-70-6
Quinine Sulphate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0019].
Preparation
Quinine Sulphate Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Quinine sulphate contains not less than 99.0 per cent and not more than the equivalent of 101.0 per
cent of alkaloid monosulphates, calculated as bis[(R)-(6-methoxyquinolin-4-yl)-[(2S,4S,5R)-5ethenyl-1-azabicyclo[2.2.2]oct-2-yl]methanol] sulphate, with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder or fine, colourless needles, slightly soluble in water,
sparingly soluble in boiling water and in alcohol.
IDENTIFICATION
the same solvent.
Reference solution. Dissolve 0.10 g of quinine sulphate CRS in methanol R and dilute to 10 ml with the
same solvent.
of diethylamine R, 24 volumes of ether R and 40 volumes of toluene R. Dry the plate in a current of air
for 15 min and repeat the development. Dry the plate at 105C for 30 min, allow to cool and spray
with iodoplatinate reagent R. The principal spot in the chromatogram obtained with the test solution is
reference solution.
B. Dissolve about 5 mg in 5 ml of water R. Add 0.2 ml of bromine water R and 1 ml of dilute
ammonia R2. A green colour develops.
completely on the addition of 1 ml of hydrochloric acid R.
D. Dissolve about 45 mg in 5 ml of dilute hydrochloric acid R. The solution gives reaction (a) of
sulphates (2.3.1).
E. It complies with the test for pH (see Tests).
TESTS
Solution S Dissolve 0.500 g in 0.1M hydrochloric acid and dilute to 25.0 ml with the same acid.
pH (2.2.3). The pH of a 10 g/l suspension in water R is 5.7 to 6.6.
34-39
mobile phase.
Inject 10 l each of reference solutions (a), (b), (c) and (d). The chromatogram obtained with
reference solution (a) shows a principal peak corresponding to quinine and a peak, corresponding to
with reference solution (b) shows a principal peak corresponding to quinidine and a peak, corresponding to dihydroquinidine, with a retention time relative to quinidine of about 1.2. The chromatogram obtained with reference solution (c) shows four peaks corresponding to quinidine, quinine,
dihydroquinidine and dihydroquinine which are identified by comparison of their retention times
and (b).
The test is not valid unless: in the chromatogram obtained with reference solution (c), the resolution between the peaks corresponding to quinine and quinidine is at least 3.0 and the resolution
between the peaks corresponding to dihydroquinidine and quinine is at least 2.0; and the chromatogram obtained with reference solution (d) shows a principal peak with a signal-to-noise ratio of at
least four.
Inject 10 l of the test solution. Record the chromatograms for 2.5 times the retention time of the
principal peak. Calculate the percentage content of related substances from the areas of the peaks in
the chromatogram obtained with the test solution by the normalisation procedure, disregarding any
peaks with an area less than that of the peak in the chromatogram obtained with reference solution
(d). The content of dihydroquinine is not greater than 10 per cent; the content of any related
substance eluted before quinine is not greater than 5 per cent; and the content of any other related
substance is not greater than 2.5 per cent.
100C to 105C.
ASSAY
Dissolve 0.300 g in a mixture of 10 ml of chloroform R and 20 ml of acetic anhydride R. Titrate with
STORAGE
IMPURITIES
A. quinidine,
HO H
H
CH2
N
N
B. (R)-(quinolin-4-yl)[(2S,4S,5R)-5-ethenyl-1-azabicyclo-[2.2.2]oct-2-yl]methanol
(cinchonidine),
34-40
OMe
HO H
H Et
N
C. (R)-(6-methoxyquinolin-4-yl)[(2S,4S,5R)-5-ethyl-1-azabicyclo[2.2.2]oct-2-yl]methanol
(dihydroquinine).
__________________________________________________________________________________________________________ Ph Eur
34-41
Racementhol
Racemic Menthol
Me
H
Me
OH
H
Me
and enantiomer
C10H20O
156.3
15356-70-4
Racementhol complies with the requirements of the 3rd edition of the European Pharmacopoeia for Racemic
Menthol [0623]. These requirements are reproduced after the heading Definition below.
Action and use Decongestant.
Preparation
Menthol and Benzoin Inhalation
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Racemic menthol is a mixture of equal parts of (1R,2S, 5R)-2-isopropyl-5-methylcyclohexanol and
(1S,2R,5S)-2-isopropyl-5-methylcyclohexanol.
CHARACTERS
A free-flowing or agglomerated, crystalline powder or prismatic or acicular, colourless, shiny crystals,
practically insoluble in water, very soluble in alcohol, in ether and in light petroleum, freely soluble in
fatty oils and in liquid paraffin, very slightly soluble in glycerol.
IDENTIFICATION
Second identification: B, D.
A. It complies with the test for angle of optical rotation (see Tests).
the same solvent.
Reference solution. Dissolve 25 mg of menthol CRS in methanol R and dilute to 5 ml with the same
solvent.
volumes of ethyl acetate R and 95 volumes of toluene R. Allow the plate to dry in air until the odour of
the solvents is no longer perceptible and spray with anisaldehyde solution R. Heat at 100C to 105C
for 5 min to 10 min. The principal spot in the chromatogram obtained with the test solution is
reference solution.
C. Examine the chromatograms obtained in the test for related substances. The principal peak in the
chromatogram obtained with test solution (b) is similar in position and approximate dimensions to
the principal peak in the chromatogram obtained with reference solution (c).
D. Dissolve 0.20 g in 0.5 ml of anhydrous pyridine R. Add 3 ml of a 150 g/l solution of dinitrobenzoyl
chloride R in anhydrous pyridine R. Heat on a water-bath for 10 min. Add 7.0 ml of water R in small
quantities with stirring and allow to stand in iced water for 30 min. A precipitate is formed. Allow to
stand and decant the supernatant liquid. Wash the precipitate with two quantities, each of 5 ml, of
iced water R, recrystallise from 10 ml of acetone R, wash with iced acetone R and dry at 75C at a
pressure not exceeding 2.7 kPa for 30 min. The crystals melt (2.2.14) at 130C to 131C.
TESTS
Solution S Dissolve 2.50 g in 10 ml of alcohol R and dilute to 25.0 ml with the same solvent.
34-42
Acidity or alkalinity Dissolve 1.0 g in alcohol R and dilute to 10 ml with the same solvent. Add
0.1 ml of phenolphthalein solution R; the solution is colourless. Not more than 0.5 ml of 0.01M sodium
hydroxide is required to change the colour of the indicator to pink.
Optical rotation (2.2.7). The angle of optical rotation of solution S is +0.2 to 0.2.
Test solution (b). Dilute 1.0 ml of test solution (a) to 10.0 ml with methylene chloride R.
Reference solution (a). Dissolve 40.0 mg of the substance to be examined and 40.0 mg of isomenthol R
in methylene chloride R and dilute to 100.0 ml with the same solvent.
Reference solution (b). Dilute 0.10 ml of test solution (a) to 100.0 ml with methylene chloride R.
Reference solution (c). Dissolve 40.0 mg of menthol CRS in methylene chloride R and dilute to 100.0 ml
a glass column 2.0 m long and 2 mm in internal diameter packed with diatomaceous earth for gas
chromatography R impregnated with 15 per cent m/m of macrogol 1500 R,
nitrogen for chromatography R as carrier gas at a flow rate of 30 ml per minute,
Inject separately 1 l of each solution. Record the chromatograms for twice the retention time of
the peak corresponding to menthol. In the chromatogram obtained with test solution (a), the sum of
the areas of the peaks, apart from the principal peak, is not greater than 1 per cent of the area of the
principal peak. Disregard any peak due to the solvent and any peak whose area is less than 0.05 per
cent of the area of the principal peak. The test is not valid unless: in the chromatogram obtained with
reference solution (a) the resolution between the peaks corresponding to menthol and isomenthol is
at least 1.4 and the principal peak in the chromatogram obtained with reference solution (b) has a
signal-to-noise ratio of at least 5.
Residue on evaporation Evaporate 2.00 g on a water-bath and heat in an oven at 100C to 105C
for 1 h. The residue weighs not more than 1.0 mg (0.05 per cent).
STORAGE
Store in a well-closed container, in a cool place.
__________________________________________________________________________________________________________ Ph Eur
34-43
Racephedrine Hydrochloride
Racemic Ephedrine Hydrochloride
OH
CH3
,HCl
H
NHMe
and enantiomer
C10H15NO,HCl
201.7
134-71-4
Racephedrine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
for Racemic Ephedrine Hydrochloride [0715]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Racemic ephedrine hydrochloride contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of (1RS,2SR)-2-methylamino-1-phenylpropan-1-ol hydrochloride, calculated
CHARACTERS
A white, crystalline powder or colourless crystals, freely soluble in water, soluble in alcohol,
IDENTIFICATION
A. It complies with the test for angle of optical rotation (see Tests).
obtained with racemic ephedrine hydrochloride CRS. Examine the substances prepared as discs.
D. To 0.1 ml of solution S (see Tests) add 1 ml of water R, 0.2 ml of copper sulphate solution R and
1 ml of strong sodium hydroxide solution R. A violet colour is produced. Add 2 ml of ether R and shake.
The ether layer is purple and the aqueous layer is blue.
E. To 5 ml of solution S add 5 ml of water R. The solution gives reaction (a) of chlorides (2.3.1).
TESTS
Solution S Dissolve 5.00 g in distilled water R and dilute to 50.0 ml with the same solvent.
sodium hydroxide; the solution is yellow. Add 0.2 ml of 0.01M hydrochloric acid ; the solution is red.
Angle of optical rotation (2.2.7). +0.2 to 0.2, determined on solution S.
coating substance.
Reference solution (a). Dissolve 20 mg of racemic ephedrine hydrochloride CRS in methanol R and dilute
Reference solution (b), Dilute 1 ml of test solution (a) to 200 ml with methanol R.
5 volumes of chloroform R, 15 volumes of concentrated ammonia R and 80 volumes of 2-propanol R.
34-44
Allow the plate to dry in air. Spray with ninhydrin solution R and heat at 110C for 5 min. Any spot in
than the spot in the chromatogram obtained with reference solution (b) (0.5 per cent). Disregard any
spot of lighter colour than the background.
100C to 105C.
ASSAY
1 ml of 0.1M sodium hydroxide corresponds to 20.17 mg of C10H16ClNO.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
34-45
Ramipril
EtOOC
H H
Me
O
N
H H
N
COOH
H
H
C23H32N2O5
416.5
87333-19-5
Ramipril complies with the requirements of the 3rd edition of the European Pharmacopoeia [1368]. These
Action and use Angiotensin converting enzyme inhibitor.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ramipril contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent of
(2S,3aS,6aS)-1-[(S)-2-[[(S)-1-(ethoxycarbonyl)-3-phenylpropyl]amino]propanoyl]-octahydrocyclopenta[b]pyrrole-2-carboxylic acid, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, sparingly soluble in water, freely soluble in methanol.
IDENTIFICATION
obtained with ramipril CRS.
TESTS
The solution is clear (2.2.1) and colourless (Method II, 2.2.2).
Specific optical rotation (2.2.7). Dissolve 0.250 g in a mixture of 14 volumes of hydrochloric
acid R1 and 86 volumes of methanol R and dilute to 25.0 ml with the same mixture of solvents. The
specific optical rotation is +32.0 to +38.0, calculated with reference to the dried substance.
Reference solution (a). Dissolve 5 mg each of ramipril impurity A CRS, ramipril impurity B CRS, ramipril
impurity C CRS and ramipril impurity D CRS in 5 ml of the test solution and dilute to 10 ml with
mobile phase B.
Reference solution (b). Dilute 5.0 ml of the test solution to 100.0 ml with mobile phase B. Dilute
5.0 ml of the solution to 50.0 ml with mobile phase B.
Reference solution (c). Dilute 1.0 ml of reference solution (b) to 10.0 ml with mobile phase B.
Mobile phase A. Dissolve 2.0 g of sodium perchlorate R in a mixture of 0.5 ml of triethylamine R
and 800 ml of water R; adjust to pH 3.6 with phosphoric acid R and add 200 ml of acetonitrile R,
Mobile phase B. Dissolve 2.0 g of sodium perchlorate R in a mixture of 0.5 ml of triethylamine R
and 300 ml of water R; adjust to pH 2.6 with phosphoric acid R and add 700 ml of acetonitrile R,
34-46
Interval
(min)
Mobile phase A
(per cent V/V)

(per cent V/V)
06
67
90
10
9075
1025
isocratic
linear gradient
720
7565
2535
linear gradient
2030
6525
25
3575
75
linear gradient
2590
90
7510
10
3040
4045
4555
isocratic
linear gradient
re-equilibration

Equilibrate the column with a mixture of 90 per cent mobile phase A and 10 per cent mobile phase
B for at least 35 min. If a suitable baseline cannot be obtained, use another grade of triethylamine.
Inject 10 l of reference solution (c). Adjust the sensitivity of the system so that the chromatogram
obtained shows a visible peak. Inject 10 l of reference solution (a), 10 l of reference solution (b)
and 10 l of the test solution. The test is not valid unless: in the chromatogram obtained with
reference solution (a) the resolution between the peaks corresponding to ramipril impurity A and
ramipril is at least 3.0; in the chromatogram obtained with reference solution (c) the principal peak
has a signal-to-noise ratio of at least three; and in the chromatogram obtained with the test solution,
the symmetry factor of the principal peak is 0.8 to 2.0.
When the chromatograms are recorded in the prescribed conditions, the retention times are:
ramipril impurity A about 14 min, ramipril about 18 min, ramipril impurity B about 22 min, toluene
about 24 min, ramipril impurity C about 26 min and ramipril impurity D about 28 min.
In the chromatogram obtained with the test solution, multiply the area of any peak corresponding
to ramipril impurity C by a correction factor of 2.4.
In the chromatogram obtained with the test solution: the area of each peak corresponding to
ramipril impurity A, ramipril impurity B, ramipril impurity C and ramipril impurity D is not greater
than the area of the principal peak in the chromatogram obtained with reference solution (b) (0.5 per
cent); the area of any peak, apart from the principal peak and any peak corresponding to the
impurities A, B, C and D, is not greater than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.1 per cent); the sum of the areas of all peaks, apart
from the principal peak, is not greater than twice the area of the principal peak in the chromatogram
obtained with reference solution (b) (1 per cent). Disregard any peak with an area less than that of
the principal peak in the chromatogram obtained with reference solution (c).
Palladium Not more than 20 ppm of Pd, determined by atomic absorption spectrometry (Method I,
2.2.23).
Test solution. Dissolve 0.200 g of the substance to be examined in a mixture of 0.3 volumes of nitric
acid R and 99.7 volumes of water R and dilute to 100.0 ml with the same mixture of solvents.
Reference solutions. Use solutions containing 0.02 g, 0.03 g and 0.05 g of palladium per millilitre,
freshly prepared by dilution of palladium standard solution (0.5 ppm Pd) R with a mixture of 0.3
volumes of nitric acid R and 99.7 volumes of water R.
Modifier solution. Dissolve 0.150 g of magnesium nitrate R in a mixture of 0.3 volumes of nitric acid R
and 99.7 volumes of water R and dilute to 100.0 ml with the same mixture of solvents.
Inject separately 20 l of the test solution and 20 l of the reference solution, and 10 l of the
modifier solution. Measure the absorbance at 247.6 nm using a palladium hollow-cathode lamp as a
source of radiation, a transmission band of preferably 1 nm and a graphite tube.
under high vacuum at 60C for 4 h.
ASSAY
Dissolve 0.300 g in 25 ml of methanol R and add 25 ml of water R. Titrate with 0.1M sodium
hydroxide, determining the end-point potentiometrically (2.2.20). Carry out a blank titration.
STORAGE
34-47
IMPURITIES
Qualified impurities
MeOOC
Me
O
N
H H
N
COOH
A. (2S,3aS,6aS)-1-[(S)-2-[[(S)-1-(methoxycarbonyl)-3-phenylpropyl]amino]propanoyl]octahydrocyclopenta-[b]pyrrole-2-carboxylic acid (ramipril methyl ester),

Pr i OOC
Me
O
N
H H
N
COOH
B. (2S,3aS,6aS)-1-[(S)-2-[[(S)-1-[(1-methylethoxy)carbonyl]-3-phenylpropyl]amino]propanoyl]octahydrocyclopenta[b]pyrrole-2-carboxylic acid (ramipril isopropyl ester),

Me
EtOOC
N
H H
O
N
COOH
C. (2S,3aS,6aS)-1-[(S)-2-[[(S)-1-(ethoxycarbonyl)-3-cyclohexylpropyl]amino]propanoyl]octahydrocyclopenta[b]pyrrole-2-carboxylic acid (hexahydroramipril),

Me
EtOOC
N
O
H
H
D. ethyl (2S)-2-[(3S,5aS,8aS,9aS)-3-methyl-1,4-dioxodecahydro-1H-cyclopenta[e]pyrrol[1,2a]pyrazin-2-yl]-4-phenyl-butanoate (ramipril diketopiperazine),

Other detectable impurities
HOOC
Me
N
H H
O
N
COOH
E. (2S,3aS,6aS)-1-[(S)-2-[[(S)-1-carboxy-3-phenylpropyl]amino]propanoyl]octahydrocyclopenta[b]pyrrole-2-carboxylic acid (ramipril diacid),

Me
EtOOC
N
H
COOH
F. (S)-2-[[(S)-1-(ethoxycarbonyl)-3-phenylpropyl]amino]propanoic acid,
CH3
G. toluene,
34-48
Me
EtOOC
N
H H
N
COOH
H. (2S,3aS,6aS)-1-[(R)-2-[[(S)-1-(ethoxycarbonyl)-3-phenylpropyl]amino]propanoyl]octahydrocyclopenta[b]pyrrole-2-carboxylic acid ((R,S-S,S,S) isomer of ramipril),

Me
EtOOC
N
H H
N
COOH
I. (2S,3aS,6aS)-1-[(S)-2-[[(R)-1-(ethoxycarbonyl)-3-phenylpropyl]amino]propanoyl]octahydrocyclopenta[b]pyrrole-2-carboxylic acid ((S,R-S,S,S) isomer of ramipril),

Me
EtOOC
N
H H
N
COOH
J. (2R,3aR,6aR)-1-[(R)-2-[[(R)-1-(ethoxycarbonyl)-3-phenylpropyl]amino]propanoyl]octahydrocyclopenta[b]pyrrole-2-carboxylic acid ((R,R-R,R,R) isomer of ramipril),

HOOC
Me
O
N
O
H
H
K. (2S)-2-[(3S,5aS,8aS,9aS)-3-methyl-1,4-dioxodecahydro-1H-cyclopenta[e]pyrrolo[1,2a]pyrazin-2-yl]-4-phenylbutanoic acid (ramipril diketopiperazine acid),

Me
EtOOC
N
N
O
HO
L. ethyl (2S)-2-[(3S,5aS,8aS,9aS)-9a-hydroxy-3-methyl-1,4-dioxodecahydro-1H-cyclopenta[e]pyrrolo[1,2-a]pyrazin-2-yl]-4-phenylbutanoate (ramipril hydroxydiketopiperazine),

O
COOH
O
O
HOOC
M. (2R,3R)-2,3-di(benzoyloxy)butanedioic acid (dibenzoyltartric acid),

EtOOC
Me
N
H H
O
N
COOH
N. (2R,3aR,6aR)-1-[(S)-2-[[(S)-1-(ethoxycarbonyl)-3-phenylpropyl]amino]propanoyl]octahydrocyclopenta[b]pyrrole-2-carboxylic acid ((S,S-R,R,R) isomer of ramipril).
34-49
The following type chromatogram is given for information and guidance only; it does not form a mandatory
part of the monograph.
retention times (min):

1. ramipril impurity E
2. ramipril impurity F
3. ramipril impurity A
5. ramipril
6.01
6.90
14.44
17.50
5. ramipril impurity B
6. toluene (impurity G)
7. ramipril impurity C
8. ramipril impurity D
22.32
24.34
26.37
27.70
Fig. 1368.1 Type chromatogram for the test for related substances
A. chromatogram of a test solution spiked with the listed substnces
B. chromatogram of a blank solution
__________________________________________________________________________________________________________ Ph Eur
34-50
Ranitidine Hydrochloride
Me2N
NO2
H
N
O
S
,HCl
NHMe
C13H22N4O3S,HCl
350.9
71130-06-8
Ranitidine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparations
Ranitidine Injection
Ranitidine Oral Solution
Ranitidine Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ranitidine hydrochloride contains not less than 98.5 per cent and not more than the equivalent of
101.0 per cent of N-[2-[[[5-[(dimethylamino)methyl]furan-2-yl]methyl]thio]ethyl]-N-methyl-2nitroethene-1,1-diamine hydrochloride, calculated with reference to the dried substance.
CHARACTERS
A white or pale yellow, crystalline powder, freely soluble in water and in methanol, sparingly soluble
in ethanol, very slightly soluble in methylene chloride.
IDENTIFICATION
A. Dissolve 10 mg in water R and dilute to 100.0 ml with the same solvent. Dilute 5.0 ml of the
obtained with ranitidine hydrochloride CRS. Examine the substances as mulls in liquid paraffin R. If the
spectra show differences, dissolve 20 mg of the substance to be examined and 20 mg of the reference
substance separately in 5 ml of methanol R. Evaporate to dryness in a water-bath at 40C under
reduced pressure and with constant stirring. Dry the residues under high vacuum at 60C for 1 h and
record new spectra using the residues.
C. Examine the chromatograms obtained in the test for related substances (see Tests). The principal
TESTS
coating substance.
Reference solution (a). Dissolve 20 mg of ranitidine hydrochloride CRS in methanol R and dilute to 10 ml
Reference solution (c). Dilute 2.0 ml of test solution (b) to 100 ml with methanol R.
34-51
Reference solution (d). Dilute 1.0 ml of test solution (b) to 100 ml with methanol R.
Reference solution (e). Dilute 0.5 ml of test solution (b) to 100 ml with methanol R.
Reference solution (f). Dissolve 10 mg of ranitidine impurity A CRS in methanol R and dilute to 100 ml
Reference solution (g). Dissolve 10 mg of ranitidine impurity B CRS in methanol R and dilute to 10 ml
Reference solution (h). Dissolve 10 mg of ranitidine impurity B CRS in test solution (a) and dilute to
2 volumes of water R, 4 volumes of concentrated ammonia R1, 15 volumes of 2-propanol R and 25
volumes of ethyl acetate R. Allow the plate to dry in air and expose to iodine vapour until the spots are
clearly visible. Examine in daylight. In the chromatogram obtained with test solution (a): any spot
corresponding to ranitidine impurity A is not more intense than the spot in the chromatogram
obtained with reference solution (f) (0.5 per cent); any spot apart from the principal spot and any
spot corresponding to ranitidine impurity A is not more intense than the spot in the chromatogram
obtained with reference solution (b) (0.3 per cent); at most three such spots are more intense than
the spot in the chromatogram obtained with reference solution (d) (0.1 per cent) and at most one of
these is more intense than the spot in the chromatogram obtained with reference solution (c) (0.2 per
cent). The test is not valid unless the chromatogram obtained with reference solution (h) shows two
clearly separated spots, corresponding to ranitidine impurity B (whose Rf value is obtained from the
chromatogram obtained with reference solution (g)) and ranitidine, and the chromatogram obtained
with reference solution (e) shows a clearly visible spot.
Loss on drying (2.2.32). Not more than 0.75 per cent, determined on 1.000 g by drying under high
vacuum at 60C.
ASSAY
Dissolve 0.280 g in 35 ml of water R. Titrate with 0.1M sodium hydroxide, determining the end-point
1 ml of 0.1M sodium hydroxide is equivalent to 35.09 mg of C13H23ClN4O3S.
STORAGE
IMPURITIES
A. N,N-bis[2-[[[5-[(dimethylamino)methyl]furan-2-yl]methyl]thio]ethyl]-2-nitroethene-1,1diamine,
B. [[5-[[(2-aminoethyl)thio]methyl]furan-2-yl]methyl]dimethylamine,
C. N-[2-[[[5-[(dimethylamino)methyl]furan-2-yl]methyl]sulphinyl]ethyl]-N-methyl-2nitroethene-1,1-diamine,
D. N-[2-[[[5-[(dimethylamino)methyl]furan-2-yl]methyl]thio]ethyl]-2-nitroacetamide,
E. N-[2-[[[5-[(dimethylamino)methyl]furan-2-yl]methyl]thio]ethyl]-N-methyl-2-nitroethene1,1-diamine N-oxide,
F. [5-[(dimethylamino)methyl]furan-2-yl]methanol,
G. 3-(methylamino)-5,6-dihydro-2H-1,4-thiazin-2-one oxime,
H. N-methyl-2-nitroacetamide.
__________________________________________________________________________________________________________ Ph Eur
34-52
Refined Rapeseed Oil

Refined Rapeseed Oil complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Rapeseed oil is the fatty oil obtained from the seeds of Brassica napus L. and Brassica campestris L. by
mechanical expression or by extraction. It is then refined. A suitable antioxidant may be added.
CHARACTERS
A clear, light yellow liquid, practically insoluble in water and in alcohol, miscible with light petroleum
(bp: 40C to 60C).
IDENTIFICATION
Carry out the identification of fatty oils by thin-layer chromatography (2.3.2). The chromatogram
obtained is similar to the typical chromatogram for rapeseed oil.
TESTS
Composition of fatty acids Carry out the test for foreign fatty oils in fatty oils by gas chromatography (2.4.22). The fatty acid fraction of the oil has the following composition:
linolenic acid: 6.0 per cent to 14.0 per cent,
eicosenoic acid: not more than 5.0 per cent,
erucic acid: not more than 2.0 per cent.
STORAGE
Store in an airtight, well-filled container, protected from light.
LABELLING
The label states:
__________________________________________________________________________________________________________ Ph Eur
34-53
Products of Recombinant DNA Technology{1045}
Products of Recombinant DNA Technology comply with the requirements of the 3rd edition of the European
Pharmacopoeia [0784]. These requirements are reproduced below.
Ph Eur ___________________________________________________________________________________________________________
The statements in this monograph are intended to be read in conjunction with the individual monographs on
products of recombinant DNA (rDNA) technology in the Pharmacopoeia. The requirements do not
necessarily apply to products that are not the subject of such monographs. The monograph is not applicable to
modified live organisms that are intended to be used directly in man and animals, for example as live
vaccines.
DEFINITION
Products of rDNA technology are produced by genetic modification in which DNA coding for the
required product is introduced, usually by means of a plasmid or a viral vector, into a suitable microorganism or cell line, in which that DNA is expressed and translated into protein. The desired
product is then recovered by extraction and purification. The cell or micro-organism before harbouring the vector is referred to as the host cell, and the stable association of the two used in the
manufacturing process is referred to as the host-vector system.
PRODUCTION
Production is based on a validated seed-lot system using a host-vector combination that has been
shown to be suitable to the satisfaction of the competent authority. The seed-lot system uses a master
cell bank and a working cell bank derived from the master seed lot of the host-vector combination. A
detailed description of cultivation, extraction and purification steps and a definition of the production
batch shall be established.
The determination of the suitability of the host-vector combination and the validation of the seedlot system include the following elements.
CLONING AND EXPRESSION
The suitability of the host-vector system, particularly as regards microbiological purity, is demonstrated by:
Characterisation of the host cell, including source, phenotype and genotype, and of the cell-culture media.
Documentation of the strategy for the cloning of the gene and characterisation of the recombinant vector
including:
i the origin and characterisation of the gene;
ii nucleotide-sequence analysis of the cloned gene and the flanking control regions of the
expression vector. The cloned sequences are kept to a minimum and all relevant expressed
sequences are clearly identified and confirmed at the RNA level.
The DNA sequence of the cloned gene is normally confirmed at the seed-lot stage, up to and
beyond the normal level of population doubling for full-scale fermentation. In certain systems,
for example, where multiple copies of the gene are inserted into the genome of a continuous cell
line, it may be inappropriate to sequence the cloned gene at the production level. Under these
circumstances, Southern blot analysis of total cellular DNA or sequence analysis of the
messenger RNA (mRNA) may be helpful, particular attention being paid to the characterisation
of the expressed protein;
iii the construction, genetics and structure of the complete expression vector.
Characterisation of the host-vector system including:
i mechanism of transfer of the vector into the host cells;
ii copy number, physical state and stability of the vector inside the host cell;
iii measures used to promote and control the expression.
CELL-BANK SYSTEM
The master cell bank is a homogeneous suspension of the original cells already transformed by the
expression vector containing the desired gene, distributed in equal volumes into individual containers
for storage (for example, in liquid nitrogen). In some cases it may be necessary to establish separate
master cell banks for the expression vector and the host cells.
The working cell bank is a homogeneous suspension of the cell material derived from the master cell
bank(s) at a finite passage level, distributed in equal volumes into individual containers for storage
(for example, in liquid nitrogen).
In both cell banks, all containers are treated identically during storage and, once removed from
storage, the containers are not returned to the cell stock.
34-54
The cell bank may be used for production at a finite passage level or for continuous-culture
production.
Production at a finite passage level
This cultivation method is defined by a limited number of passages or population doublings which
must not be exceeded during production. The maximum number of cell doublings, or passage levels,
during which the manufacturing process routinely meets the criteria described below must be stated.
Continuous-culture production
By this cultivation method the number of passages or population doublings is not restricted from the
beginning of production. Criteria for the harvesting as well as for the termination of production have
to be defined by the manufacturer. Monitoring is necessary throughout the life of the culture; the
required frequency and type of monitoring will depend on the nature of the production system and
the product.
Information is required on the molecular integrity of the gene being expressed and on the
phenotypic and genotypic characteristics of the host cell after long-term cultivation. The acceptance
of harvests for further processing must be clearly linked to the schedule of monitoring applied and a
clear definition of a batch of product for further processing is required.
VALIDATION OF THE CELL BANKS
Validation of the cell banks includes:
i stability by measuring viability and the retention of the vector;
ii identity of the cells by phenotypic features;
iii where appropriate, evidence that the cell banks are free from potentially oncogenic or infective
adventitious agents (viral, bacterial, fungal or mycoplasmal). Special attention has to be given to
viruses that can commonly contaminate the species from which the cell line has been derived.
Certain cell lines contain endogenous viruses, for example, retroviruses, which may not readily
be eliminated. The expression of these organisms, under a variety of conditions known to cause
their induction, shall be tested for;
iv for mammalian cells, details of the tumorigenic potential of the cell bank shall be obtained.
CONTROL OF THE CELLS
The origin, form, storage, use and stability at the anticipated rate of use must be documented in full
for all cell banks under conditions of storage and recovery. New cell banks must be fully validated.
VALIDATION OF THE PRODUCTION PROCESS
Extraction and purification
The capacity of each step of the extraction and purification procedure to remove and/or inactivate
contaminating substances derived from the host cell or culture medium, including, in particular, virus
particles, proteins, nucleic acids and added substances, must be validated.
Validation studies are carried out to demonstrate that the production process routinely meets the
following criteria:
exclusion of extraneous agents from the product. Studies including, for example, viruses with
relevant physico-chemical features are undertaken, and a reduction capacity for such
contaminants at each relevant stage of purification is established;
adequate removal of vector, host-cell, culture medium and reagent-derived contaminants from
the product. The reduction capacity for DNA is established by spiking. The reduction of
proteins of animal origin can be determined by immunochemical methods;
maintenance within stated limits of the yield of product from the culture;
adequate stability of any intermediate of production and/or manufacturing when it is intended to
use intermediate storage during the process.
Characterisation of the substance
The identity, purity, potency and stability of the final bulk product are established initially by carrying out a wide range of chemical, physical, immunochemical and biological tests. Prior to release,
each batch of the product is tested by the manufacturer for identity and purity and an appropriate
assay is carried out.
Production consistency
Suitable tests for demonstrating the consistency of the production and purification are performed.
The tests include, especially characterisation tests, in-process controls and final-product tests, for
example:
Amino-acid composition
Partial amino-acid sequence analysis The sequence data permit confirmation of the correct
N-terminal processing and detection of loss of the C-terminal amino acids.
Peptide mapping Peptide mapping using chemical and/or enzymatic cleavage of the protein
product and analysis by a suitable method such as two-dimensional gel electrophoresis, capillary
electrophoresis or liquid chromatography must show no significant difference between the test
34-55
protein and the reference preparation. Peptide mapping can also be used to demonstrate correct
disulphide bonding.
Determination of molecular mass
Cloned-gene retention The minimum amount in percentage of the cells containing the vector or
the cloned gene after cultivation is approved by the relevant authority.
Total protein The yield of protein is determined.
Chemical purity The purity of the protein product is analysed in comparison with a reference
preparation by a suitable method such as liquid chromatography, capillary electrophoresis or sodium
dodecyl sulphate polyacrylamide gel electrophoresis.
Host-cell-derived proteins Host-cell-derived proteins are detected by immunochemical methods,
using, for example, polyclonal antisera raised against protein components of the host-vector system
used to manufacture the product, unless otherwise prescribed. The following types of procedure may
be used: liquid-phase displacement assays (for example, radio-immunoassay), liquid-phase directbinding assays and direct-binding assays using antigens immobilised on nitrocellulose (or similar)
membranes (for example, dot-immunoblot assays, Western blots). General requirements for the
validation of immunoassay procedures are given under 2.7.1. Immunochemical Methods. In addition,
immunoassay methods for host-cell contaminants meet the following criteria:
Antigen preparations. Antisera are raised against a preparation of antigens derived from the host
organism, into which has been inserted the vector used in the manufacturing process that lacks the
specific gene coding for the product. This host cell is cultured, and proteins are extracted, using
conditions identical to those used for culture and extraction in the manufacturing process. Partly
purified preparations of antigens, using some of the purification steps in the manufacturing process,
may also be used for the preparation of antisera.
Calibration and standardisation. Quantitative data are obtained by comparison with dose-response
curves obtained using standard preparations of host-derived protein antigens. Since these preparations are mixtures of poorly defined proteins, a standard preparation is prepared and calibrated by a
suitable protein determination method. This preparation is stored in a stable state suitable for use
over an extended period of time.
Antisera. Antisera contain high-avidity antibodies recognising as many different proteins in the
antigen mixture as possible, and do not cross-react with the product.
Host-cell-and vector-derived DNA Residual DNA is detected by hybridisation analysis, using
suitably sensitive, sequence-independent analytical techniques or other suitably sensitive analytical
techniques.
Hybridisation analysis
DNA in the test sample is denatured to give single-stranded DNA, immobilised on a nitrocellulose or
other suitable filter and hybridised with labelled DNA prepared from the host-vector manufacturing
system (DNA probes). Although a wide variety of experimental approaches is available, hybridisation
methods for measurement of host-vector DNA meet the following criteria:
DNA probes. Purified DNA is obtained from the host-vector system grown under the same conditions
as those used in the manufacturing process. Host chromosomal DNA and vector DNA may be
separately prepared and used as probes.
Calibration and standardisation. Quantitative data are obtained by comparison with responses obtained
using standard preparations. Chromosomal DNA probes and vector DNA probes are used with
chromosomal DNA and vector DNA standards, respectively. Standard preparations are calibrated by
spectroscopic measurements and stored in a state suitable for use over an extended period of time.
Hybridisation conditions. The stringency of hybridisation conditions is such as to ensure specific
hybridisation between probes and standard DNA preparations and the drug substances must not
interfere with hybridisation at the concentrations used.
Sequence-independent techniques
Suitable procedures include: detection of sulphonated cytosine residues in single-stranded DNA
(where DNA is immobilised on a filter and cytosines are derivatised in situ, before detection and
quantitation using an antibody directed against the sulphonated group); detection of single-stranded
DNA using a fragment of single-stranded DNA bound to a protein and an antibody of this protein.
Neither procedure requires the use of specific host or vector DNA as an assay standard. However, the
method used must be validated to ensure parallelism with the DNA standard used, linearity of
response and non-interference of either the drug substance or excipients of the formulation at the
dilutions used in the assay.
IDENTIFICATION, TESTS AND ASSAY
The requirements with which the final product (bulk material or dose form) must comply throughout
its period of validity, as well as specific test methods, are stated in the individual monograph.
34-56
STORAGE
See the individual monographs.
LABELLING
See the individual monographs.
__________________________________________________________________________________________________________________________________ Ph Eur
34-57
Reserpine
N
MeO
N
H H
H
O
H
H
OMe
MeOOC
O
H
OMe
OMe
OMe
C33H40N2O9
609
50-55-5
Reserpine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0528]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Reserpine contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of
total alkaloids and not less than 98.0 per cent and not more than the equivalent of 102.0 per cent of
methyl 11,17-dimethoxy-18-[(3,4,5-trimethoxybenzoyl)oxy]-3,20-yohimbane-16-carboxylate,
both calculated with reference to the dried substance.
CHARACTERS
A crystalline powder or small, white to slightly yellow crystals, darkening slowly on exposure to light,
practically insoluble in water and in ether, very slightly soluble in alcohol.
IDENTIFICATION
A. Dissolve 20.0 mg in chloroform R and dilute to 10.0 ml with the same solvent. Dilute 1.0 ml of this
solution to 100.0 ml with alcohol R. Examined immediately between 230 nm and 350 nm (2.2.25),
Over the range 288 nm to 295 nm, the curve shows a slight absorption minimum followed by a
shoulder or a slight absorption maximum; over this range, the specific absorbance is about 170.
obtained with reserpine CRS. Examine the substances prepared as discs.
C. To about 1 mg add 0.1 ml of a 1 g/l solution of sodium molybdate R in sulphuric acid R. A yellow
colour is produced which becomes blue within 2 min.
D. To about 1 mg add 0.2 ml of a freshly prepared 10 g/l solution of vanillin R in hydrochloric acid R.
A pink colour develops within 2 min.
E. Mix about 0.5 mg with 5 mg of dimethylaminobenzaldehyde R and 0.2 ml of glacial acetic acid R and
add 0.2 ml of sulphuric acid R. A green colour is produced. Add 1 ml of glacial acetic acid R. The
colour becomes red.
TESTS
Specific optical rotation (2.2.7). Carry out the determination immediately after preparing the solution.
Dissolve 0.250 g in chloroform R and dilute to 25.0 ml with the same solvent. The specific optical
rotation is 116 to 128, calculated with reference to the dried substance.
Oxidation products Dissolve 20 mg in glacial acetic acid R and dilute to 100.0 ml with the same
acid. The absorbance (2.2.25) measured immediately at 388 nm is not greater than 0.10.
ASSAY
Total alkaloids Dissolve 0.500 g in a mixture of 6 ml of acetic anhydride R and 40 ml of anhydrous
acetic acid R. Titrate with 0.1M perchloric acid, determining the end-point potentiometrically (2.2.20).
1 ml of 0.1M perchloric acid is equivalent to 60.9 mg of total alkaloids.
34-58
Reserpine Protect the solutions from light. Moisten 25.0 mg with 2 ml of alcohol R, add 2 ml of 0.25M
sulphuric acid and 10 ml of alcohol R, and warm gently to effect solution. Cool and dilute to 100.0 ml
with alcohol R. Dilute 5.0 ml of this solution to 50.0 ml with alcohol R. Prepare a reference solution in
the same manner using 25.0 mg of reserpine CRS. Place 10.0 ml of each solution separately in two
boiling-tubes, add 2.0 ml of 0.25M sulphuric acid and 2.0 ml of a freshly prepared 3 g/l solution of
sodium nitrite R. Mix and heat in a water-bath at 55C for 35 min. Cool, add 1.0 ml of a freshly
prepared 50 g/l solution of sulphamic acid R and dilute to 25.0 ml with alcohol R. Measure the absorbance (2.2.25) of each solution at the maximum at 388 nm, using as the compensation liquid 10.0 ml
of the same solution treated at the same time in the same manner, but omitting the sodium nitrite.
the solutions.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
34-59
Resorcinol
OH
OH
C6H6O2
110.1
108-46-3
Resorcinol complies with the requirements of the 3rd edition of the European Pharmacopoeia [0290]. These
Action and use Keratolytic.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Resorcinol contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of
benzene-1,3-diol, calculated with reference to the dried substance.
CHARACTERS
A colourless or slightly pinkish-grey, crystalline powder or crystals, turning red on exposure to light
and air, very soluble in water and in alcohol, freely soluble in ether.
IDENTIFICATION
B. Dissolve 0.1 g in 1 ml of water R, add 1 ml of strong sodium hydroxide solution R and 0.1 ml of
chloroform R, heat and allow to cool. An intense, deep-red colour develops which becomes pale yellow
on the addition of a slight excess of hydrochloric acid R.
C. Thoroughly mix about 10 mg with about 10 mg of potassium hydrogen phthalate R, both finely
powdered. Heat over a naked flame until an orange-yellow colour is obtained. Cool and add 1 ml of
dilute sodium hydroxide solution R and 10 ml of water R and shake to dissolve. The solution shows an
intense green fluorescence.
TESTS
solution B5 or R5 (Method II, 2.2.2) and remains so when heated in a water-bath for 5 min.
Acidity or alkalinity To 10 ml of solution S add 0.05 ml of bromophenol blue solution R2. Not more
than 0.05 ml of 0.1M hydrochloric acid or 0.1M sodium hydroxide is required to change the colour of the
indicator.
coating substance.
the same solvent.
40 volumes of ethyl acetate R and 60 volumes of hexane R. Allow the plate to dry in air for 15 min and
expose it to iodine vapour. Any spot in the chromatogram obtained with the test solution, apart from
Pyrocatechol To 2 ml of solution S add 1 ml of ammonium molybdate solution R2 and mix. Any
yellow colour in the solution is not more intense than that in a standard prepared at the same time in
the same manner using 2 ml of a 0.1 g/l solution of pyrocatechol R.
Loss on drying (2.2.32). Not more than 1.0 per cent, determined on 1.00 g of powdered substance
by drying in a desiccator for 4 h.
ASSAY
in a ground-glass-stoppered flask add 1.0 g of potassium bromide R, 50.0 ml of 0.0167M potassium
34-60
bromate, 15 ml of chloroform R and 15.0 ml of hydrochloric acid R1. Stopper the flask, shake and allow
to stand in the dark for 15 min, shaking occasionally. Add 10 ml of a 100 g/l solution of potassium
iodide R, shake thoroughly, allow to stand for 5 min and titrate with 0.1M sodium thiosulphate, using
1 ml of starch solution R as indicator.
1 ml of 0.0167M potassium bromate is equivalent to 1.835 mg of C6H6O2.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
34-61
Rhatany Root
Krameria
Rhatany Root complies with the requirements of the 3rd edition of the European Pharmacopoeia [0289].
Action and use Astringent.
When Powdered Rhatany Root is prescribed or demanded, material complying with the requirements
below with the exception of Identification test A and the test for Foreign matter shall be dispensed or
supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Rhatany root, known as Peruvian rhatany, consists of the dried, usually fragmented, underground
organs of Krameria triandra Ruiz and Pavon. It contains not less than 5.0 per cent of tannins,
expressed as pyrogallol (C6H6O3; Mr 126.1), calculated with reference to the dried drug.
CHARACTERS
IDENTIFICATION
A. The taproot is dark red-brown and has a thick, knotty crown. The secondary roots are the same
colour and nearly straight or somewhat tortuous. The bark is rugged to scaly in the older pieces and
smooth with sharp, transverse fissures in the younger pieces; it separates readily from the wood. The
fracture is fibrous in the bark and splintery in the wood. The smooth, transversely cut surface shows a
dark brown-red bark about one third of the radius in thickness; a dense, pale red-brown and finely
porous wood is present with numerous fine medullary rays; the central heartwood is often darker.
B. Reduce to a powder (355). The powder is brown-red. Examine under a microscope using chloral
hydrate solution R. The powder shows cork cells containing dark brown phlobaphenes; fragments of
unlignified phloem fibres, usually 12 m to 30 m in diameter with moderately thick walls; phloem
parenchyma cells in files containing prisms and microcrystals of calcium oxalate; fragments of vessels
usually 20 m to 60 m in diameter with bordered pits; fragments of tracheids up to 20 m wide with
slit-shaped pits. Examine under a microscope using a 50 per cent V/V solution of glycerol R. The
powder shows rounded starch granules, simple or two- to four-compound, an individual granule
measuring up to 30 m in diameter and some granules being found in the cells of the medullary rays
and in the parenchyma.
C. Examine by thin layer chromatography (2.2.27), using a TLC silica gel plate R.
Test solution. To 1.0 g of the powdered drug (355) add 10 ml of a mixture of 3 volumes of water R
and 7 volumes of alcohol R, shake for 10 min and filter. To the filtrate add 10 ml of light petroleum R
and shake. Separate the light petroleum layer, add 2 g of anhydrous sodium sulphate R, shake and
filter. Evaporate the filtrate to dryness. Dissolve the residue in 0.5 ml of methanol R.
Reference solution. Dissolve 5.0 mg of Sudan red G R in 10 ml of methanol R.
Apply to the plates as bands 10 l of each solution. Develop over a path of 15 cm using a mixture of
2 volumes of ethyl acetate R and 98 volumes of toluene R. Allow the plate to dry in air and spray the
plate with a 5 g/l solution of fast blue B salt R. Allow the plate to dry in air and spray the plate with
0.1M ethanolic sodium hydroxide. Examine in daylight. The chromatogram obtained with the reference
solution shows in the lower third a red zone due to Sudan red G. The chromatogram obtained with
the test solution shows a violet zone due to rhatany phenol I similar in position to the zone of Sudan
red G in the chromatogram obtained with the reference solution, below it the brownish zone due to
rhatany phenol II and below it the bluish-grey zone due to rhatany phenol III. Further zones may be
present.
TESTS
Foreign matter (2.8.2). Not more than 2 per cent of foreign matter and not more than 5 per cent of
fragments of crown or root exceeding 25 mm in diameter. Root without bark may be present in very
small quantities.
ASSAY
Carry out the determination of tannins in herbal drugs (2.8.14). Use 0.750 g of powdered drug
(180).
34-62
STORAGE
__________________________________________________________________________________________________________ Ph Eur
34-63
Rhubarb
Rhubarb complies with the requirements of the 3rd edition of the European Pharmacopoeia [0291]. These
Preparation
Compound Rhubarb Tincture
When Powdered Rhubarb is prescribed or demanded, material complying with the requirements
supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Rhubarb consists of the whole or cut, dried underground parts of Rheum palmatum L. or of Rheum
officinale Baillon or of hybrids of these two species or of a mixture. The underground parts are often
divided; the stem and most of the bark with the rootlets are removed. It contains not less than 2.2 per
cent of hydroxyanthracene derivatives, expressed as rhein (C15H8O6, Mr 284.2), calculated with
CHARACTERS
Rhubarb has a characteristic, aromatic odour.
IDENTIFICATION
A. The appearance is variable: disc-shaped pieces up to 10 cm in diameter and 1 cm to 5 cm in
thickness; cylindrical pieces; oval or planoconvex pieces. The surface has a pinkish tinge and is
usually covered with a layer of brownish-yellow powder. It shows, especially after moistening, a
reticulum of darker lines. This structure causes the marbled appearance of the drug. The fracture is
granular. The transverse section of the rhizome shows a narrow outer zone of radiating brownish-red
lines. These medullary rays are crossed perpendicularly by a dark cambial ring. Inside this zone is a
ring of small star-spot formations of anomalous vascular bundles. The root shows a more radiate
structure.
B. Reduce to a powder (355). The powder is orange to brownish-yellow. Examine under a
microscope using chloral hydrate solution R. The powder shows the following diagnostic characters:
large calcium oxalate cluster crystals, which may measure more than 100 m, and their fragments;
reticulately thickened non-lignified vessels measuring up to 175 m. Numerous groups of rounded or
polygonal, thin-walled parenchyma cells. Sclereids and fibres are absent. Examine under a
microscope using a 50 per cent V/V solution of glycerol R. The powder shows simple, rounded or
compound (2 to 4) starch granules with a star-shaped hilum.
substance.
Test solution. Heat 50 mg of the powdered drug (180) in a water-bath for 15 min with a mixture of
1 ml of hydrochloric acid R and 30 ml of water R. Allow to cool and shake the liquid with 25 ml of
ether R. Dry the ether layer over anhydrous sodium sulphate R and filter. Evaporate the ether layer to
dryness and dissolve the residue in 0.5 ml of ether R.
Reference solution. Dissolve 5 mg of emodin R in 5 ml of ether R.
mixture of 1 volume of anhydrous formic acid R, 25 volumes of ethyl acetate R and 75 volumes of light
petroleum R. Allow the plate to dry in air and examine in ultraviolet light at 365 nm. The chromatogram obtained with the reference solution shows in its central part a zone of orange fluorescence
(emodin). The chromatogram obtained with the test solution shows: a zone due to emodin; above
the emodin zone, two zones of similar fluorescence (physcione and chrysophanol, in order of increasing Rf value); below the emodin zone, also two zones of similar fluorescence (rhein and aloe-emodin,
in order of decreasing Rf value). Spray with a 100 g/l solution of potassium hydroxide R in methanol R.
All the zones become red to violet.
D. To about 50 mg of the powdered drug (180) add 25 ml of dilute hydrochloric acid R and heat the
mixture on a water-bath for 15 min. Allow to cool, shake with 20 ml of ether R and discard the
aqueous layer. Shake the ether layer with 10 ml of dilute ammonia R1. The aqueous layer becomes red
to violet.
34-64
TESTS
Rheum rhaponticum Examine by thin-layer chromatography (2.2.27), using silica gel G R as the
coating substance.
Test solution. To 0.2 g of the powdered drug (180) add 2 ml of methanol R and boil for 5 min under a
reflux condenser. Allow to cool and filter. Use the filtrate as the test solution.
Reference solution. Dissolve 10 mg of rhaponticin R in 10 ml of methanol R.
Apply separately to the plate, as bands not more than 20 mm by 3 mm, 20 l of each solution.
Develop over a path of 12 cm using a mixture of 20 volumes of methanol R and 80 volumes of
methylene chloride R. Allow the plate to dry in air and spray with phosphomolybdic acid solution R. The
chromatogram obtained with the test solution does not show a blue zone near the starting-line
(rhaponticin) corresponding to the zone in the chromatogram obtained with the reference solution.
ASSAY
Carry out the assay protected from bright light.
Introduce 0.100 g of the powdered drug (180) into a 100 ml flask. Add 30.0 ml of water R, mix and
weigh. Heat in a water-bath under a reflux condenser for 15 min. Allow to cool, add 50 mg of sodium
hydrogen carbonate R, weigh and adjust to the original mass with water R. Centrifuge and transfer
10.0 ml of the liquid to a 100 ml round-bottomed flask with a ground-glass neck. Add 20 ml of ferric
chloride solution R1 and mix. Heat under a reflux condenser on a water-bath for 20 min, add 1 ml of
hydrochloric acid R and heat for a further 20 min, shaking frequently. Cool, transfer to a separating
funnel and shake with three quantities, each of 25 ml, of ether R previously used to rinse the flask.
Combine the ether extracts and wash with two quantities, each of 15 ml, of water R. Filter the ether
extracts through a plug of absorbent cotton into a volumetric flask and dilute to 100.0 ml with
ether R. Evaporate 10.0 ml carefully to dryness on a water-bath and dissolve the residue in 10.0 ml of
a 5 g/l solution of magnesium acetate R in methanol R. Measure the absorbance (2.2.25) at 515 nm,
using methanol R as the compensation liquid.
Calculate the percentage content of rhein from the expression:
A 0.64
m
taking the specific absorbance of rhein to be 468, calculated on the basis of the specific absorbance of
barbaloin.
m = mass of the drug used in grams.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
34-65
Ribavirin
O
H2N
N
N
N
HO
O
OH
C8H12N4O5
OH
244.2
36791-04-5
Definition Ribavirin is 1--D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide. It contains not less

than 98.0% and not more than 102.0% of C8H12N4O5, calculated with reference to the dried
substance.
Characteristics. A white, crystalline powder.
Freely soluble in water; slightly soluble in ethanol (96%).
spectrum of ribavirin (RS 349).
Specific optical rotation In a freshly prepared 1% w/v solution, 33 to 37, Appendix V F,
test A for heavy metals, Appendix VII (10 ppm). Prepare the standard using 2 ml of lead standard
solution (10 ppm Pb).
Related substances Carry out the method for liquid chromatography, Appendix III D, using three
solutions in the mobile phase containing (1) 0.050% w/v of the substance being examined, (2)
0.000125% w/v of the substance being examined and (3) 0.050% w/v of ribavirin impurity standard
BPCRS.
(10 cm 7.8 mm) packed with a strong cation-exchange resin of sulphonated, cross-linked
styrenedivinylbenzene copolymer in the hydrogen form (7 to 11 m) (Aminex HPAH is suitable)
and maintained at 40, (b) water adjusted to pH 2.5 with sulphuric acid as the mobile phase with a
flow rate of 1 ml per minute and (c) a detection wavelength of 207 nm. Inject separately 10 l of
each solution. For solution (1) allow the chromatography to proceed for ten times the retention
time of the principal peak.
The test is not valid unless the symmetry factor of the principal peak in the chromatogram
obtained with solution (1) is at least 0.7 and not more than 1.5 and unless the chromatogram
obtained with solution (3) closely resembles the reference chromatogram supplied with ribavirin
impurity standard BPCRS.
In the chromatogram obtained with solution (1), the area of any secondary peak is not greater than
that of the principal peak in the chromatogram obtained with solution (2) (0.25%) and the sum of
the areas of any such peaks is not greater than 4 times the area of the principal peak in the chromatogram obtained with solution (2) (1%).
Assay Carry out the method for liquid chromatography, Appendix III D, using two solutions in the
mobile phase containing (1) 0.0025% w/v of the solution being examined and (2) 0.0025% w/v of
ribavirin BPCRS.
The chromatographic procedure described under Related substances may be used.
Calculate the content of C8H12N4O5 using the declared content of C8H12N4O5 in ribavirin
BPCRS.
Storage Store in a well-closed container protected from light.
Action and use Antiviral.
Preparation
Ribavirin Nebuliser Solution
When tribavirin is prescribed or demanded, Ribavirin shall be dispensed or supplied.
34-66
IMPURITIES
HOOC
N
N
HO
O
OH OH
A. 1--D-ribofuranosyl-1H-1,2,4-triazole-3-carboxylic acid
HO
O
HO HO
N
N
H2N
O
B. 1--D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide (anomer)
O
HO
N
N
N
H
C. 1H-1,2,4-triazole-3-carboxylic acid
O
H2N
N
N
N
H
D. 1H-1,2,4-triazole-3-carboxamide
O
H2N
N
N
PhCOO
OH OH
E. O5-benzoyltribavirin
34-67
Riboflavin
CH2OH
HO
HO
HO
Me
Me
O
NH
O
C17H20N4O6
376.4
83-88-5
Riboflavin complies with the requirements of the 3rd edition of the European Pharmacopoeia for Riboflavine
When riboflavine is prescribed or demanded, Riboflavin shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Riboflavine contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent
of 7,8-dimethyl-10-[(2S,3S,4R)-2,3,4,5-tetrahydroxypentyl]-3H,10H-benzopteridine-2,4-dione,
CHARACTERS
A yellow or orange-yellow, crystalline powder, very slightly soluble in water, practically insoluble in
alcohol and in ether. It is more soluble in a 9 g/l solution of sodium chloride than in water. Solutions
deteriorate on exposure to light, especially in the presence of alkali.
IDENTIFICATION
obtained with riboflavine CRS.
C. Dissolve about 1 mg in 100 ml of water R. The solution has, by transmitted light, a pale greenishyellow colour, and, by reflected light, an intense yellowish-green fluorescence which disappears on
the addition of mineral acids or alkalis.
TESTS
Acidity or alkalinity To 0.5 g add 25 ml of water R and boil for 2 min. Allow to cool and filter. To
10 ml of the filtrate add 0.05 ml of phenolphthalein solution R1 and 0.4 ml of 0.01M sodium hydroxide.
The solution is orange. Add 0.5 ml of 0.01M hydrochloric acid. The solution is yellow. Add 0.15 ml of
methyl red solution R. The solution is orange.
Specific optical rotation (2.2.7). Dissolve 50.0 mg in 0.05M sodium hydroxide free from carbonate
and dilute to 10.0 ml with the same solvent. Measure the optical rotation within 30 min of dissolution. The specific optical rotation is 115 to 135, calculated with reference to the dried substance.
Absorbance (2.2.25). Dilute the final solution prepared for the assay with an equal volume of
water R. The solution shows four maxima, at 223 nm, 267 nm, 373 nm and 444 nm. The ratio of the
absorbance at the maximum at 373 nm to that at the maximum at 267 nm is 0.31 to 0.33, and the
ratio of the absorbance at the maximum at 444 nm to that at the maximum at 267 nm is 0.36 to
0.39.
Lumiflavine Shake 25 mg with 10 ml of chloroform R for 5 min and filter. The filtrate is not more
intensely coloured than reference solution BY6 (Method II, 2.2.2).
34-68
100C to 105C.
for loss on drying.
ASSAY
Carry out the assay in subdued light. In a brown-glass 500 ml volumetric flask, suspend 65.0 mg in
5 ml of water R ensuring that it is completely wetted and dissolve in 5 ml of dilute sodium hydroxide
solution R. As soon as dissolution is complete, add 100 ml of water R and 2.5 ml of glacial acetic acid R
and dilute to 500.0 ml with water R. Place 20.0 ml of this solution in a brown-glass 200 ml
volumetric flask, add 3.5 ml of a 14 g/l solution of sodium acetate R and dilute to 200.0 ml with
water R. Measure the absorbance (2.2.25) at the maximum at 444 nm.
Calculate the content of C17H20N4O6 taking the specific absorbance to be 328.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
34-69
Riboflavin Sodium Phosphate

O
CH 2 O
HO
HO
HO
Me
Me
ONa
OH
O
NH
O
C17H20N4NaO9P,2H2O
514.4
130-40-5
Riboflavin Sodium Phosphate complies with the requirements of the 3rd edition of the European Pharmacopoeia for Riboflavine Sodium Phosphate [0786]. These requirements are reproduced after the heading Definition below.
Preparation
When riboflavine sodium phosphate is prescribed or demanded, Riboflavin Sodium Phosphate shall
be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Riboflavine sodium phosphate is a mixture containing riboflavine 5-(sodium hydrogen phosphate) as
the main component and other riboflavine sodium monophosphates. It contains not less than
73.0 per cent and not more than 79.0 per cent of riboflavine (C17H20N4O6; Mr 376.4), calculated
with reference to the dried substance. It contains a variable amount of water.
CHARACTERS
A yellow or orange-yellow, crystalline powder, hygroscopic, soluble in water, very slightly soluble in
IDENTIFICATION
A. Dissolve 50.0 mg in phosphate buffer solution pH 7.0 R and dilute to 100.0 ml with the same buffer
solution. Dilute 2.0 ml of the solution to 100.0 ml with phosphate buffer solution pH 7.0 R. Examined
between 230 nm and 350 nm (2.2.25), the solution shows an absorption maximum at 266 nm. The
specific absorbance at the maximum is 580 to 640.
B. Examine the chromatograms obtained in the test for related substances. The principal peak in the
chromatogram obtained with the test solution is similar in position and approximate size to the
principal peak in the chromatogram obtained with the reference solution (b).
C. Dissolve about 10 mg in dilute sodium hydroxide solution R and dilute to 100 ml with the same
solution. Expose 1 ml to ultraviolet light at 254 nm for 5 min, add sufficient acetic acid R to make the
solution acidic to blue litmus paper R and shake with 2 ml of methylene chloride R. The lower layer
shows yellow fluorescence.
D. To 0.5 g add 10 ml of nitric acid R, evaporate the mixture to dryness on a water-bath. Ignite the
residue until it becomes white, dissolve the residue in 5 ml of water R and filter. The filtrate gives
reaction (a) of sodium and reaction (b) of phosphates (2.3.1).
TESTS
Specific optical rotation (2.2.7). Dissolve 0.300 g in 18.2 ml of hydrochloric acid R1 and dilute to
25.0 ml with water R. The specific optical rotation is +38.0 to +43.0, calculated with reference to
34-70
Lumiflavine. To about 35 mg add 10 ml of methylene chloride R, shake for 5 min and filter. The
filtrate is not more intensely coloured than reference solution BY6 (Method II, 2.2.2).
Related substances. Examine by liquid chromatography (2.2.29). Carry out the test protected from
actinic light.
Reference solution (a). Dissolve 60 mg of riboflavine CRS in 1 ml of hydrochloric acid R and dilute to
250.0 ml with water R. Dilute 4.0 ml to 100.0 ml with the mobile phase.
Reference solution (b). Dissolve 0.100 g of riboflavine sodium phosphate CRS in 50 ml of water R and
dilute to 100.0 ml with the mobile phase. Dilute 8.0 ml of this solution to 50.0 ml with the mobile
phase.
as mobile phase at a flow rate of 2 ml per minute a mixture of 150 volumes of methanol R and
850 volumes of a 7.35 g/l solution of potassium dihydrogen phosphate R,
riboflavine 5-monophosphate is about 20 min and the relative retention times are: riboflavine
3,4-diphosphate, about 0.2; riboflavine 3,5-diphosphate, about 0.3; riboflavine 4,5-diphosphate,
about 0.5; riboflavine 3-monophosphate, about 0.7; riboflavine 4-mono-phosphate, about 0.9;
riboflavine 5-monophosphate, 1.0; riboflavine, about 2.
Inject 100 l of reference solution (b). Continue the chromatography until the peak corresponding to
riboflavine can be clearly evaluated. The test is not valid unless: in the chromatogram obtained with
reference solution (b), the resolution between the peaks corresponding to riboflavine 4-monophosphate and riboflavine 5-monophosphate is at least 1.5.
Inject 100 l of the test solution, 100 l of reference solution (a) and 100 l of reference solution
(b). Calculate the percentage content of free riboflavine and of riboflavine in the form of the diphosphates of riboflavine from the areas of the peaks in the chromatogram obtained with the test solution
and the amount of free riboflavine in reference solution (a). The content of free riboflavine is not
greater than 6.0 per cent and the content of riboflavine in the form of the diphosphates of riboflavine
is not greater than 6.0 per cent, both calculated with reference to dried material.
Inorganic phosphate Dissolve 0.10 g in water R and dilute to 100 ml with the same solvent. To
5 ml of the solution, add 10 ml of water R, 5 ml of buffered copper sulphate solution pH 4.0 R, 2 ml of a
30 g/l solution of ammonium molybdate R, 1 ml of a freshly prepared solution containing 20 g/l of
4-methylaminophenol sulphate R and 50 g/l of sodium metabisulphite R and 1 ml of a 3 per cent V/V
solution of perchloric acid R. Dilute to 25.0 ml with water R and measure, within 15 min of its preparation, the absorbance of the solution at 800 nm (2.2.25) using as the compensation liquid a solution
prepared in the same manner but without the substance to be examined. The absorbance is not
greater than that of a solution prepared as follows: to 15 ml of phosphate standard solution (5 ppm
PO4) R, add 5 ml of buffered copper sulphate solution pH 4.0 R, 2 ml of a 30 g/l solution of ammonium
molybdate R, 1 ml of a freshly prepared solution containing 20 g/l of 4-methylaminophenol sulphate R
and 50 g/l of sodium metabisulphite R and 1 ml of a 3 per cent V/V solution of perchloric acid R; dilute
to 25.0 ml with water R (1.5 per cent).
Heavy metals (2.4.8). To 2.0 g in a silica crucible add 2 ml of nitric acid R, dropwise, followed by
0.25 ml of sulphuric acid R. Heat cautiously until white fumes are evolved and ignite. Extract the
cooled residue with two quantities, each of 2 ml, of hydrochloric acid R and evaporate the extracts to
dryness. Dissolve the residue in 2 ml of dilute acetic acid R and dilute to 20 ml with water R. 12 ml of
the solution complies with limit test A for heavy metals (10 ppm). Prepare the standard using 10 ml
of lead standard solution (1 ppm Pb) R.
100C to 105C at a pressure not exceeding 0.7 kPa for 5 h.
ASSAY
Carry out the assay protected from light. Dissolve 0.100 g in 150 ml of water R, add 2 ml of glacial acetic
acid R and dilute to 1000.0 ml with water R. To 10.0 ml add 3.5 ml of a 14 g/l solution of sodium
acetate R and dilute to 50.0 ml with water R. Measure the absorbance (2.2.25) at the maximum at
444 nm.
Calculate the content of C17H20N4O6 taking the specific absorbance to be 328.
STORAGE
34-71
IMPURITIES
CH2OR
R1O
R2O
HO
H
CH2
Me
Me
O
NH
A. R = H, R1 = R2 = PO3H2:
riboflavine 3,4-diphosphate,
B. R1 = H, R = R2 = PO3H2:
C. R2 = H, R = R1 = PO3H2:
D. R = R1 = R2 = H: riboflavine,
Me
Me
Me
O
NH
E. lumiflavine.
__________________________________________________________________________________________________________ Ph Eur
34-72
Rice Starch
Rice Starch complies with the requirements of the 3rd edition of the European Pharmacopoeia [0349]. These
or used.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Rice starch is obtained from the caryopsis of Oryza sativa L.
CHARACTERS
A very fine, white powder which creaks when pressed between the fingers, tasteless, practically
insoluble in cold water and in alcohol. The presence of granules with cracks or irregularities on the
edge is exceptional.
DESCRIPTION
Examined under a microscope it presents polyhedral granules 2 m to 5 m in size, either isolated or
aggregated in ovoid masses of 10 m to 20 m in size. The granules have a poorly visible central
hilum and there are no concentric striations. Between crossed nicol prisms, the starch granules show
a distinct black cross intersecting at the hilum.
IDENTIFICATION
A. Suspend 1 g in 50 ml of water R, boil for 1 min and cool. A thin, cloudy mucilage is formed.
B. To 1 ml of the mucilage obtained in identification test A add 0.05 ml of iodine solution R1. A darkblue colour is produced which disappears on heating and reappears on cooling.
TESTS
Acidity Add 10 g to 100 ml of alcohol (70 per cent V/V) R previously neutralised to 0.5 ml of phenolphthalein solution R and shake for 1 h. Filter and take 50 ml of the filtrate. Not more than 2.0 ml of
100C to 105C.
fungi per gram, determined by plate count. It complies with the test for Escherichia coli (2.6.13).
STORAGE
__________________________________________________________________________________________________________ Ph Eur
35-1
Rifampicin
Me
O
H
OH
O
O
N
N
Me
N
HN
Me
Me
OH
Me
O
HO
OMe
OH
Me
OAc
OH
Me
C43H58N4O12
823
Me
13292-46-1
Rifampicin complies with the requirements of the 3rd edition of the European Pharmacopoeia [0052]. These
Preparations
Rifampicin Capsules
Rifampicin Oral Suspension
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Rifampicin is (12Z,14E,24E)-(2S,16S,17S,18R,19R,20R, 21S,22R,23S)-5,6,9,17,19-pentahydroxy23-methoxy-2,4,12,16,18,20,22-heptamethyl-8-[N-(4-methyl-1-piperazinyl)formimidoyl]-1,11dioxo-2,7-[epoxy(1,11,13-pentadecatrieno)imino]-1,2-dihydronaphtho[2,1-b]furan-21-yl acetate, a
semisynthetic antibiotic obtained from rifamycin SV. It contains not less than 97.0 per cent and not
more than the equivalent of 102.0 per cent of C43H58N4O12, calculated with reference to the dried
substance.
CHARACTERS
A reddish-brown or brownish-red, crystalline powder, slightly soluble in water, soluble in methanol,
slightly soluble in acetone, in alcohol and in ether.
IDENTIFICATION
A. Dissolve 50 mg in 50 ml of methanol R. Dilute 1 ml of the solution to 50 ml with phosphate buffer
solution pH 7.4 R. Examined between 220 nm and 500 nm, the solution shows four absorption
maxima (2.2.25), at 237 nm, 254 nm, 334 nm and 475 nm. The ratio of the absorbance at the
maximum at 334 nm to that at 475 nm is about 1.75.
obtained with rifampicin CRS. Examine the substances prepared as mulls in liquid paraffin R.
C. Suspend about 25 mg in 25 ml of water R, shake for 5 min and filter. To 5 ml of the filtrate add
1 ml of a 100 g/l solution of ammonium persulphate R in phosphate buffer solution pH 7.4 R and shake
for a few minutes. The colour changes from orange-yellow to violet-red and no precipitate is formed.
TESTS
pH (2.2.3). The pH of a 10 g/l suspension in carbon dioxide-free water R is 4.5 to 6.5.
Solvent mixture. To 10 volumes of a 210.1 g/l solution of citric acid R add 23 volumes of a 136.1 g/l
solution of potassium dihydrogen phosphate R, 77 volumes of a 174.2 g/l solution of dipotassium
hydrogen phosphate R, 250 volumes of acetonitrile R and 640 volumes of water R.
Prepare the test solution and the reference solution immediately before use.
Test solution. Dissolve 20.0 mg of the substance to be examined in acetonitrile R and dilute to 10.0 ml
with the same solvent. Dilute 5.0 ml to 50.0 ml with the solvent mixture.
35-2
Reference solution. Dissolve 20.0 mg of rifampicin quinone CRS in acetonitrile R and dilute to 100.0 ml
with the same solvent. To 1.0 ml of the solution add 1.0 ml of the test solution and dilute to
65 volumes of a solution containing 0.1 per cent V/V of phosphoric acid R, 1.9 g/l of sodium
perchlorate R, 5.9 g/l of citric acid R and 20.9 g/l of potassium dihydrogen phosphate R,
Inject the reference solution. Adjust the sensitivity of the detector so that the height of the two
principal peaks is not less than half the full scale of the recorder. The test is not valid unless the
resolution between the two principal peaks is at least 4.0. Adjust the concentration of acetonitrile in
the mobile phase, if necessary. Inject the test solution and continue the chromatography for at least
twice the retention time of rifampicin: the area of any peak corresponding to rifampicin quinone is
not greater than 1.5 times the area of the corresponding peak in the chromatogram obtained with the
reference solution (1.5 per cent); the area of any peak, apart from the principal peak and the peak
corresponding to rifampicin quinone, is not greater than the area of the rifampicin peak in the chromatogram obtained with the reference solution (1.0 per cent) and the sum of the areas of any such
peaks is not greater than 3.5 times the area of the rifampicin peak in the chromatogram obtained with
the reference solution (3.5 per cent). Disregard any peak due to the solvent and any peak with an
area less than 0.05 times that of the peak corresponding to rifampicin in the chromatogram obtained
ASSAY
Dissolve 0.100 g in methanol R and dilute to 100.0 ml with the same solvent. Dilute 2.0 ml of the
solution to 100.0 ml with phosphate buffer solution pH 7.4 R. Measure the absorbance (2.2.25) at the
maximum at 475 nm, using phosphate buffer solution pH 7.4 R as the compensation liquid.
Calculate the content of C43H58N4O12, taking the specific absorbance to be 187.
STORAGE
Store under nitrogen in an airtight container, protected from light at a temperature not exceeding
25C.
IMPURITIES
H
N
Me
Me
O
O
N
HN
Me
Me
Me
O HO
Me
O HO
OMe
OAc
OH
Me
Me
A. rifampicin quinone,
N
Me
N
O
B. rifampicin N-oxide.
__________________________________________________________________________________________________________ Ph Eur
35-3
Rifamycin Sodium
OH
Me
Me
Me
Me
OH
AcO
NH
HO
Me
Me
Na+
MeO
O
OH
O
C37H46NNaO12
Me
720
O
14897-39-3
Rifamycin Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia [0432].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Rifamycin sodium is sodium (12Z,14E,24E)-(2S,16S,17S, 18R,19R,20R,21S,22R,23S)-21(acetyloxy)-6,9,17,19-tetrahydroxy-23-methoxy-2,4,12,16,18,20,22-heptamethyl-1,11-dioxo-1,2dihydro-2,7-(epoxypentadeca[1,11,13]trienimino)naphtho-[2,1-b]furan-5-olate, the monosodium
salt of rifamycin SV, a substance obtained by chemical transformation of rifamycin B, which is
produced during the growth of certain strains of Amycolatopsis mediterranei. Rifamycin SV may also be
obtained directly from certain A. mediterranei mutants. The potency is not less than 900 I.U. per
milligram, calculated with reference to the anhydrous substance.
PRODUCTION
It is produced by methods of manufacture designed to minimise or eliminate substances lowering
blood pressure. The method of manufacture is validated to demonstrate that the product if tested
would comply with the following test:
Abnormal toxicity (2.6.9). Inject into each mouse 4 mg dissolved in 0.5 ml of water for injections R.
CHARACTERS
A fine or slightly granular, red powder, soluble in water, freely soluble in ethanol, practically insoluble
in ether.
IDENTIFICATION
obtained with rifamycin sodium CRS. Examine the substances as discs prepared using potassium
bromide R.
TESTS
The pH is 6.5 to 8.0.
Absorbance (2.2.25). Dissolve 20.0 mg in 5 ml of methanol R and dilute to 100.0 ml with freshly
prepared phosphate buffer solution pH 7.0 R1 to which 1 g/l of ascorbic acid R has been added immediately before use. Dilute 5.0 ml of the solution to 50.0 ml with the same phosphate buffer solution
containing ascorbic acid. Allow to stand for 30 min. The solution shows an absorption maximum at
445 nm. The specific absorbance at the maximum is 190 to 210, calculated with reference to the
Rifamycin B, rifamycin S and other related substances Examine by liquid chromatography
(2.2.29). Prepare the solutions immediately before use.
3.9 g/l solution of sodium dihydrogen phosphate R, adjusted to pH 3.0 with phosphoric acid R, and
acetonitrile R and dilute to 50.0 ml with the same mixture of solvents.
35-4
Reference solution (a). Dissolve 10.0 mg of rifamycin B CRS and 40.0 mg of rifamycin S CRS in a
mixture of equal volumes of a 3.9 g/l solution of sodium dihydrogen phosphate R, adjusted to pH 3.0
with phosphoric acid R, and acetonitrile R and dilute to 200.0 ml with the same mixture of solvents.
Dilute 5.0 ml of the solution to 50.0 ml with the same mixture of solvents.
Reference solution (b). Dissolve 25 mg of the substance to be examined and 8 mg of rifamycin S CRS
in a mixture of equal volumes of a 3.9 g/l solution of sodium dihydrogen phosphate R, adjusted to pH
3.0 with phosphoric acid R, and acetonitrile R and dilute to 250.0 ml with the same mixture of solvents.
a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with octyldecylsilyl
as mobile phase at a flow rate of 1 ml per minute the following solutions, prepared and
maintained at a temperature not lower than 20C:
Mobile phase A. Mix 10 volumes of acetonitrile R and 90 volumes of a 3.9 g/l solution of sodium
dihydrogen phosphate R, adjusted to pH 7.5 with dilute sodium hydroxide solution R,
Mobile phase B. Mix 70 volumes of acetonitrile R and 30 volumes of a 3.9 g/l solution of sodium
dihydrogen phosphate R, adjusted to pH 7.5 with dilute sodium hydroxide solution R,
Time
(min)
Mobile phase A
(per cent V/V)

(per cent V/V)
040
4045
4547
4755
8020
20
2080
80
2080
80
8020
20
linear gradient
isocratic
linear gradient
re-equilibration

Inject reference solution (a). When the chromatogram is recorded in the prescribed conditions, the
substances elute in the following order: rifamycin B, rifamycin SV, rifamycin S. Inject reference
solution (b). Adjust the sensitivity of the system so that the height of the peak corresponding to
rifamycin S in the chromatogram obtained is at least 50 per cent of the full scale of the recorder. The
test is not valid unless the resolution between the peaks corresponding to rifamycin SV and rifamycin
S is at least 5.0. Inject the test solution and reference solution (a). In the chromatogram obtained
with the test solution: the area of any peak corresponding to rifamycin B is not greater than that of
the peak due to rifamycin B in the chromatogram obtained with reference solution (a) (0.5 per cent);
the area of any peak corresponding to rifamycin S is not greater than that of the peak due to
rifamycin S in the chromatogram obtained with reference solution (a) (2 per cent); the sum of the
areas of all the peaks, apart from the principal peak and any peaks corresponding to rifamycin B and
rifamycin S, is not greater than the area of the peak corresponding to rifamycin S in the chromatogram obtained with reference solution (a) (2 per cent). Disregard any peak with an area less than
0.05 times that of the area of the peak corresponding to rifamycin S in the chromatogram obtained
ASSAY
STORAGE
Store in an airtight container, protected from light at a temperature of 2C to 8C. If the substance is
sterile, store in a sterile, airtight, tamper-proof container.
LABELLING
The label states:
35-5
IMPURITIES
O Me
R
O
OMe
HN
R'
OH
Me
Me
OAc
HO
Me
Me
Me
Me
OH
A. R = -O-CH2-CO2H, R = -OH: rifamycin B,

B. R = O, R = O: rifamycin S,
C. R = -O-CO-CH2-O-, R = O: rifamycin O.
__________________________________________________________________________________________________________ Ph Eur
35-6
Risperidone
1/01
N
Me
N
N
O
N
O
C23H27FN4O2
410.5
106266-06-2
Risperidone complies with the requirements of the 3rd edition of the European Pharmacopoeia [1559]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Risperidone contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of 3-[2-[4-(6-fluoro-1,2-benzisoxazol-3-yl)piperidin-1-yl]ethyl]-2-methyl-6,7,8,9-tetrahydro-4Hpyrido[1,2-a]pyrimidin-4-one, calculated with reference to the dried substance.
CHARACTERS
A white or almost white powder, practically insoluble in water, freely soluble in methylene chloride,
sparingly soluble in alcohol. It dissolves in dilute acid solutions.
IDENTIFICATION
with risperidone CRS. Examine the substances prepared as discs. If the spectra obtained show differences, dissolve the substance to be examined and the reference substance separately in the minimum
volume of acetone R, evaporate to dryness and record new spectra using the residues.
TESTS
Appearance of solution Dissolve 0.1 g in a 7.5 g/l solution of tartaric acid R and dilute to 100 ml
with the same acid solution. The solution is clear (2.2.1) and colourless (Method II, 2.2.2).
Reference solution (a). Dissolve 5.0 mg of risperidone CRS and 5.0 mg of haloperidol CRS in methanol R
the solution to 25.0 ml with methanol R.
Mobile phase A. A 5 g/l solution of ammonium acetate R,
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
0 15
70 30
30 70
Linear gradient
15 20
30
70
20 21
30 70
70 30
Isocratic
Switch to initial
eluent
composition
21 25
70
30
Re-equilibration
25 = 0
70
30
Re-start gradient
35-7
Equilibrate the column with the initial eluent composition at a flow rate of 1 ml/min for at least
10 min.
two peaks in the chromatogram obtained is at least 50 per cent of the full scale of the recorder. When
the chromatogram is recorded in the prescribed conditions, the retention times are: risperidone,
about 10.5 min and haloperidol, about 11 min. The test is not valid unless the resolution between
the peaks due to risperidone and haloperidol is not less than 3.0. If necessary, adjust the concentration of methanol in the mobile phase or adjust the time programme for the linear gradient elution.
Inject 10 l of methanol R as a blank, 10 l of the test solution and 10 l of reference solution (b).
solution (b) (0.2 per cent); the sum of the areas of all the peaks, apart from the principal peak, is not
solution (b) (0.3 per cent). Disregard any peak obtained with the blank and any peak with an area
less than 0.25 times the area of the principal peak in the chromatogram obtained with reference
solution (b).
100C to 105C for 4 h.
ASSAY
Dissolve 0.160 g in 70 ml of a mixture of 1 volume of glacial acetic acid R and 7 volumes of methyl
ethyl ketone R and titrate with 0.1M perchloric acid. Determine the end-point potentiometrically
(2.2.20).
STORAGE
IMPURITIES
N
Me
N
O
HO
A. 3-[2-[4-[(E)-(2,4-difluorophenyl)(hydroxyimino)methyl]piperidin-1-yl]ethyl]-2-methyl6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one,
N
Me
N
O
N
F
OH
B. 3-[2-[4-[(Z)-(2,4-difluorophenyl)(hydroxyimino)methyl]piperidin-1-yl]ethyl]-2-methyl6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one,
H OH
N
N
Me
N
O
N
and enantiomer
C. (9RS)-3-[2-[4-(6-fluoro-1,2-benzisoxazol-3-yl)piperidin-1-yl]ethyl]-9-hydroxy-2-methyl6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one,
35-8
N
Me
N
O
F
N
D. 3-[2-[4-(5-fluoro-1,2-benzisoxazol-3-yl)piperidin-1-yl]ethyl]-2-methyl-6,7,8,9-tetrahydro4H-pyrido[1,2-a]pyrimidin-4-one,
N
N
Me
N
H Me O
N
and enantiomer
E. (6RS)-3-[2-[4-(6-fluoro-1,2-benzisoxazol-3-yl)piperidin-1-yl]ethyl]-2,6-dimethyl-6,7,8,9tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one.
__________________________________________________________________________________________________________ Ph Eur
35-9
Ritodrine Hydrochloride
H
OH
H
N
,HCl
Me
HO
OH
and/or enantiomer
C17H21NO3,HCl
323.8
23239-51-2
Definition Ritodrine Hydrochloride is erythro-1-(p-hydroxyphenyl)-2-(4-hydroxyphenethylamino)propan-1-ol hydrochloride. It contains not less than 97.0% and not more than 103.0% of
C17H21NO3,HCl, calculated with reference to the dried substance.
Freely soluble in water; soluble in absolute ethanol; practically insoluble in acetone and in ether.
Identification
ritodrine hydrochloride (RS 313).
B. A 1% w/v solution yields reaction A characteristic of chlorides, Appendix VI.
Heavy metals 12 ml of a 10.0% w/v solution complies with limit test A for heavy metals, Appendix
VII. Use lead standard solution (2 ppm Pb) to prepare the standard (20 ppm).
phase. For solution (2) dilute 1 volume of solution (1) to 100 volumes with the mobile phase. For
solution (3) dissolve about 20 mg of the substance being examined in 50 ml of the mobile phase, add
5.6 ml of sulphuric acid and sufficient of the mobile phase to produce 100 ml, mix and heat at a
temperature of 85 for 2 hours. Cool to room temperature, carefully mix 10 ml of the cooled solution
with 8 ml of a 10% w/v solution of sodium hydroxide and allow to cool (generation of threodiastereoisomer).
(25 cm 4.6 mm) packed with stationary phase B (7 m) (Zorbax C8 is suitable), (b) as the mobile
phase with a flow rate of 2 ml per minute a mixture of a solution containing 6.6 g of diammonium
hydrogen orthophosphate and 1.1 g of sodium heptanesulphonate in 700 ml of water and 300 ml of
methanol; the mixture adjusted to pH 3.0 with an 85% v/v solution of orthophosphoric acid and (c) a
between the two principal peaks (ritodrine and the threo-diastereoisomer) is at least 1.5.
In the chromatogram obtained with solution (1), identify any peaks corresponding to tyramine
(relative retention time to ritodrine, 0.3), hexahydroketone II (0.65), hexahydroketone I (0.85),
threo-diastereoisomer (1.15) and aminoketone (2.3). In the chromatogram obtained with solution
(1), (a) the area of any peak corresponding to hexahydroketone II is not greater than 0.1 times the
area of the principal peak in the chromatogram obtained with solution (2) (0.3% [response relative to
ritodrine 0.35]), (b) the areas of any peaks corresponding to tyramine and aminoketone are not
greater than 0.2 times the area of the principal peak in the chromatogram obtained with solution (2)
(0.2% each), (c) the area of any peak corresponding to hexahydroketone I is not greater than 0.2
times the area of the principal peak in the chromatogram obtained with solution (2) (0.4% [response
relative to ritodrine 0.5]), (d) the area of any peak corresponding to the threo-diastereoisomer is not
(0.4%) and (e) the area of any other secondary peak is not greater than 0.1 times the area of the
principal peak in the chromatogram obtained with solution (2) (0.1%).
Solution (1) contains 0.02% w/v of the substance being examined in the mobile phase. Solution (2)
contains 0.02% w/v of ritodrine hydrochloride BPCRS in the mobile phase. For solution (3) dissolve
about 20 mg of the substance being examined in 50 ml of the mobile phase, add 5.6 ml of sulphuric
acid and sufficient of the mobile phase to produce 100 ml, mix and heat at a temperature of 85 for 2
hours. Cool to room temperature, carefully mix 10 ml of the cooled solution with 8 ml of a 10% w/v
solution of sodium hydroxide and allow to cool (generation of threo-diastereoisomer).
35-10
The chromatographic procedure described under Related substances may be used.
between the two principal peaks (ritodrine and the threo-diastereoisomer) is at least 1.5.
Calculate the content of C17H21NO3,HCl in the substance being examined from the chromatograms obtained and from the declared content of C17H21NO3, HCl in ritodrine hydrochloride BPCRS.
Storage Ritodrine Hydrochloride should be kept in an airtight container.
Preparations
Ritodrine Injection
Ritodrine Tablets
IMPURITIES
NH2
HO
A. tyramine
H OH H
N
HO
H Me
B. erythro-p-hydroxy--{1-{2-(4-oxocyclo-hexyl)ethylamino]ethyl}benzyl alcohol
(hexahydroketone I)
H OH
H
N
H Me
O
OH
C. erythro-1-(4-oxocyclohexyl)-2-[(p-hydroxy-phenethyl)amino]propan-1-ol (hexahydroketone
II)
H OH H
N
HO
H Me
OH
D. threo-1-(4-hydroxyphenyl)-2-(4-hydroxyphenethylamino)propan-1-ol (threo-diastereoisomer)
O
H
N
H Me
HO
OH
E. p-hydroxy--[(p-hydroxyphenethyl)-amino]propiophenone (aminoketone)
35-11
Rosemary Leaf
1/01
Rosemary Leaf complies with the requirements of the 3rd edition of the European Pharmacopoeia [1560].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Rosemary leaf consists of the whole or cut dried leaf of Rosmarinus officinalis L. It contains not less
than 12 ml/kg of essential oil, calculated with reference to the anhydrous drug.
CHARACTERS
Rosemary leaf has a strongly aromatic odour.
IDENTIFICATION
A. The leaves are sessile, tough, linear to linear-lanceolate, 10 mm to 40 mm long and 2 mm to
4 mm wide, and have recurved edges. The upper surface is dark green and glabrous, the lower
surface is greyish-green and densely tomentose with a prominent midrib.
B. Reduce to a powder (355). The powder is greyish-green to yellowish-green. Examine under a
microscope using chloral hydrate solution R. The powder shows fragments of lower epidermis with
beaded, straight to sinuous-walled cells and numerous diacytic stomata (2.8.3); fragments of the
upper epidermis with the cells straight-walled, slightly thickened and pitted, with an underlying
hypodermis composed of from one to several rows of large, irregular cells with thickened anticlinal
walls; below the hypodermis is a one or two-layered palisade arranged to form a large, crescentshaped area; numerous multicellular, covering trichomes which are mainly branched; glandular
trichomes of two types, the majority with a short, unicellular stalk and a radiate head composed of
eight cells, others, less abundant, with a unicellular stalk and a spherical, unicellular or bicellular
head.
Test solution. Dissolve 20 l of the oil obtained in the assay in 1 ml of hexane R.
Reference solution. Dissolve 5 mg of borneol R, 5 mg of bornyl acetate R and 10 l of cineole R in 1 ml of
hexane R.
volumes of ethyl acetate R and 95 volumes of toluene R. Allow the plate to dry in air. Spray with
vanillin reagent R. Heat the plate at 100C to 105C for 10 min and examine in daylight within
10 min. The chromatogram obtained with the reference solution shows in the lower third the violetblue zone due to borneol, above it the blue zone due to cineole and in the middle third the bluishgrey zone due to bornyl acetate. The chromatogram obtained with the test solution shows three zones
similar in position and colour to those in the chromatogram obtained with the reference solution.
The chromatogram obtained with the test solution also shows several violet-blue to violet-grey zones
in the lower third, a violet-pink zone in the middle third, a violet-grey zone in the upper third and an
intense violet zone near the solvent front.
TESTS
Foreign matter (2.8.2). Not more than 5 per cent of stems and not more than 2 per cent of other
foreign matter.
Water (2.2.13). Not more than 10.0 per cent, determined on the powdered drug (355) by distillation.
Total ash (2.4.16). Not more than 9 per cent.
ASSAY
Carry out the determination of essential oils in vegetable drugs (2.8.12). Use 25.0 g of the crushed
drug, a 1000 ml flask and 300 ml of water R as the distillation liquid. Distil at a rate of 2 ml/min to
3 ml/min for 3 h.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
35-12
Roxithromycin
H3C
N
Me
Me
Me
HO
HO
OH
Me
Me
Me
O
NMe2
OH
O
HO
Et
Me
Me
OMe
Me
C41H76N2O15
837
80214-83-1
Roxithromycin complies with the requirements of the 3rd edition of the European Pharmacopoeia [1146].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Roxithromycin contains not less than 97.0 per cent and not more than the equivalent of 101.0 per
cent of (3R,4S,5S,6R,7R,9R,11S,12R,13S,14R)-4-[(2,6-dideoxy-3-C-methyl-3-O-methyl--L-ribohexopyranosyl)oxy]-14-ethyl-7,12,13-trihydroxy-10-[(E)-[(2-methoxyethoxy)methoxy]imino]3,5,7,9,11,13-hexamethyl-6-[[3,4,6-trideoxy-3-(dimethylamino)--D-xylo-hexopyranosyl]oxy]oxacyclotetradecan-2-one, calculated with reference to the anhydrous and solvent-free substance.
CHARACTERS
A white, crystalline powder, very slightly soluble in water, freely soluble in acetone, in alcohol and in
methylene chloride. It is slightly soluble in dilute hydrochloric acid.
IDENTIFICATION
obtained with roxithromycin CRS. If the spectra obtained shows differences, prepare further spectra
using 90 g/l solutions in methylene chloride R.
B. Examine the chromatograms obtained in the assay. The retention time and size of the principal
peak in the chromatogram obtained with test solution (b) are approximately the same as those of the
Specific optical rotation (2.2.7). Dissolve 0.500 g in acetone R and dilute to 50.0 ml with the same
solvent. The specific optical rotation is 93 to 96, calculated with reference to the anhydrous,
solvent-free substance.
Related substances Examine by liquid chromatography (2.2.29) as prescribed under Assay using:
as mobile phase at a flow rate of 1.0 ml/min the following solutions:
Mobile phase A. To 510 ml of water R add 200 ml of a 170 g/l solution of ammonium dihydrogen
phosphate R; adjust to pH 5.3 with dilute sodium hydroxide solution R and add 315 ml of acetonitrile R,
Mobile phase B. A mixture of 300 volumes of water R and 700 volumes of acetonitrile R,
with the following elution programme:
35-13
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
038
3839
3990
100
100
10090
90
0
0
010
10
equilibration
isocratic
linear gradient
isocratic
Before each analysis equilibrate the chromatographic system with mobile phase A for 20 min.
N-demethylroxithromycin (roxithromycin impurity F), 15 min to 17 min and roxithromycin, 20 min
to 22 min. Inject 20 l of reference solution (c). The test is not valid unless in the chromatogram
obtained with reference solution (c), the resolution between the peaks corresponding to N-demethylroxithromycin and roxithromycin is at least 6.0, and the symmetry factor of the peak corresponding
to roxithromycin is at most 1.5. Adjust the flow rate of the mobile phase if necessary.
Inject 20 l of test solution (a) and 20 l of reference solution (b). In the chromatogram obtained
with test solution (a): the area of any peak, apart from the principal peak, is not greater than the area
sum of the areas of all the peaks, apart from the principal peak, is not greater than six times the area
of the principal peak in the chromatogram obtained with reference solution (b) (3 per cent).
obtained with reference solution (b). Any peak eluting, when using mobile phase B, with a relative
retention time of 3 (to the roxithromycin peak) corresponding to toluene, should be disregarded for
the purposes of the related substances test.
Ethanol and toluene (2.4.24). Not more than 0.2 per cent of ethanol and 0.1 per cent of toluene.
the limit test B for heavy metals (10 ppm). Prepare the standard using lead standard solution (1 ppm
Pb) obtained by diluting lead standard solution (100 ppm Pb) R with a mixture of 15 volumes of
water R and 85 volumes of acetone R.
ASSAY
Test solution (a). Dissolve 40.0 mg of the substance to be examined in mobile phase A and dilute to
Test solution (b). Dilute 5.0 ml of test solution (a) to 10.0 ml with mobile phase A.
Reference solution (a). Dissolve 20.0 mg of roxithromycin CRS in mobile phase A and dilute to 10.0 ml
with mobile phase A.
Reference solution (b). Dilute 1.0 ml of reference solution (a) to 100.0 ml with mobile phase A.
Reference solution (c). Dissolve 5.0 mg of roxithromycin CRS and 5.0 mg of N-demethylroxithromycin
CRS in mobile phase A and dilute to 50.0 ml with mobile phase A.
silica get for chromatography R (5 m),
as mobile phase at a flow rate of 1.5 ml/min a mixture of 510 ml of water R, 200 ml of a 170 g/l
solution of ammonium dihydrogen phosphate R adjusted to pH 5.3 with dilute sodium hydroxide
solution R and 315 ml of acetonitrile R,
N-demethylroxithromycin, 7 min to 10 min and roxithromycin, 10 min to 13 min. Inject 20 l of
reference solution (c). The assay is not valid unless: in the chromatogram obtained with reference
solution (c), the resolution between the peaks corresponding to N-demethylroxithromycin and
roxithromycin is at least 6.0; the symmetry factor of the peak corresponding to roxithromycin is at
most 1.5. Adjust the flow rate of the mobile phase if necessary. Inject alternately test solution (b) and
STORAGE
35-14
IMPURITIES
HO
Me
OMe
R=
Me
Me
Me
OH
Me
HO
Me
Me
Me
O
OH
O
RO
Et
O
NMe2
Me
OH
A. erythromycin A,
O
MeO
Me
Me
OH
Me
HO
Me
Me
Me
O
OH
O
HO
NMe2
Et
O
Me
OH
B. 4-O-de(2,6-dideoxy-3-C-methyl-3-O-methyl--ribo-hexopyranosyl)erythromycin 10-(E)[O-[(2-methoxyethoxy)methyl]oxime],
HO
Me
Me
OH
Me
HO
Me
Me
Me
O
OH
O
RO
NMe2
Et
O
Me
OH
C. erythromycin 10-(E)-oxime,
Me
OH
Me
Me
Me
O
NMe2
OMe
Me
HO
Me
RO
OH
O
Et
O
Me
OH
D. erythromycin 10-(Z)-[[(2-methoxyethoxy)methyl]oxime],
35-15
MeO
Me
Me
OH
Me
HO
Me
Me
Me
O
OH
O
Et
RO
NMe2
Me
OH
E. 3-O-demethylerythromycin 10-(E)-[O-[(2-methoxyethoxy)methyl]oxime] (roxithromycin C),

O
MeO
Me
Me
OH
Me
HO
Me
Me
Me
O
OH
O
Et
RO
NHMe
Me
OH
F. N-demethylerythromycin 10-(E)-[[O-(2-methoxyethoxy)-methyl]oxime],
O
MeO
Me
Me
OH
Me
HO
Me
Me
Me
O
OH
O
RO
Et
O
NMe2
Me
OH
G. erythromycin 10-(E)-[O-[[(2-methoxyethoxy)methoxy]methyl]oxime],
O
MeO
Me
Me
OH
Me
HO
Me
Me
Me
O
OH
O
RO
Et
O
NMe2
Me
OH
H. 13-deoxyerythromycin 10-(E)-[O-[(2-methoxyethoxy)methyl]oxime] (roxithromycin B),

O
MeO
Me
Me
OH
Me
HO
Me
O
NMe2
MeO
Me
Me
RO
OH
O
Et
O
Me
O
I. 2-O-[(2-methoxyethoxy)methyl]erythromycin 10-(E)-[O-[(2-methoxyethoxy)methyl]oxime].
__________________________________________________________________________________________________________ Ph Eur
35-16
Saccharin
O
NH
S
O
C7H5NO3S
O
183.2
81-07-2
Saccharin complies with the requirements of the 3rd edition of the European Pharmacopoeia [0947]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Saccharin contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent of
1,2-benzisothiazol-3(2H)-one 1,1-dioxide, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder or colourless crystals, sparingly soluble in boiling water and in alcohol,
slightly soluble in cold water and in ether. It dissolves in dilute solutions of alkali hydroxides and
carbonates.
IDENTIFICATION
A. A saturated solution, prepared without heating, turns blue litmus paper R red.
obtained with saccharin CRS. Examine the substances prepared as discs.
D. Mix about 10 mg with about 10 mg of resorcinol R, add 0.25 ml of sulphuric acid R and carefully
heat the mixture over a naked flame until a dark green colour is produced. Allow to cool, add 10 ml
of water R and dilute sodium hydroxide solution R until an alkaline reaction is produced. An intense
green fluorescence develops.
E. To 0.2 g add 1.5 ml of dilute sodium hydroxide solution R, evaporate to dryness and heat the residue
carefully until it melts, avoiding carbonisation. Allow to cool, dissolve the mass in about 5 ml of
water R, add dilute hydrochloric acid R until a weak acid reaction is produced and filter, if necessary.
To the filtrate add 0.2 ml of ferric chloride solution R2. A violet colour develops.
TESTS
Solution S Dissolve 5.0 g in 20 ml of a 200 g/l solution of sodium acetate R and dilute to 25 ml with
the same solution.
o- and p-Toluenesulphonamide Examine by gas chromatography (2.2.28), using caffeine R as the
internal standard.
Internal standard solution. Dissolve 25 mg of caffeine R in methylene chloride R and dilute to 100 ml
Test solution. Suspend 10.0 g of the substance to be examined in 20 ml of water R and dissolve using
5 ml to 6 ml of strong sodium hydroxide solution R. If necessary adjust the solution to pH 7 to 8 with
1M sodium hydroxide or 1M hydrochloric acid and dilute to 50 ml with water R. Shake the solution with
four quantities, each of 50 ml, of methylene chloride R. Combine the lower layers, dry over anhydrous
sodium sulphate R and filter. Wash the filter and the sodium sulphate with 10 ml of methylene
chloride R. Combine the solution and the washings and evaporate almost to dryness in a water-bath at
a temperature not exceeding 40C. Using a small quantity of methylene chloride R, quantitatively
transfer the residue into a suitable 10 ml tube, evaporate to dryness in a current of nitrogen and
dissolve the residue in 1.0 ml of the internal standard solution.
Blank solution. Evaporate 200 ml of methylene chloride R to dryness in a water-bath at a temperature
not exceeding 40C. Dissolve the residue in 1 ml of methylene chloride R.
35-17
Reference solution. Dissolve 20.0 mg of o-toluenesulphonamide R and 20.0 mg of p-toluenesulphonamide R in methylene chloride R and dilute to 100.0 ml with the same solvent. Dilute 5.0 ml of the
solution to 50.0 ml with methylene chloride R. Evaporate 5.0 ml of the final solution to dryness in a
current of nitrogen. Dissolve the residue in 1.0 ml of the internal standard solution.
a fused-silica column 10 m long and 0.53 mm in internal diameter, coated with polymethylphenylsiloxane R (film thickness 2 m),
a split injector (1/2),
detector at 250C.
Inject 1 l of the reference solution. Adjust the sensitivity of the detector so that the height of the
peak due to caffeine is not less than 50 per cent of the full scale of the recorder. The substances are
eluted in the following order: o-toluenesulphonamide, p-toluenesulphonamide and caffeine. The test
is not valid unless the resolution between the peaks due to o-toluenesulphonamide and p-toluenesulphonamide is at least 1.5.
Inject 1 l of the blank solution. In the chromatogram obtained, verify that there are no peaks with
the same retention times as the internal standard, o-toluenesulphonamide and
p-toluenesulphonamide.
Inject 1 l of the test solution and 1 l of the reference solution. If any peaks due to o-toluenesulphonamide and p-toluenesulphonamide appear in the chromatogram obtained with the test solution, the ratio of their areas to that of the internal standard is not greater than the corresponding ratio
in the chromatogram obtained with the reference solution (10 ppm of o-toluenesulphonamide and
10 ppm of p-toluenesulphonamide).
100C to 105C for 4 h.
ASSAY
Dissolve 0.150 g in 25 ml of alcohol R, with slight heating if necessary. Add 25 ml of water R and
0.25 ml of phenolphthalein solution R1. Titrate with 0.1M sodium hydroxide. Carry out a blank titration.
1 ml of 0.1M sodium hydroxide is equivalent to 18.32 mg of C7H5NO3S.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
35-18
Saccharin Sodium
Soluble Saccharin
NNa
S
O
C7H4NNaO3S
O
205.2
128-44-9
Saccharin Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia [0787].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Saccharin sodium contains not less than 99.0 per cent and not more than the equivalent of 101.0 per
cent of 2-sodio-1,2-benzisothiazol-3(2H)-one 1,1-dioxide, calculated with reference to the anhydrous
substance. It may contain a variable quantity of water.
CHARACTERS
A white, crystalline powder or colourless crystals, efflorescent in dry air, freely soluble in water,
sparingly soluble in alcohol, practically insoluble in ether.
IDENTIFICATION
A. To 5 ml of solution S (see Tests) add 3 ml of dilute hydrochloric acid R. A white precipitate is
formed. Filter and wash with water R. Dry the precipitate at 100C to 105C. The melting point
(2.2.14) is 226C to 230C.
obtained with saccharin sodium CRS. Dry the substances before use at 100C to 105C. Examine the
substances prepared as discs.
C. Mix about 10 mg with about 10 mg of resorcinol R, add 0.25 ml of sulphuric acid R and carefully
heat the mixture over a naked flame until a dark green colour is produced. Allow to cool, add 10 ml
of water R and dilute sodium hydroxide solution R until an alkaline reaction is produced. An intense
green fluorescence develops.
D. To 0.2 g add 1.5 ml of dilute sodium hydroxide solution R, evaporate to dryness and heat the residue
carefully until it melts, avoiding carbonisation. Allow to cool, dissolve the mass in about 5 ml of
water R, add dilute hydrochloric acid R until a weak acid reaction is produced and filter, if necessary.
To the filtrate add 0.2 ml of ferric chloride solution R2. A violet colour develops.
E. 0.5 ml of solution S gives reaction (b) of sodium (2.3.1).
TESTS
Appearance of solution Dissolve 5.0 g in 25 ml of carbon dioxide-free water R. The solution is clear
Acidity or alkalinity To 10.0 ml of solution S add 5.0 ml of 0.005M sulphuric acid. Heat to boiling
and cool. Add 0.1 ml of phenolphthalein solution R. Not less than 4.5 ml and not more than 5.5 ml of
0.01M sodium hydroxide is required to change the colour of the indicator to pink.
o- and p-Toluenesulphonamide Examine by gas chromatography (2.2.28), using caffeine R as the
internal standard.
Internal standard solution. Dissolve 25 mg of caffeine R in methylene chloride R and dilute to 100 ml
Test solution. Dissolve 10.0 g of the substance to be examined in 50 ml of water R. If necessary adjust
the solution to pH 7 to 8 by addition of 1M sodium hydroxide or 1M hydrochloric acid. Shake the solution with four quantities, each of 50 ml, of methylene chloride R. Combine the lower layers, dry over
anhydrous sodium sulphate R and filter. Wash the filter and the sodium sulphate with 10 ml of
35-19
methylene chloride R. Combine the solution and the washings and evaporate almost to dryness in a
water-bath at a temperature not exceeding 40C. Using a small quantity of methylene chloride R,
quantitatively transfer the residue into a suitable 10 ml tube, evaporate to dryness in a current of
nitrogen and add 1.0 ml of the internal standard solution.
Blank solution. Evaporate 200 ml of methylene chloride R to dryness in a water-bath at a temperature
not exceeding 40C. Dissolve the residue in 1 ml of methylene chloride R.
Reference solution. Dissolve 20.0 mg of o-toluenesulphonamide R and 20.0 mg of p-toluenesulphonamide R in methylene chloride R and dilute to 100.0 ml with the same solvent. Dilute 5.0 ml of the
solution to 50.0 ml with methylene chloride R. Evaporate 5.0 ml of the final solution to dryness in a
current of nitrogen. Take up the residue using 1.0 ml of the internal standard solution.
a fused-silica column 10 m long and 0.53 mm in internal diameter, coated with polymethylphenylsiloxane R (film thickness 2 m),
a split injector (1/2),
detector at 250C.
Inject 1 l of the reference solution. Adjust the sensitivity of the detector so that the height of the
peak due to caffeine is not less than 50 per cent of the full scale of the recorder. The substances are
eluted in the following order: o-toluenesulphonamide, p-toluenesulphonamide and caffeine. The test
is not valid unless the resolution between the peaks due to o-toluenesulphonamide and p-toluenesulphonamide is at least 1.5.
Inject 1 l of the blank solution. In the chromatogram obtained, verify that there are no peaks with
the same retention times as the internal standard, o-toluenesulphonamide and p-toluenesulphonamide.
Inject separately 1 l of the test solution and 1 l of the reference solution. If any peaks due to
o-toluenesulphonamide and p-toluenesulphonamide appear in the chromatogram obtained with the
test solution, the ratio of their areas to that of the internal standard is not greater than the corresponding ratio in the chromatogram obtained with the reference solution (10 ppm of o-toluenesulphonamide and 10 ppm of p-toluenesulphonamide).
ASSAY
Dissolve 0.150 g in 50 ml of anhydrous acetic acid R, with slight heating if necessary. Titrate with
1 ml of 0.1M perchloric acid is equivalent to 20.52 mg of C7H4NNaO3S.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
35-20
Sage Leaf
Sage Leaf complies with the requirements of the 3rd edition of the European Pharmacopoeia for Sage Leaf
(Salvia officinalis) [1370]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sage leaf (Salvia officinalis) consists of the whole or cut dried leaves of Salvia officinalis L. The whole
drug contains not less than 15 ml/kg of essential oil and the cut drug not less than 10 ml/kg of
essential oil, both calculated with reference to the anhydrous drug.
CHARACTERS
Sage leaf (Salvia officinalis) oil is rich in thujone.
Sage leaf (Salvia officinalis) has the macroscopic and microscopic characters described under
identification tests A and B.
IDENTIFICATION
A. The lamina of whole sage leaf (Salvia officinalis) is about 2 cm to 10 cm long and 1 cm to 2 cm
wide, oblong-ovate to elliptical. The margin is finely crenate to smooth. The apex is rounded or
subacute and the base is shrunken at the petiole and rounded or cordate. The upper surface is
greenish-grey and finely granular; the lower surface is white and pubescent and shows a dense
network of raised veinlets.
B. Reduce to a powder (355). The powder is light grey to brownish-green. Examine under a
microscope, using chloral hydrate solution R. The powder shows the following diagnostic characters:
very numerous articulated and bent trichomes with narrow elongated cells and a very thick cell at the
base as well as fragments of these trichomes; fragments of the upper epidermis with pitted, somewhat
polygonal cells; fragments of the lower epidermis with sinuous cells and numerous diacytic stomata
(2.8.3); rare single glandular trichomes with a uni- or bicellular head and a stalk consisting of one to
four cells; abundant glandular trichomes with a unicellular stalk and a head composed of eight
radiating cells with a raised common cuticle.
substance.
Test solution. Shake 0.30 g of the freshly powdered drug (355) with 5.0 ml of ether R for 5 min and
filter through 2 g of anhydrous sodium sulphate R.
Reference solution. Dissolve 5 l of thujone R and 2 l of cineole R in 20.0 ml of ether R.
Spray with anisaldehyde solution R and heat at 100C to 105C for 10 min. Examine the plate in
ultraviolet light at 365 nm. The chromatogram obtained with the reference solution shows in the
lower third the bright fluorescent zone of cineole and in the middle third the red fluorescent zone of
thujone. The chromatogram obtained with the test solution shows a bright fluorescent zone corresponding to cineole and a red fluorescent zone corresponding to thujone, approximatively equal or
greater in size and intensity to the zones in the chromatogram obtained with the reference solution.
TESTS
foreign matter.
ASSAY
Carry out the determination of essential oils in vegetable drugs (2.8.12). Use 20.0 g of drug, if
necessary cut immediately before the assay, a 500 ml flask, 250 ml of water R as the distillation liquid
and 0.5 ml of xylene R in the graduated tube. Distil at a rate of 2 ml/min to 3 ml/min for 2 h.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
35-21
Three-lobed Sage Leaf

1/01
Three-lobed Sage complies with the requirements of the 3rd edition of the European Pharmacopoeia [1561].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Three-lobed sage leaf consists of the whole or cut, dried leaves of Salvia fructicosa Mill.(S. triloba L.
fil). The whole drug contains not less than 18 ml/kg of essential oil, and the cut drug not less than
12 ml/kg of essential oil, both calculated with reference to the anhydrous drug.
CHARACTERS
Three-lobed sage leaf has a spicy odour when ground, similar to eucalyptus oil.
IDENTIFICATION
A. The lamina of the whole three-lobed sage leaf is about 8 mm to 50 mm long and about 4 mm to
20 mm wide, and oblong-ovate to lanceolate. The margin is finely crenate and undulate but
indistinct owing to the dense hairy covering on both surfaces. The base is obtuse and sometimes
bears one or two more or less developed lobes. The upper surface is grey-tomentose pubescent, the
lower surface is densely white-tomentose pubescent; the venation is indistinct. The densely whitetomentose pubescent petiole is about 1 mm in diameter.
B. Reduce to a powder (355). The powder is greyish-green and tomentose. Examine under a
microscope using chloral hydrate solution R. The powder shows very numerous, whole and fragmented, covering and glandular trichomes, scattered and attached to fragments of the epidermises;
covering trichomes articulated, uniseriate, thick-walled and bluntly tapering, those on the upper
epidermis straight, those on the lower epidermis longer, tortuous and more densely packed; glandular
trichomes, some with a unicellular or bicellular head and a stalk consisting of from one to four cells,
the majority having a short, unicellular stalk and a head composed of eight radiating cells with a
raised common cuticle; the upper epidermis with pitted and beaded cells, somewhat polygonal, with
a few diacytic stomata (2.8.3); the lower epidermis with sinuous-to wavy-walled cells and numerous
diacytic stomata.
C. Examine the chromatogram obtained in the test for thujone. The chromatogram obtained with
the test solution shows a blue zone corresponding to cineole, equal or greater in size and intensity to
the zone in the chromatogram obtained with the reference solution. Further zones are present.
TESTS
Thujone Examine by thin-layer chromatography (2.2.27), using a TLC silica gel plate R.
Test solution. Shake 0.3 g of the freshly powdered drug (355) with 5.0 ml of ethanol R for 5 min.
Reference solution. Dissolve 20 l of thujone R and 25 l of cineole R in 20 ml of ethanol R.
volumes of ethyl acetate R and 95 volumes of toluene R. Allow the plate to dry in air. Spray with a
200 g/l solution of phosphomolybdic acid R in ethanol R and heat at 100C to 105C for 10 min.
Examine in daylight. The chromatogram obtained with the reference solution shows in the middle
part a blue zone (cineole) and in the upper part a pink-blue zone (thujone). The chromatogram
obtained with the test solution shows no zone or a very faint pink-blue zone corresponding to
thujone.
foreign matter.
ASSAY
Carry out the determination of essential oils in vegetable drugs (2.8.12). Use 20.0 g of drug, if
necessary cut immediately before the assay, a 500 ml flask, 250 ml of water R as the distillation liquid
and 0.50 ml of xylene R in the graduated tube. Distil at a rate of 2 ml/min to 3 m/min for 2 h.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
35-22
Salbutamol
H
OH
NHBut
HO
CH2OH
and enantiomer
C13H21NO3
239.3
18559-94-9
Salbutamol complies with the requirements of the 3rd edition of the European Pharmacopoeia [0529]. These
Preparation
Salbutamol Pressurised Inhalation
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Salbutamol contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent
of (RS)-2-(1,1-dimethyl)ethylamino-1-[4-hydroxy-3-(hydroxymethyl)phenyl]ethanol, calculated with
CHARACTERS
A white or almost white, crystalline powder, sparingly soluble in water, soluble in alcohol, slightly
soluble in ether.
IDENTIFICATION
obtained with salbutamol CRS.
D. Dissolve about 10 mg in 50 ml of a 20 g/l solution of disodium tetraborate R. Add 1 ml of a 30 g/l
solution of aminopyrazolone R, 10 ml of methylene chloride R and 10 ml of a 20 g/l solution of
potassium ferricyanide R. Shake and allow to separate. An orange-red colour develops in the methylene
chloride layer.
TESTS
2.2.2).
coating substance.
Reference solution. Dissolve 10 mg of salbutamol CRS in methanol R and dilute to 100 ml with the same
solvent.
35-23
3 volumes of concentrated ammonia R, 18 volumes of water R, 35 volumes of ethyl acetate R, 45
volumes of 2-propanol R and 50 volumes of methyl isobutyl ketone R. Allow the plate to dry in air until
the odour of the solvent is no longer perceptible and spray with a 1 g/l solution of
methylbenzothiazolinone hydrazone hydrochloride R in a 90 per cent V/V solution of methanol R,
followed by a 20 g/l solution of potassium ferricyanide R in a mixture of 1 volume of concentrated
ammonia R1 and 3 volumes of water R, followed by a further spraying with a 1 g/l solution of methylbenzothiazolinone hydrazone hydrochloride R in a 90 per cent V/V solution of methanol R. Any spot in
than the spot in the chromatogram obtained with the reference solution (0.5 per cent).
Boron
Test solution. To 50 mg of the substance to be examined add 5 ml of a solution containing 13 g/l of
anhydrous sodium carbonate R and 17 g/l of potassium carbonate R. Evaporate to dryness on a waterbath and dry at 120C. Ignite the residue rapidly until the organic matter has been destroyed, allow
to cool and add 0.5 ml of water R and 3.0 ml of a freshly prepared 1.25 g/l solution of curcumin R in
glacial acetic acid R. Warm gently to effect solution, allow to cool and add 3.0 ml of a mixture
prepared by adding 5 ml of sulphuric acid R, slowly and with stirring, to 5 ml of glacial acetic acid R.
Mix and allow to stand for 30 min. Dilute to 100.0 ml with alcohol R, filter and use the filtrate.
Reference solution. Dissolve 0.572 g of boric acid R in 1000.0 ml of water R. Dilute 1.0 ml to 100.0 ml
with water R. To 2.5 ml of the solution add 5 ml of a solution containing 13 g/l of anhydrous sodium
carbonate R and 17 g/l of potassium carbonate R, and treat this mixture in the same manner as the test
solution.
Measure the absorbance (2.2.25) of the test solution and of the reference solution at the maximum at
about 555 nm. The absorbance of the test solution is not greater than that of the reference solution
(50 ppm).
100C to 105C.
ASSAY
STORAGE
__________________________________________________________________________________________________________ Ph Eur
35-24
Salbutamol Sulphate
H
OH
NHBut
,H2SO4
HO
CH2OH
and enantiomer
(C13H21NO3)2,H2SO4
576.7
51022-70-9
Salbutamol Sulphate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparations
Salbutamol Injection
Salbutamol Nebuliser Solution
Salbutamol Oral Solution
Salbutamol Pressurised Inhalation
Salbutamol Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Salbutamol sulphate contains not less than 98.0 per cent and not more than the equivalent of
101.0 per cent of di[(RS)-2-(1,1-dimethyl)ethylamino-1-[4-hydroxy-3-(hydroxymethyl)phenyl]ethanol] sulphate, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, freely soluble in water, slightly soluble in alcohol and in
ether, very slightly soluble in methylene chloride.
IDENTIFICATION
obtained with salbutamol sulphate CRS. Examine the substances prepared as discs using potassium
bromide R.
D. Dissolve about 10 mg in 50 ml of a 20 g/l solution of disodium tetraborate R. Add 1 ml of a 30 g/l
solution of aminopyrazolone R, 10 ml of a 20 g/l solution of potassium ferricyanide R and 10 ml of
methylene chloride R. Shake and allow to separate. An orange-red colour develops in the methylene
chloride layer.
TESTS
Acidity or alkalinity To 10 ml of solution S add 0.15 ml of methyl red solution R and 0.2 ml of
0.01M sodium hydroxide. The solution is yellow. Not more than 0.4 ml of 0.01M hydrochloric acid is
required to change the colour of the indicator to red.
35-25
coating substance.
the same solvent.
Test solution (b). Dilute 0.5 ml of test solution (a) to 100 ml with water R.
Reference solution. Dissolve 12 mg of salbutamol sulphate CRS in water R and dilute to 100 ml with the
same solvent.
3 volumes of concentrated ammonia R, 18 volumes of water R, 35 volumes of ethyl acetate R, 45
volumes of 2-propanol R and 50 volumes of methyl isobutyl ketone R. Allow the plate to dry in air until
the odour of the solvent is no longer perceptible and spray with 1 g/l solution of methylbenzothiazolinone hydrazone hydrochloride R in a 90 per cent V/V solution of methanol R, followed by a 20 g/l
solution of potassium ferricyanide R in a mixture of 1 volume of concentrated ammonia R1 and 3
volumes of water R, followed by a further spraying with a 1 g/l solution of methylbenzothiazolinone
hydrazone hydrochloride R in a 90 per cent V/V solution of methanol R. Any spot in the chromatogram
obtained with test solution (a) apart from the principal spot, is not more intense than the spot in the
Boron
Test solution. To 50 mg of the substance to be examined add 5 ml of a solution containing 13 g/l of
anhydrous sodium carbonate R and 17 g/l of potassium carbonate R. Evaporate to dryness on a waterbath and dry at 120C. Ignite the residue rapidly until the organic matter has been destroyed, allow
to cool and add 0.5 ml of water R and 3.0 ml of a freshly prepared 1.25 g/l solution of curcumin R in
glacial acetic acid R. Warm gently to effect solution, allow to cool and add 3.0 ml of a mixture
prepared by adding 5 ml of sulphuric acid R, slowly and with stirring, to 5 ml of glacial acetic acid R.
Mix and allow to stand for 30 min. Dilute to 100.0 ml with alcohol R, filter and use the filtrate.
Reference solution. Dissolve 0.572 g of boric acid R in 1000.0 ml of water R. Dilute 1.0 ml to 100.0 ml
with water R. To 2.5 ml of the solution add 5 ml of a solution containing 13 g/l of anhydrous sodium
carbonate R and 17 g/l of potassium carbonate R, and treat this mixture in the same manner as the test
solution.
Measure the absorbance (2.2.25) of the test solution and of the reference solution at the maximum at
about 555 nm. The absorbance of the test solution is not greater than that of the reference solution
(50 ppm).
100C to 105C.
ASSAY
STORAGE
__________________________________________________________________________________________________________ Ph Eur
35-26
Salicylic Acid
COOH
OH
C7H6O3
138.1
69-72-7
Salicylic Acid complies with the requirements of the 3rd edition of the European Pharmacopoeia [0366].
Preparations
Salicylic Acid Collodion
Salicylic Acid Ointment
Coal Tar and Salicylic Acid Ointment
Zinc and Salicylic Acid Paste
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Salicylic acid contains not less than 99.0 per cent and not more than the equivalent of 100.5 per cent
of 2-hydroxybenzenecarboxylic acid, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder or white or colourless, acicular crystals, slightly soluble in water, freely
soluble in alcohol and in ether, sparingly soluble in methylene chloride.
IDENTIFICATION
obtained with salicylic acid CRS.
C. Dissolve about 30 mg in 5 ml of 0.05M sodium hydroxide, neutralise if necessary and dilute to
20 ml with water R. 1 ml of the solution gives reaction (a) of salicylates (2.3.1).
TESTS
Solution S Dissolve 2.5 g in 50 ml of boiling distilled water R, cool and filter.
Appearance of solution Dissolve 1 g in 10 ml of alcohol R. The solution is clear (2.2.1) and colourless (Method II, 2.2.2).
Reference solution (a). Dissolve 10 mg of phenol R in the mobile phase and dilute to 100.0 ml with the
mobile phase.
Reference solution (b). Dissolve 25 mg of 4-hydroxyisophthalic acid R in the mobile phase and dilute to
Reference solution (c). Dissolve 50 mg of 4-hydroxybenzoic acid R in the mobile phase and dilute to
Reference solution (d). Dilute 1.0 ml of reference solution (a) to 10.0 ml with the mobile phase.
Reference solution (e). Dilute a mixture of 1.0 ml of each of reference solutions (a), (b) and (c) to
Reference solution (f). Dilute a mixture of 0.1 ml of each of reference solutions (a), (b) and (c) to
a stainless steel column 0.15 m long and 4.6 mm in internal diameter packed with nondeactivated octadecylsilyl silica gel for chromatography R (5 m),
as mobile phase at a flow rate of 0.5 ml per minute a mixture of 1 volume of glacial acetic acid R,
40 volumes of methanol R and 60 volumes of water R,
35-27
Inject 10 l of reference solutions (d) and (e). When the chromatograms are recorded in the
prescribed conditions, the retention times relative to phenol are: 4-hydroxybenzoic acid, about 0.70;
4-hydroxyisophthalic acid, about 0.90. Adjust the sensitivity of the detector so that the height of the
principal peak in the chromatogram obtained with reference solution (f) is at least 70 per cent of the
full scale of the recorder. The test is not valid unless: in the chromatogram obtained with reference
solution (e), the third peak corresponds to the phenol peak in the chromatogram obtained with
reference solution (d) and the resolution between the peaks corresponding to 4-hydroxyisophthalic
acid and to phenol is at least 1.0. If this resolution is not obtained adjust the quantity of acetic acid in
the mobile phase.
Inject 10 l of the test solution and 10 l of reference solution (f). In the chromatogram obtained
with the test solution: the areas of the peaks due to 4-hydroxybenzoic acid, 4-hydroxyisophthalic acid
and phenol are not greater than the areas of the corresponding peaks in the chromatogram obtained
with reference solution (f) (0.1 per cent for 4-hydroxybenzoic acid; 0.05 per cent for 4-hydroxyisophthalic acid and 0.02 per cent for phenol).
peak and the peaks due to 4-hydroxybenzoic acid, 4-hydroxyisophthalic acid and phenol, is not
greater than that of the peak due to 4-hydroxyisophthalic acid in the chromatogram obtained with
reference solution (f) (0.05 per cent); the sum of the areas of all the peaks, apart from the principal
peak, is not greater than twice the area of the peak due to 4-hydroxybenzoic acid in the chromatogram obtained with reference solution (f) (0.2 per cent). Disregard any peak with an area less than
0.01 times that of the principal peak in the chromatogram obtained with reference solution (f).
Sulphates Not more than 200 ppm. Dissolve 1.0 g in 5 ml of dimethylformamide R and add 4 ml of
water R. Mix thoroughly. Add 0.2 ml of dilute hydrochloric acid R and 0.5 ml of a 25 per cent m/m
solution of barium chloride R. After 15 min any opalescence in the solution is not more intense than
that in a standard prepared as follows: to 2 ml of sulphate standard solution (100 ppm SO4) R add
0.2 ml of dilute hydrochloric acid R, 0.5 ml of a 25 per cent m/m solution of barium chloride R, 3 ml of
water R and 5 ml of dimethylformamide R.
Heavy metals (2.4.8). Dissolve 2.0 g in 15 ml of alcohol R and add 5 ml of water R. 12 ml of the
solution complies with limit test B for heavy metals (20 ppm). Prepare the standard using lead
standard solution (2 ppm Pb) prepared by diluting lead standard solution (100 ppm Pb) R with a
mixture of 5 volumes of water R and 15 volumes of alcohol R.
desiccator.
ASSAY
Dissolve 0.120 g in 30 ml of alcohol R and add 20 ml of water R. Titrate with 0.1M sodium hydroxide,
using 0.1 ml of phenol red solution R as indicator.
STORAGE
IMPURITIES
COOH
HO
A. 4-hydroxybenzoic acid,
HOOC
COOH
OH
B. 4-hydroxyisophthalic acid,
C. phenol.
__________________________________________________________________________________________________________ Ph Eur
35-28
Selegiline Hydrochloride
1/01
Me
N
H
C13H18ClN
Me
CH
, HCl
223.7
14611-52-0
Selegiline Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparations
Selegiline Oral Solution
Selegiline Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Selegiline hydrochloride contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of N-methyl-N-[(1R)-1-methyl-2-phenylethyl]prop-2-yn-1-amine hydrochloride,
CHARACTERS
A white or almost white, crystalline powder, freely soluble in water and in methanol, slightly soluble
in acetone.
IDENTIFICATION
A. Dissolve 2.000 g in carbon dioxide-free water R and dilute to 20.0 ml with the same solvent. The
specific optical rotation (2.2.7) is 10.0 to 12.0, calculated with reference to the dried substance.
obtained with selegiline hydrochloride CRS. Examine the substances as discs prepared using potassium
chloride R.
TESTS
Reference solution (a). Dissolve 50.0 mg of selegiline hydrochloride CRS and 10.0 mg of butyl parahydroxybenzoate R in the mobile phase and dilute to 50.0 ml with the mobile phase. Dilute 1.0 ml to
as mobile phase at a flow rate of 1 ml/min a mixture prepared as follows: dilute 500 ml of
acetonitrile R to 1000.0 ml with a butylammonium acetate buffer solution pH 6.5 prepared by
dissolving 4 ml of butylamine R in 900 ml of water R and adjusting to pH 6.5 with acetic acid R
before diluting to 1000.0 ml with water R,
Inject 20 l of reference solution (a). The test is not valid unless, in the chromatogram obtained
with reference solution (a), the resolution between the peaks corresponding to selegiline and butyl
parahydroxybenzoate is greater than three. Inject 20 l of the test solution and 20 l of reference
solution (b). Continue the chromatography for 1.7 times the retention time of selegiline. In the
35-29
chromatogram obtained with the test solution: the area of any peak apart from the principal peak is
not greater than that of the peak in the chromatogram obtained with reference solution (b) (0.2 per
cent); and the sum of the areas of such peaks is not greater than 2.5 times the area of the principal
peak in the chromatogram obtained with reference solution (b) (0.5 per cent). Disregard any peak
due to chlorides and any peak with an area less than 0.1 times that of the principal peak in the
(S)-Selegiline Examine by liquid chromatography (2.2.29).
Test solution. Dissolve 20.0 mg of the substance to be examined in a mixture of 1 ml of 2-propanol R
and 10 l of butylamine R and dilute to 10.0 ml with the mobile phase.
Reference solution (a). Dissolve 8.0 mg of (RS)-selegiline hydrochloride CRS in a mixture of 10 l of
butylamine R and 1 ml of 2-propanol R and dilute to 10.0 ml with the mobile phase.
a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with silica gel OD
for chiral separation R,
as the mobile phase at a flow rate of 1 ml/min a mixture of 0.2 volumes of 2-propanol R and 99.8
volumes of cyclohexane R,
Inject 20 l of each solution. When the chromatograms are recorded in the prescribed conditions,
the retention time of (S)-selegiline is about 10 min. Adjust the sensitivity of the system so that the
height of the peaks in the chromatogram obtained with reference solution (b) is about 10 per cent of
the full scale of the recorder. The test is not valid unless in the chromatogram obtained with reference solution (a) the resolution between the peaks corresponding to (S)-selegiline and (R)-selegiline
is at least 1.5. If necessary, adjust the concentration of 2-propanol in the mobile phase. In the
chromatogram obtained with the test solution, any peak due to (S)-selegiline is not greater than the
area of the corresponding peak in the chromatogram obtained with reference solution (b) (0.5 per
cent).
pressure not exceeding 0.5 kPa.
ASSAY
1 ml of 0.1M perchloric acid is equivalent to 22.37 mg of C13H18ClN.
STORAGE
IMPURITIES
NHMe
H Me
and enantiomer
A. (2RS)-N-methyl-1-phenylpropan-2-amine [(RS)-metamphetamine],
B. (2R)-1-phenylpropan-2-amine (amphetamine),
C. (1RS,2SR)-2-amino-1-phenylpropan-1-ol (phenylpropanolamine),
H
N
CH
H Me
D. N-[(1R)-1-methyl-2-phenylethyl]prop-2-yn-1-amine (demethylselegiline),
Me
N
H
CH
Me
E. N-methyl-N-[(1S)-1-methyl-2-phenylethyl]prop-2-yn-1-amine [(S)-selegiline].
__________________________________________________________________________________________________________ Ph Eur
35-30
Selenium Sulphide
corrected 1/01
SeS2
143.1
7488-56-4
Selenium Sulphide complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Selenium Disulphide [1147]. These requirements are reproduced after the heading Definition below.
Action and use Used in treatment of dandruff and seborrhoeic dermatitis of the scalp.
Preparation
Selenium Sulphide Scalp Application
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Selenium disulphide contains not less than 52.0 per cent and not more than 55.5 per cent of Se.
CHARACTERS
A bright-orange or reddish-brown powder, practically insoluble in water.
IDENTIFICATION
A. Gently boil about 50 mg with 5 ml of nitric acid R for 30 min. Dilute to 50 ml with water R and
filter. To 5 ml of the filtrate add 10 ml of water R and 5 g of urea R. Heat to boiling, cool and add
1.5 ml of potassium iodide solution R. A yellow to orange colour is produced which darkens rapidly on
standing. This solution is used in identification test B.
B. Allow the coloured solution obtained under identification A to stand for 10 min and filter through
kieselguhr G R. 5 ml of the filtrate give reaction (a) of sulphates (2.3.1).
TESTS
Soluble selenium compounds To 10 g add 100 ml of water R, mix well, allow to stand for 1 h with
frequent shaking and filter. To 10 ml of the filtrate add 2 ml of a 115 g/l solution of anhydrous formic
acid R, dilute to 50 ml with water R and adjust to pH 2.0 to 3.0 with an 115 g/l solution of anhydrous
formic acid R. Add 2 ml of a 5 g/l solution of 3,3-diaminobenzidine tetrahydrochloride R. Allow to stand
for 45 min and then adjust to pH 6.0 to 7.0 with dilute ammonia R1. Shake the solution for 1 min
with 10 ml of toluene R and allow the phases to separate. The absorbance (2.2.25) of the upper layer
measured at 420 nm is not greater than that of a standard prepared at the same time and in the same
manner beginning at the words add 2 ml of an 115 g/l solution of anhydrous formic acid R and using
5 ml of selenium standard solution (1 ppm Se) R (5 ppm, calculated as Se).
ASSAY
To 0.100 g add 25 ml of fuming nitric acid R and heat on a water-bath for 1 h; a small insoluble
residue may remain. Cool and dilute to 100.0 ml with water R. To 25.0 ml add 50 ml of water R and
5 g of urea R and heat to boiling. Cool, add 7 ml of potassium iodide solution R, 3 ml of starch solution R and titrate immediately with 0.1M sodium thiosulphate. Carry out a blank titration.
1 ml of 0.1M sodium thiosulphate is equivalent to 1.974 mg of Se.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
35-31
Senega Root
Senega
Senega Root complies with the requirements of the 3rd edition of the European Pharmacopoeia [0202]. These
Action and use Expectorant.
When Powdered Senega Root is prescribed or demanded, material complying with the requirements
supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Senega root consists of the dried and usually fragmented root and root-crown of Polygala senega L. or
of certain other closely related species or of a mixture of these Polygala species.
CHARACTERS
Senega root has a faint, sweet odour, slightly rancid or reminiscent of methyl salicylate.
Reduced to a powder, it is irritant and sternutatory. Shaken with water, the powder produces a
copious froth.
It has the macroscopic and microscopic characters described under identification tests A, B and C.
IDENTIFICATION
A. The root crown is greyish-brown and wider than the root; it forms an irregular head consisting of
numerous remains of stems and tightly packed purplish-brown buds. The taproot is brown to yellow,
occasionally branched, sometimes flexuous, usually tortous without secondary roots, except in the
Japanese varieties and species, which contain numerous fibrous rootlets. The diameter is usually
1 mm to 8 mm at the crown, gradually tapering to the tip; the surface is transversely and
longitudinally striated and often shows a more or less distinct decurrent, elongated spiral keel. The
fracture is short and shows a yellowish cortex of varying thickness surrounding a paler central woody
area somewhat circular or irregular in shape depending on the species.
B. Examine under a microscope using chloral hydrate solution R. The transverse section of the root
shows cork formed from several layers of thin-walled cells, phelloderm of slightly collenchymatous
cells containing droplets of oil; the phloem and xylem arrangement is usually normal, especially near
the crown but where a keel is present this is formed by increased development of phloem; other
anomalous secondary development sometimes occurs, resulting in the formation of one or two large
wedge-shaped rays in the phloem and xylem, the parenchymatous cells of which contain droplets of
oil. The xylem is usually central and consists of vessels up to 60 m in diameter associated with
numerous thin-walled tracheids and a few small lignified parenchymatous cells.
C. Reduce to a powder (355). The powder is light brown. Examine under a microscope using chloral
hydrate solution R. The powder shows the following diagnostic characters: longitudinal fragments of
lignified tissue made up of pitted tracheids and somewhat larger vessels with numerous bordered pits
or with reticulate thickening; yellowish parenchyma and collenchymatous cells containing droplets of
oil; occasional fragments of cork, and of epidermal tissue with stomata and unicellular trichomes
from the bud scales. Crystals and stone cells are absent.
D. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance.
Test solution. To 1.0 g of the powdered drug (355) add 10 ml of alcohol (70 per cent V/V) R, boil
under a reflux condenser for 15 min, filter and allow to cool.
Reference solution. Dissolve 10 mg of aescin R in alcohol (70 per cent V/V) R and dilute to 10 ml with
the same solvent.
Apply separately to the plate as bands 20 mm by 3 mm 10 l of the test solution and 10 l and 40 l
of the reference solution. Develop over a path of 12 cm using the upper layer of a mixture of 10
volumes of glacial acetic acid R, 40 volumes of water R and 50 volumes of butanol R. Dry the plate at
100C to 105C, spray with about 10 ml of anisaldehyde solution R for a plate 200 mm square and
heat again at 100C to 105C, observing the chromatogram obtained with the test solution until red
zones (corresponding to saponosides) appear. In the chromatogram obtained with the test solution,
three to five red zones appear in the lower and middle parts, similar in position to the grey-violet
zones corresponding to aescin in the chromatogram obtained with the reference solution. Spray the
plate with about 10 ml of a 200 g/l solution of phosphomolybdic acid R in ethanol R and heat at 100C
to 105C, observing the chromatograms, until the zones corresponding to saponosides become blue.
The intensity and size of the zones in the chromatogram obtained with the test solution are between
those of the two bands corresponding to aescin in the chromatograms obtained respectively with
10 l and 40 l of the reference solution.
35-32
TESTS
STORAGE
Store protected from light and humidity.
__________________________________________________________________________________________________________ Ph Eur
35-33
Alexandrian Senna Fruit

Alexandrian Senna Fruit complies with the requirements of the 3rd edition of the European Pharmacopoeia
for Alexandrian Senna Pods [0207]. These requirements are reproduced after the heading Definition below.
Preparations
Senna Liquid Extract
Standardised Senna Granules
Senna Tablets
When Powdered Alexandrian Senna Fruit is prescribed or demanded, material complying with the
requirements below with the exception of Identification test A and the test for Foreign matter shall be
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Alexandrian senna pods consist of the dried fruit of Cassia senna L. (C. acutifolia Delile). They
contain not less than 3.4 per cent of hydroxyanthracene glycosides, calculated as sennoside B
(C42H38O20; Mr 863) with reference to the dried drug.
CHARACTERS
Alexandrian senna pods have a slight odour.
They have the macroscopic and microscopic characters described under identification tests A and
B.
IDENTIFICATION
A. They occur as flattened reniform pods, green to greenish-brown with brown patches at the positions corresponding to the seeds, usually 40 mm to 50 mm long and at least 20 mm wide. At one end
is a stylar point and at the other a short stalk. The pods contain six or seven flattened and obovate
seeds, green to pale brown, with a continuous network of prominent ridges on the testa.
B. Reduce to a powder (355). The powder is brown. Examine under a microscope using chloral
hydrate solution R. The powder shows the following diagnostic characters: epicarp with polygonal cells
and a small number of conical warty trichomes and occasional anomocytic or paracytic stomata
(2.8.3); fibres in two crossed layers accompanied by a crystal sheath of calcium oxalate prisms;
characteristic palisade cells in the seed and stratified cells in the endosperm; clusters and prisms of
calcium oxalate.
Test solution. To 0.5 g of the powdered drug (180) add 5 ml of a mixture of equal volumes of
alcohol R and water R and heat to boiling. Centrifuge and use the supernatant liquid.
Reference solution. Dissolve 10 mg of senna extract CRS in 1 ml of a mixture of equal volumes of
alcohol R and water R (a slight residue remains).
Apply separately to the plate as bands 20 mm by 2 mm, 10 l of each solution. Develop over a path
of 10 cm using a mixture of 1 volume of glacial acetic acid R, 30 volumes of water R, 40 volumes of
ethyl acetate R and 40 volumes of propanol R. Allow the plate to dry in air, spray with a 20 per cent
V/V solution of nitric acid R and heat at 120C for 10 min. Allow to cool and spray with a 50 g/l
solution of potassium hydroxide R in alcohol (50 per cent V/V) R until the zones appear. The principal
zones in the chromatogram obtained with the test solution are similar in position (sennosides B, A, D
and C in the order of increasing Rf value), colour and size to the principal zones in the chromatogram
obtained with the reference solution. Between the zones corresponding to sennosides D and C, a red
zone corresponding to rhein-8-glucoside may be visible. The zones corresponding to sennosides D
and C are faint in the chromatogram obtained with the test solution.
D. Place about 25 mg of the powdered drug (180) in a conical flask and add 50 ml of water R and
2 ml of hydrochloric acid R. Heat in a water-bath for 15 min, cool and shake with 40 ml of ether R.
Separate the ether, dry over anhydrous sodium sulphate R, evaporate 5 ml to dryness and to the cooled
residue add 5 ml of dilute ammonia R1. A yellow or orange colour develops. Heat on a water-bath for
2 min. A reddish-violet colour develops.
TESTS
Foreign matter (2.8.2). Not more than 1 per cent.
35-34
ASSAY
Place 0.150 g of the powdered drug (180) in a 100 ml flask. Add 30.0 ml of water R, mix, weigh and
place in a water-bath. Heat under a reflux condenser for 15 min. Allow to cool, weigh and adjust to
the original mass with water R. Centrifuge and transfer 20.0 ml of the supernatant liquid to a 150 ml
separating funnel. Add 0.1 ml of dilute hydrochloric acid R and shake with three quantities, each of
15 ml, of chloroform R. Allow to separate and discard the chloroform layer. Add 0.10 g of sodium
hydrogen carbonate R and shake for 3 min. Centrifuge and transfer 10.0 ml of the supernatant liquid
to a 100 ml round-bottomed flask with a ground-glass neck. Add 20 ml of ferric chloride solution R1
and mix. Heat for 20 min under a reflux condenser in a water-bath with the water level above that of
the liquid in the flask; add 1 ml of hydrochloric acid R and heat for a further 20 min, with frequent
shaking, to dissolve the precipitate. Cool, transfer the mixture to a separating funnel and shake with
three quantities, each of 25 ml, of ether R previously used to rinse the flask. Combine the ether layers
and wash with two quantities, each of 15 ml, of water R. Transfer the ether layers to a volumetric
flask and dilute to 100.0 ml with ether R. Evaporate 10.0 ml carefully to dryness and dissolve the
residue in 10.0 ml of a 5 g/l solution of magnesium acetate R in methanol R. Measure the absorbance
(2.2.25) at 515 nm using methanol R as the compensation liquid.
Calculate the percentage content of sennoside B from the expression:
A 125
.
m
i.e. taking the specific absorbance to be 240.
m = mass of the substance to be examined in grams.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
35-35
Tinnevelly Senna Fruit

Tinnevelly Senna Fruit complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Tinnevelly Senna Pods [0208]. These requirements are reproduced after the heading Definition below.
Preparations
Senna Liquid Extract
Senna Tablets
When Powdered Tinnevelly Senna Fruit is prescribed or demanded, material complying with the
requirements below with the exception of Identification test A and the test for Foreign matter shall be
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Tinnevelly senna pods consist of the dried fruit of Cassia angustifolia Vahl. They contain not less than
2.2 per cent of hydroxyanthracene glycosides, calculated as sennoside B (C42H38O20; Mr 863) with
CHARACTERS
Tinnevelly senna pods have a slight odour.
They have the macroscopic and microscopic characters described under identification tests A and
B.
IDENTIFICATION
A. They occur as flattened, slightly reniform pods, yellowish-brown to brown with dark brown
patches at the positions corresponding to the seeds, usually 35 mm to 60 mm long and 14 mm to
18 mm wide. At one end is a stylar point and at the other a short stalk. The pods contain five to eight
flattened and obovate seeds, green to pale brown, with incomplete, wavy, transverse ridges on the
testa.
B. Reduce to a powder (355). The powder is brown. Examine under a microscope using chloral
hydrate solution R. The powder shows the following diagnostic characters: epicarp with polygonal cells
and a small number of conical warty trichomes and occasional anomocytic or paracytic stomata
(2.8.3); fibres in two crossed layers accompanied by a crystal sheath of calcium oxalate prisms;
characteristic palisade cells in the seed and stratified cells in the endosperm; clusters and prisms of
calcium oxalate.
10 cm using a mixture of 1 volume of glacial acetic acid R, 30 volumes of water R, 40 volumes of ethyl
acetate R and 40 volumes of propanol R. Allow the plate to dry in air, spray with a 20 per cent V/V
solution of nitric acid R and heat at 120C for 10 min. Allow to cool and spray with a 50 g/l solution
of potassium hydroxide R in alcohol (50 per cent V/V) R until the zones appear. The principal zones in
the chromatogram obtained with the test solution are similar in position (sennosides B, A, D and C
in the order of increasing Rf value), colour and size to the principal zones in the chromatogram
obtained with the reference solution. Between the zones corresponding to sennosides D and C a red
zone corresponding to rhein-8-glucoside may be visible. The zones corresponding to sennosides D
and C are faint in the chromatogram obtained with the test solution.
TESTS
Foreign matter (2.8.2). Not more than 1 per cent.
Total ash (24.16). Not more than 9.0 per cent.
35-36
ASSAY
15 ml, of chloroform R. Allow to separate and discard the chloroform layer. Add 0.10 g of sodium
(2.2.25) at 515 nm using methanol R as the compensation liquid.
A 125
.
m
STORAGE
__________________________________________________________________________________________________________ Ph Eur
35-37
Senna Leaf
Senna Leaf complies with the requirements of the 3rd edition of the European Pharmacopoeia [0206]. These
Action and use Stimulant laxative.
Preparation
Standardised Senna Leaf Dry Extract.
When Powdered Senna Leaf is prescribed or demanded, material complying with the requirements
supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Senna leaf consists of the dried leaflets of Cassia senna L. (C. acutifolia Delile), known as Alexandrian
or Khartoum senna, or Cassia angustifolia Vahl, known as Tinnevelly senna, or a mixture of the two
species. It contains not less than 2.5 per cent of hydroxyanthracene glycosides, calculated as
sennoside B (C42H38O20; Mr 863) with reference to the dried drug.
CHARACTERS
Senna leaf has a slight characteristic odour.
IDENTIFICATION
A. C. senna occurs as greyish-green to brownish-green, thin, fragile leaflets, lanceolate, mucronate,
asymmetrical at the base, usually 15 mm to 40 mm long and 5 mm to 15 mm wide, the maximum
width being at a point slightly below the centre; the lamina is slightly undulant with both surfaces
covered with fine, short trichomes. Pinnate venation is visible mainly on the lower surface, with
lateral veins leaving the midrib at an angle of about 60 and anastomosing to form a ridge near the
margin.
Stomatal index (2.8.3): 1012.515.
C. angustifolia occurs as yellowish-green to brownish-green leaflets, elongated and lanceolate, slightly
asymmetrical at the base, usually 20 mm to 50 mm long and 7 mm to 20 mm wide at the centre.
Both surfaces are smooth with a very small number of short trichomes and are frequently marked
with transverse or oblique lines.
Stomatal index (2.8.3): 1417.520
B. Reduce to a powder (355). The powder is light green to greenish-yellow. Examine under a
microscope using chloral hydrate solution R. The powder shows the following diagnostic characters:
polygonal epidermal cells showing paracytic stomata (2.8.3); unicellular trichomes, conical in shape,
with warted walls, isolated or attached to fragments of epidermis; fragments of vascular bundles with
a crystal sheath of prismatic crystals of calcium oxalate; cluster crystals isolated or in fragments of
parenchyma.
10 cm using a mixture of 1 volume of glacial acetic acid R, 30 volumes of water R, 40 volumes of ethyl
acetate R and 40 volumes of propanol R. Allow the plate to dry in air, spray with a 20 per cent V/V
solution of nitric acid R and heat at 120C for 10 min. Allow to cool and spray with a 50 g/l solution
of potassium hydroxide R in alcohol (50 per cent V/V) R until the zones appear. The principal zones in
the chromatogram obtained with the test solution are similar in position (sennosides B, A, D and C
in the order of increasing Rf value), colour and size to the principal zones in the chromatogram
obtained with the reference solution. Between the zones corresponding to sennosides D and C a red
zone corresponding to rhein-8-glucoside may be visible.
35-38
TESTS
Foreign matter (2.3.2). Not more than 3 per cent of foreign organs and not more than 1 per cent of
foreign elements.
ASSAY
15 ml of chloroform R. Allow to separate and discard the chloroform layer. Add 0.10 g of sodium
(2.2.25) at 515 nm, using methanol R as the compensation liquid.
A 125
.
m
STORAGE
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35-39
Serine
H
HO
C3H7NO3
NH2
COOH
105.1
56-45-1
Serine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0788]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Serine contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of
(S)-2-amino-3-hydroxypropanoic acid, calculated with reference to the dried substance.
PRODUCTION
When Serine is produced by a process involving fermentation steps, it complies with the requirements of the monograph on Products of fermentation (1468).
CHARACTERS
A white or almost white, crystalline powder or colourless crystals, freely soluble in water, practically
insoluble in alcohol and in ether.
IDENTIFICATION
obtained with serine CRS. Examine the substances prepared as discs.
D. To 1 ml of a 10 g/l solution of the substance to be examined in a test-tube, add 5 ml of a 20 g/l
solution of sodium periodate R. Heat on a water-bath and collect the vapour on glass wool moistened
with water R and inserted in the opening of the test tube. After heating for 5 min, transfer the glass
wool to a test-tube containing 1 ml of a 15 g/l solution of chromotropic acid, sodium salt R and 3 ml of
sulphuric acid R. Heat on a water-bath for 10 min. A violet-red colour is produced.
TESTS
Specific optical rotation (2.2.7). Dissolve 2.50 g in dilute hydrochloric acid R and dilute to 25.0 ml
with the same acid. The specific optical rotation is +14.0 to +16.0, calculated with reference to the
dried substance.
gel plate R.
Test solution (a). Dissolve 0.10 g of the substance to be examined in 0.1M hydrochloric acid and dilute
Reference solution (a). Dissolve 10 mg of serine CRS in 0.1M hydrochloric acid and dilute to 50 ml with
the same acid.
Reference solution (c). Dissolve 10 mg of methionine CRS and 10 mg of serine CRS in 0.1M hydrochloric
acid and dilute to 25 ml with the same acid.
35-40
spots.
To the inner wall of the upper watch glass stick a piece of red litmus paper R, 5 mm square and wetted
Briefly triturate with a glass rod. Immediately close the cell by putting the two watch glasses together.
(100 ppm NH4) R, 0.5 ml of water R and 0.30 g of heavy magnesium oxide R (200 ppm).
100C to 105C.
ASSAY
Dissolve 0.100 g in 3 ml of anhydrous formic acid R. Add 30 ml of anhydrous acetic acid R. Using
0.1 ml of naphtholbenzein solution R as indicator, titrate with 0.1M perchloric acid until the colour
changes from brownish-yellow to green.
STORAGE
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35-41
Sertaconazole Nitrate
Cl
Cl
H
N
O
,HNO3
S
Cl
and enantiomer
C20H15Cl3N2OS,HNO3
500.8
99592-39-9
Sertaconazole Nitrate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sertaconazole nitrate contains not less than 98.5 per cent and not more than the equivalent of
101.0 per cent of (RS)-1-[2-[(7-chloro-1-benzothiophen-3-yl)methoxy]-2-(2,4-dichlorophenyl)ethyl]-1H-imidazole nitrate, calculated with reference to the anhydrous substance.
CHARACTERS
A white or almost white powder, practically insoluble in water, soluble in methanol, sparingly soluble
in alcohol and in methylene chloride.
IDENTIFICATION
B. Dissolve 0.1 g in methanol R and dilute to 100 ml with the same solvent. Dilute 10 ml of the
solution to 100 ml with methanol R. Examined between 240 nm and 320 nm (2.2.25), the solutions
shows three absorption maxima, at 260 nm, 293 nm and 302 nm. The ratio of the absorbance
measured at the maximum at 302 nm to that measured at the maximum at 293 nm is 1.16 to 1.28.
obtained with sertaconazole nitrate CRS. Dry the substances at 100C to 105C for 2 h and examine
as discs prepared using potassium bromide R.
D. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance.
Test solution. Dissolve 40 mg of the substance to be examined in a mixture of 1 volume of concentrated
ammonia R and 9 volumes of methanol R and dilute to 10 ml with the same mixture of solvents.
Reference solution (a). Dissolve 40 mg of sertaconazole nitrate CRS in a mixture of 1 volume of concentrated ammonia R and 9 volumes of methanol R and dilute to 10 ml with the same mixture of solvents.
Reference solution (b). Dissolve 20 mg of miconazole nitrate CRS in reference solution (a) and dilute to
volume of concentrated ammonia R, 40 volumes of toluene R and 60 volumes of dioxan R. Dry the plate
in a current of air for 15 min and expose the plate to iodine vapour for 30 min. The principal spot in
the chromatogram obtained with the test solution is similar in position, colour and size to the spot in
E. About 1 mg gives the reaction of nitrates (2.3.1).
TESTS
2.2.2).
35-42
Reference solution (b). Dissolve 5.0 mg of sertaconazole nitrate CRS and 5.0 mg of miconazole nitrate
a stainless steel column 0.25 m long and 4.0 mm in internal diameter packed with nitrile silica gel
for chromatography R1 (10 m),
63 volumes of a 1.5 g/l solution of sodium dihydrogen phosphate R,
Inject 20 l of reference solution (a) and 20 l of reference solution (b). When the chromatograms
are recorded in the prescribed conditions, the retention times are: nitrate ion, about 1 min;
miconazole, about 17 min; sertaconazole, about 19 min. When using a recorder, adjust the sensitivity
of the system so that the height of the principal peak in the chromatogram obtained with reference
solution (a) is not less than 25 per cent of the full scale of the recorder. The test is not valid unless in
the chromatogram obtained with reference solution (b), the resolution between the peaks due to
miconazole and sertaconazole is at least 2.0.
Inject separately 20 l of the test solution and 20 l of reference solution (a). Continue the chromatography for 1.3 times the retention time of the principal peak. In the chromatogram obtained with
the test solution: the area of any peak, apart from the principal peak and the peak due to nitrate ion,
is not greater than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.25 per cent) and the sum of the areas of such peaks, is not greater than twice the area of
the principal peak in the chromatogram obtained with the reference solution (a) (0.5 per cent).
Disregard any peak with an area less than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (a).
of water.
ASSAY
Dissolve 0.400 g in 50 ml of a mixture of equal volumes of anhydrous acetic acid R and methyl ethyl
ketone R. Titrate with 0.1M perchloric acid, determining the end-point potentiometrically (2.2.20).
1 ml of 0.1M perchloric acid is equivalent to 50.08 mg of C20H16Cl3N3O4S.
STORAGE
IMPURITIES
A. 1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethanol,
B. 3-(bromomethyl)-7-chloro-1-benzothiophene,
C. (7-chloro-1-benzothiophen-3-yl)methanol.
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35-43
Refined Sesame Oil

Sesame Oil
Refined Sesame Oil complies with the requirements of the 3rd edition of the European Pharmacopoeia [0433].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sesame oil is the fatty oil obtained from the ripe seeds of Sesamum indicum L. by expression or
extraction. It is then refined. Improved colour and odour may be obtained by further refining. It may
contain a suitable antioxidant.
CHARACTERS
A clear, light yellow liquid, almost colourless, practically insoluble in alcohol, miscible with light
petroleum.
It solidifies to a soft mass at about 4C.
IDENTIFICATION
Second identification: A, B.
A. It complies with the test for refractive index (see Tests).
B. Carry out the identification of fatty oils by thin-layer chromatography (2.3.2). The chromatogram
obtained is similar to the type chromatogram for sesame oil.
C. It complies with the test for composition of triglycerides (see Tests).
TESTS
Acid value (2.5.1). Not more than 0.6, determined on 10.0 g. If intended for use in the manufacture
of parenteral dosage forms, not more than 0.3.
Cottonseed oil Mix 5 ml in a test-tube with 5 ml of a mixture of equal volumes of pentanol R and a
10 g/l solution of sulphur R in carbon disulphide R. Warm the mixture carefully until the carbon
disulphide is expelled, and immerse the tube to one-third of its depth in a boiling, saturated solution
of sodium chloride R. No reddish colour develops within 15 min.
Composition of triglycerides Examine by liquid chromatography (2.2.29).
Test solution. Into a 10 ml volumetric flask, weigh 0.200 g and dilute to 10.0 ml with the mobile phase.
two stainless steel columns 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 m) in series,
as mobile phase at a flow rate of 1.0 ml/min a mixture of 1 volume of methylene chloride R and 2
volumes of acetonitrile R,
as detector a refractometer.
Inject 20 l of the test solution. Identify the peaks from the type chromatogram (Figure 433.-1).
The fatty acid radicals are designated as linolenic (Ln), linoleic (L), oleic (O), palmitic (P) and stearic
(S).
Calculate the percentage content of triglycerides from the areas of the peaks in the chromatogram
obtained with the test solution by the normalisation procedure.
The composition of triglycerides is the following:
LLL: 7.0 per cent to 19.0 per cent,
OLL: 13.0 per cent to 30.0 per cent,
PLL: 5.0 per cent to 9.0 per cent,
OOL: 14.0 per cent to 25.0 per cent,
POL: 8.0 per cent to 16.0 per cent,
OOO: 5.0 per cent to 14.0 per cent,
SOL: 2.0 per cent to 8.0 per cent,
POO: 2.0 per cent to 10.0 per cent.
35-44
0.05 per cent, determined on 5.0 g by the semi-micro determination of water.
STORAGE
Refined sesame oil intended for use in the manufacture of parenteral dosage forms is stored under
an inert gas in an airtight container.
When the container has been opened, its contents are to be used as soon as possible. Any part of
the contents not used at once is protected by an atmosphere of an inert gas.
LABELLING
The label states:
whether the oil is obtained by expression or extraction,
where applicable, the name and amount of any added antioxidant,
forms,
where applicable, the name of the inert gas used.
The type chromatogram is given for information and guidance only; it does not form a mandatory part of the
monograph.
Fig. 433-1 Sesame oil. Type chromatogram for the composition of triglycerides
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35-45
Shellac
corrected 1/01
Shellac complies with the requirements of the 3rd edition of the European Pharmacopoeia [1149]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Shellac is a purified material obtained from the resinous secretion of the female insect Kerria lacca
(Kerr) Lindinger (Laccifer lacca Kerr). There are four types of shellac depending on the nature of the
treatment of crude secretion (seedlac): wax-containing shellac, bleached shellac, dewaxed shellac and
bleached, dewaxed shellac.
Wax-containing shellac is obtained from seedlac; it is purified by filtration of the molten substance
and/or by hot extraction using a suitable solvent.
Bleached shellac is obtained from seedlac by treatment with sodium hypochlorite after dissolution in
a suitable alkaline solution, precipitation by dilute acid and drying.
Dewaxed shellac is obtained from wax-containing shellac or seedlac by treatment with a suitable
solvent and removal of the insoluble wax by filtering.
Bleached, dewaxed shellac is obtained from wax-containing shellac or seedlac by treatment with
sodium hypochlorite after dissolution in a suitable alkaline solution; the insoluble wax is removed by
filtration. It is precipitated by dilute acid and dried.
CHARACTERS
Shellac occurs as brownish-orange or yellow, shining, translucent, hard or brittle, more or less thin
flakes (wax-containing shellac and dewaxed shellac), or a creamy white or brownish-yellow powder
(bleached shellac and bleached, dewaxed shellac).
Shellac is practically insoluble in water, partly soluble in ether. With ethanol, it gives a more or less
opalescent solution (wax containing shellac and bleached shellac) or a clear solution (dewaxed shellac
and bleached, dewaxed shellac). When warmed, it is sparingly soluble or soluble in alkaline solutions.
IDENTIFICATION
A. Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable silica gel
Test solution. Heat 0.25 g of the powdered substance (500) on a water-bath with 2 ml of dilute sodium
hydroxide solution R for 5 min. Cool, add 5 ml of ethyl acetate R and slowly, with stirring, 2 ml of dilute
acetic acid R. Shake and filter the upper layer through anhydrous sodium sulphate R.
Reference solution. Dissolve 6.0 mg of aleuritic acid R in 1.0 ml of methanol R, heating slightly if
necessary.
Apply to the plate as bands 10 l of each solution. Develop twice over a path of 15 cm using a
mixture of 1 volume of acetic acid R, 8 volumes of methanol R, 32 volumes of methylene chloride R and
60 volumes of ethyl acetate R. Allow the plate to dry in air. Spray the plate with anisaldehyde solution R. Heat the plate at 100C to 105C for 5 min to 10 min and examine in daylight. The chromatogram obtained with the test solution shows several coloured zones, one of which is similar in
position and colour to the zone in the chromatogram obtained with the reference solution. Above this
zone the chromatogram obtained with the test solution shows a pink zone and below it several violet
zones. Below the zone corresponding to aleuritic acid, there is a light blue zone (shellolic acid)
accompanied by zones of the same colour but of lower intensity. Other faint grey and violet zones
may be visible.
B. Examine the chromatograms obtained in the test for colophony. For wax-containing shellac, in the
chromatogram obtained with the test solution, a more or less strong bluish-grey zone is visible, just
above the zone due to thymolphthalein in the chromatogram obtained with the reference solution.
For dewaxed shellac, no such zone is visible just above the zone due to thymolphthalein in the
TESTS
Colophony Examine by thin-layer chromatography (2.2.27), as described under identification test A
with the following modifications:
Test solution. Dissolve 50 mg of the powdered substance (500), while heating, in a mixture of 0.5 ml
of methylene chloride R and 0.5 ml of methanol R.
35-46
Reference solution. Dissolve 2.0 mg of thymolphthalein R in 1.0 ml of methanol R.
Examine the plate in ultraviolet light at 254 nm. Mark the quenching zones in the chromatogram
obtained with the test solution that have similar Rf values to that of the quenching zone of
thymolphthalein in the chromatogram obtained with the reference solution. Spray with anisaldehyde
solution R, heat the plate at 100C to 105C for 5 min to 10 min and examine in daylight. The
chromatogram obtained with the reference solution shows a principal zone with a reddish-violet
colour (thymolphthalein). None of the quenching zones in the chromatogram obtained with the test
solution that have an Rf value similar to the zone due to thymolphthalein in the reference solution
show a more or less strong violet or brownish colour (colophony). Disregard any faint violet zone at
this level that does not show quenching before spraying and heating.
Acid value (2.5.1). 65 to 95, calculated with reference to the dried substance. Examine 1.00 g of the
coarsely ground substance. Determine the end-point by potentiometry (2.2.20).
Arsenic (2.4.2). Introduce 0.33 g of the substance to be examined and 5 ml of sulphuric acid R into a
combustion flask. Carefully add a few millilitres of strong hydrogen peroxide solution R and heat to
boiling until a clear, colourless solution is obtained. Continue heating to eliminate the water and as
much sulphuric acid as possible and dilute to 25 ml with water R. The solution complies with limit
test A for arsenic (3 ppm).
Loss on drying (2.2.32). Not more than 2.0 per cent for unbleached shellac and not more than
6.0 per cent for bleached shellac, determined on 1.000 g of the powdered substance (500) by drying
in an oven at 40C to 45C for 24 h.
STORAGE
Store in a well-closed container, protected from light. Store bleached shellac and bleached, dewaxed
shellac at a temperature not exceeding 15C.
LABELLING
The label indicates the type of shellac.
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35-47
Colloidal Anhydrous Silica

SiO2
60.1
7631-86-9
Colloidal Anhydrous Silica complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Colloidal anhydrous silica contains not less than 99.0 per cent and not more than the equivalent of
100.5 per cent of SiO2, determined on the ignited substance.
CHARACTERS
A light, fine, white, amorphous powder, with a particle size of about 15 nm, practically insoluble in
water and in mineral acids except hydrofluoric acid. It dissolves in hot solutions of alkali hydroxides.
IDENTIFICATION
About 20 mg gives the reaction of silicates (2.3.1).
TESTS
pH (2.2.3). Shake 1.0 g with 30 ml of carbon dioxide-free water R. The pH of the suspension is 3.5 to
5.5.
Chlorides (2.4.4). To 1.0 g add a mixture of 20 ml of dilute nitric acid R and 30 ml of water R and
heat on a water-bath for 15 min, shaking frequently. Dilute to 50 ml with water R if necessary, filter
and cool. 10 ml of the filtrate diluted to 15 ml with water R complies with the limit test for chlorides
(250 ppm).
Heavy metals (2.4.8). Suspend 2.5 g in sufficient water R to produce a semi-fluid slurry. Dry at
140C. When the dried substance is white, break up the mass with a glass rod. Add 25 ml of 1M
hydrochloric acid and boil gently for 5 min, stirring frequently with the glass rod. Centrifuge for
20 min and filter the supernatant liquid through a membrane filter. To the residue in the centrifuge
tube add 3 ml of dilute hydrochloric acid R and 9 ml of water R and boil. Centrifuge for 20 min and
filter the supernatant liquid through the same membrane filter. Wash the residue with small quantities of water R, combine the filtrates and washings and dilute to 50 ml with water R. To 20 ml of the
solution add 50 mg of ascorbic acid R and 1 ml of concentrated ammonia R. Neutralise with dilute
ammonia R2. Dilute to 25 ml with water R. 12 ml of the solution complies with limit test A for heavy
Loss on ignition Not more than 5.0 per cent, determined on 0.200 g by ignition in a platinum
crucible at 900C for 2 h. Allow to cool in a desiccator before weighing.
ASSAY
To the residue obtained in the test for loss on ignition add 0.2 ml of sulphuric acid R and sufficient
alcohol R to moisten the residue completely. Add 6 ml of hydrofluoric acid R and evaporate to dryness
on a hot-plate at 95C to 105C, taking care to avoid loss from sputtering. Wash down the sides of
the dish with 6 ml of hydrofluoric acid R and evaporate to dryness. Ignite at 900C, allow to cool in a
desiccator and weigh.
The difference between the mass of the final residue and the mass of the residue obtained in the
test for loss on ignition gives the amount of SiO2 in the quantity of the substance to be examined
used.
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35-48
Colloidal Hydrated Silica

SiO2,xH2O
60.1(anhydrous)
63231-67-4
Colloidal Hydrated Silica complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Colloidal hydrated silica contains not less than 98.0 per cent and not more than the equivalent of
100.5 per cent of SiO2 (Mr 60.1), determined on the ignited substance.
CHARACTERS
A white or almost white, light, fine, amorphous powder, practically insoluble in water and in mineral
acids, with the exception of hydrofluoric acid. It dissolves in hot solutions of alkali hydroxides.
IDENTIFICATION
A. About 20 mg gives the reaction of silicates (2.3.1).
B. When heated in an oven at 100C to 105C for 2 h, it shows a loss of mass greater than 3 per
cent.
TESTS
Solution S To 2.5 g add 50 ml of hydrochloric acid R and mix. Heat on a water-bath for 30 min,
stirring from time to time. Maintain the original volume by adding dilute hydrochloric acid R. Evaporate to dryness. Add to the residue a mixture of 8 ml of dilute hydrochloric acid R and 24 ml of water R.
Heat to boiling and filter under reduced pressure through a sintered-glass filter (16). Wash the
residue on the filter with a hot mixture of 3 ml of dilute hydrochloric acid R and 9 ml of water R. Wash
with small quantities of water R, combine the filtrate and washings and dilute to 50 ml with water R.
pH (2.2.3). Suspend 1.0 g in 30 ml of a 75 g/l solution of potassium chloride R. The pH of the
suspension is 4.0 to 7.0.
Water-absorption capacity In a mortar, triturate 5 g with 5 ml of water R, added drop by drop.
The mixture remains powdery.
Substances soluble in hydrochloric acid In a platinum dish, evaporate to dryness 10.0 ml of
solution S and dry to constant mass at 100C to 105C. The mass of the residue is not more than
10 mg (2.0 per cent).
Chlorides (2.4.4). Heat 0.5 g with 50 ml of water R on a water-bath for 15 min. Dilute to 100 ml
with water R and centrifuge at 1500 g for 5 min. 10 ml of the supernatant solution diluted to 15 ml
with water R complies with the limit test for chlorides (0.1 per cent).
Sulphates (2.4.13). Dilute 2 ml of solution S to 100 ml with distilled water R. 15 ml of the solution
complies with the limit test for sulphates (1 per cent).
Iron (2.4.9). To 2 ml of solution S add 28 ml of water R. 10 ml of the solution complies with the
Heavy metals (2.4.8). To 20 ml of solution S add 50 mg of hydroxylamine hydrochloride R and 1 ml
of concentrated ammonia R. Adjust to pH 3.5 by adding dilute ammonia R2, monitoring the pH potentiometrically. Dilute to 25 ml with water R. 12 ml of the solution complies with limit test A for heavy
Loss on ignition Not more than 20.0 per cent, determined on 0.200 g in a platinum crucible by
heating at 100C to 105C for 1 h and then at 900C for 2 h.
ASSAY
To the residue obtained in the test for loss on ignition add 0.2 ml of sulphuric acid R and a quantity of
alcohol R sufficient to moisten the residue completely. Add 6 ml of hydrofluoric acid R and evaporate
to dryness at 95C to 105C, taking care to avoid loss from sputtering. Wash the inside of the dish
with 6 ml of hydrofluoric acid R and evaporate to dryness again. Ignite at 900C, allow to cool in a
desiccator and weigh. The difference between the mass of the final residue and that of the mass
obtained in the test for loss on ignition corresponds to the mass of SiO2 in the test sample.
STORAGE
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35-49
Dental-type Silica
1/01
Dental-type Silica complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Dental Type Silica [1562] . These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Dental type silica is an amorphous silica (precipitated, gel or obtained by flame hydrolysis). It
contains not less than 94.0 per cent and not more than 100.5 per cent of SiO2 determined on the
ignited substance.
CHARACTERS
A white or almost white, light, fine amorphous powder, practically insoluble in water and in mineral
acids. It dissolves in hydrofluoric acid and hot solutions of alkali hydroxides.
IDENTIFICATION
About 20 mg gives the reaction of silicates (2.3.1).
TESTS
Solution S To 2.5 g add 50 ml of hydrochloric acid R and mix. Heat on a water-bath for 30 min,
stirring from time to time. Evaporate to dryness. Add to the residue a mixture of 8 ml of dilute
hydrochloric acid R and 24 ml of water R. Heat to boiling and filter under reduced pressure through a
sintered-glass filter (16). Wash the residue on the filter with a hot mixture of 3 ml of dilute hydrochloric acid R and 9 ml of water R. Wash with small quantities of water R, combine the washings and
the filtrate and dilute to 50 ml with water R.
pH (2.2.3). Suspend 5 g in a mixture of 5 ml of a 7.46 g/l solution of potassium chloride R and 90 ml
of carbon dioxide-free water R. The pH of the suspension is 3.2 to 8.9.
Chlorides Examine by liquid chromatography (2.2.29) as prescribed under sulphates.
Inject 25 l of the test solution and 25 l of the reference solution. In the chromatogram obtained
with the test solution the area of any peak corresponding to chloride is not greater than that of the
corresponding peak in the chromatogram obtained with the reference solution (0.3 per cent).
Sulphates. Examine by liquid chromatography (2.2.29).
Test solution. To 0.625 g add 30 ml of water R and boil for 2 h. Allow to cool and quantitatively
transfer to a 50 ml graduated flask. Dilute to 50.0 ml with water R. Dilute 5.0 ml of the supernatant
to 50.0 ml with water R and filter through a membrane filter (nominal pore size: 0.45 m).
Reference solution. Dissolve 0.50 g of anhydrous sodium sulphate R and 0.062 g of sodium chloride R in
water R and dilute to 1000.0 ml with water R. Dilute 5.0 ml of this solution to 50.0 ml with water R.
a non-metallic column 0.25 m long and 4.6 mm in internal diameter packed with a suitable
anion-exchange resin (30 m to 50 m),
sodium carbonate R and 0.05 g of sodium hydrogen carbonate R in water R and dilute to 1000 ml
with the same solvent,
a conductivity detector,
a loop injector.
Inject 25 l of the test solution and 25 l of the reference solution. When the chromatograms are
recorded in the prescribed conditions, the retention times are: sulphate about 8 min and chloride
about 4 min.
In the chromatogram obtained with the test solution the area of any peak corresponding to
sulphate is not greater than that of the corresponding peak in the chromatogram obtained with the
reference solution (4.0 per cent calculated as sodium sulphate).
Iron (2.4.9). Dilute 2 ml of solution S to 40 ml with water R. 10 ml of the solution complies with the
Heavy metals (2.4.8). To 20 ml of solution S, add 50 mg of hydroxylamine hydrochloride R and 1 ml
of concentrated ammonia R. Adjust to pH 3.5 by adding dilute ammonia R2, monitoring the pH
potentiometrically. Dilute to 25 ml with water R. 12 ml of the solution complies with limit test A for
heavy metals (25 ppm). Prepare the standard using lead standard solution (1 ppm Pb) R.
Loss on ignition Not more than 25.0 per cent, determined on 0.200 g in a platinum crucible by
heating at 100C to 105C for 1 h and then at 1000C for 2 h.
35-50
ASSAY
To the residue obtained in the test for loss on ignition add 0.2 ml of sulphuric acid R and a quantity of
alcohol R sufficient to moisten the residue completely. Add 6 ml of hydrofluoric acid R and evaporate
to dryness at 95C to 105C, taking care to avoid loss from sputtering. Wash the inside of the
crucible with 6 ml of hydrofluoric acid R and evaporate to dryness again. Ignite at 900C, allow to cool
in a desiccator and weigh. The difference between the mass of the final residue and that of the mass
obtained in the test for loss on ignition corresponds to the mass of SiO2 in the test sample.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
35-51
Silver Nitrate
AgNO3
169.9
7761-88-8
Silver Nitrate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0009].
Preparation
Sterile Silver Nitrate Solution
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Silver nitrate contains not less than 99.0 per cent and not more than the equivalent of 100.5 per cent
of AgNO3.
CHARACTERS
A white, crystalline powder or transparent, colourless crystals, very soluble in water, soluble in
alcohol.
IDENTIFICATION
A. 10 mg gives the reaction of nitrates (2.3.1).
B. 10 mg gives the reaction of silver (2.3.1).
TESTS
Acidity or alkalinity To 2 ml of solution S add 0.1 ml of bromocresol green solution R. The solution is
blue. To 2 ml of solution S add 0.1 ml of phenol red solution R. The solution is yellow.
Foreign salts To 30 ml of solution S, add 7.5 ml of dilute hydrochloric acid R, shake vigorously, heat
for 5 min on a water-bath and filter. Evaporate 20 ml of the filtrate to dryness on a water-bath and
dry at 100C to 105C. The residue weighs not more than 2 mg (0.3 per cent).
Aluminium, lead, copper and bismuth Dissolve 1.0 g in a mixture of 4 ml of concentrated
ammonia R and 6 ml of water R. The solution is clear (2.2.1) and colourless (Method II, 2.2.2).
ASSAY
Dissolve 0.300 g in 50 ml of water R and add 2 ml of dilute nitric acid R and 2 ml of ferric ammonium
sulphate solution R2. Titrate with 0.1M ammonium thiocyanate until a reddish-yellow colour is
obtained.
1 ml of 0.1M ammonium thiocyanate is equivalent to 16.99 mg of AgNO3.
STORAGE
Store in a well-closed, non-metallic container, protected from light.
__________________________________________________________________________________________________________ Ph Eur
35-52
Simeticone
Simeticone complies with the requirements of the 3rd edition of the European Pharmacopoeia [1470]. These
Action and use Surfactant.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Simeticone is prepared by incorporation of 4 per cent to 7 per cent silica into poly(dimethylsiloxane)
with a degree of polymerisation between 20 and 400. Simeticone contains 90.5 per cent to 99.0 per
cent of poly(dimethylsiloxane).
PRODUCTION
Poly(dimethylsiloxane) is obtained by hydrolysis and polycondensation of dichlorodimethylsilane and
chlorotrimethylsilane and the silica is modified at the surface by incorporation of methylsilyl groups.
CHARACTERS
A viscous, greyish white, opalescent liquid, practically insoluble in water and in methanol, very
slightly soluble to practically insoluble in ethanol, partly miscible with ethyl acetate, with methylene
chloride, with methyl ethyl ketone and with toluene.
IDENTIFICATION
A. Examine by infrared absorption spectrophotometry (2.2.24) as described in the assay. Absorption
maxima are observed at 2964 cm-1, 2905 cm-1, 1412 cm-1, 1260 cm-1 and 1020 cm-1. In addition, the
spectrum obtained is identical to that of the substance selected for the type sample. Examine the
substances as thin films between sodium chloride R plates.
B. Heat 0.5 g in a test-tube over a small flame until white fumes begin to appear. Invert the tube over
a second tube containing 1 ml of a 1 g/l solution of chromotropic acid, sodium salt R in sulphuric acid R
so that the fumes reach the solution. Shake the second tube for about 10 s and heat on a water-bath
for 5 min. The solution is violet.
C. The residue obtained in the test for silica under Assay, gives the reaction of silicates (2.3.1).
TESTS
Acidity To 2.0 g add 25 ml of a mixture of equal volumes of ethanol R and ether R previously
neutralised to 0.2 ml of bromothymol blue solution R1, and shake. Not more than 3.0 ml of 0.01M
sodium hydroxide is required to change the colour of the solution to blue.
Defoaming activity
Foaming solution. Dissolve 5.0 g of docusate sodium R in 1 litre of water R (warm to 50C if necessary).
Defoaming solution. To 50 ml of methyl ethyl ketone R add 0.250 g of simeticone, warm to not more
than 50C with shaking.
Into a 250 ml cylindrical tube about 5 cm in diameter introduce 100 ml of foaming solution and
1 ml of defoaming solution. Close tightly and fix the tube on a suitable oscillating shaker that
complies with the following conditions:
250 to 300 oscillations per minute,
angle of oscillation of about 10 degrees,
oscillation radius of about 10 cm.
Shake for 10 s and record the time between the end of the shaking and the instant the first portion
of foam-free liquid surface appears.
This duration does not exceed 15 s.
Mineral oils Place 2.0 g in a test-tube and examine in ultraviolet light at 365 nm. The fluorescence
is not more intense than that of a solution containing 0.1 ppm of quinine sulphate R in 0.005M
sulphuric acid examined in the same conditions.
Phenylated compounds Dissolve 5.0 g with shaking in 10.0 ml of cyclohexane R. Determine the
absorbance of the solution between 200 nm and 350 nm (2.2.25) using cyclohexane R as compensation liquid. The corrected absorbance (absorbance measured at the maximum between 250 nm and
270 nm minus the absorbance measured at 300 nm) is not greater than 0.2.
Heavy metals Mix 1.0 g with methylene chloride R and dilute to 20 ml with the same solvent. Add
1.0 ml of a freshly prepared 0.02 g/l solution of dithizone R in methylene chloride R, 0.5 ml of water R
and 0.5 ml of a mixture of 1 volume of dilute ammonia R2 and 9 volumes of a 2 g/l solution of
hydroxylamine hydrochloride R. At the same time, prepare a standard as follows: to 20 ml of methylene
chloride R add 1.0 ml of a freshly prepared 0.02 g/l solution of dithizone R in methylene chloride R,
0.5 ml of lead standard solution (10 ppm Pb) R and 0.5 ml of a mixture of 1 volume of dilute
35-53
ammonia R2 and 9 volumes of a 2 g/l solution of hydroxylamine hydrochloride R. Immediately shake
each solution vigorously for 1 min. Any red colour in the test solution is not more intense than that in
Volatile matter Not more than 1.0 per cent, determined on 1.00 g by heating in an oven at 150C
for 2 h. Carry out the test using a dish 60 mm in diameter and 10 mm deep.
ASSAY
Silica Not more than 7 per cent, determined on 20.0 mg by thermogravimetry (2.2.34). Heat the
substance to be examined to 800C increasing the temperature by 20C per minute under a current
of nitrogen R at a flow rate of 200 ml/min.
Dimeticone
Test solution. Place about 50 mg (E) in a screw-capped 125 ml cylindrical tube, add 25.0 ml of
toluene R, swirl manually to disperse and add 50 ml of dilute hydrochloric acid R, close the tube and
place on a vortex mixer; shake for 5 min. Transfer the contents of the tube to a separating funnel,
allow to settle and transfer 5 ml of the upper layer to a screw-capped test tube containing 0.5 g of
anhydrous sodium sulphate R. Cap and shake vigorously manually. Centrifuge to obtain a clear test
solution.
Reference solution. Introduce about 0.20 g of dimeticone CRS in 100.0 ml of toluene R. Prepare the
reference solution in the same way as for the test solution, using 25.0 ml of the dimeticone solution
obtained above. Prepare a blank by shaking 10 ml of toluene R with 1 g of anhydrous sodium
sulphate R. Centrifuge the resulting suspension.
Record the infrared absorption spectra for the assay solution and the standard solution in 0.5 mm
cells, from 1330 cm-1 to 1180 cm-1, and determine the absorbance of the band at 1260 cm-1 (2.2.24).
Calculate the percentage content of dimeticone using the expression:
25C AM 100
AE E
AM = absorbance of the test solution,

AE = absorbance of the reference solution,
C = concentration of the reference solution in milligrams per millilitre,
E = mass of the substance to be examined, in milligrams.
__________________________________________________________________________________________________________ Ph Eur
35-54
Simeticone for Oral Use

Definition Simeticone for Oral Use contains not less than 4.5% and not more than 8.0% w/w of
silica, SiO2, and not less than 91.0% and not more than 97.0% w/w of Dimeticone 1000. It is
prepared by the addition of finely powdered silica to Dimeticone 1000.
Characteristics A viscous, grey, translucent liquid; odourless or almost odourless.
Immiscible with water and with ethanol (96%); almost completely soluble in chloroform and in
toluene, leaving a residue of silica.
Identification
A. The infrared absorption spectrum of the supernatant liquid obtained by centrifugation, Appendix
II A, is concordant with the reference spectrum of dimeticone (RS 381).
B. Ignite; dense white fumes are evolved leaving a white residue which is insoluble in hydrochloric acid.
Acidity Dissolve 15.0 g in a mixture of 15 ml of toluene and 15 ml of butan-1-ol, previously neutralised to a 0.5% w/v solution of bromophenol blue in ethanol (96%), and titrate with 0.1M ethanolic
potassium hydroxide VS using the bromophenol blue solution as indicator. Not more than 0.15 ml is
required to change the colour of the solution.
Refractive index Of the supernatant liquid obtained in Identification test A, 1.4050 to 1.4080,
Appendix V E.
Viscosity Of the supernatant liquid obtained in Identification test A, 950 to 1050 mm2 s-1, Appendix
V H, Method I, using a U-tube viscometer (size G).
Weight per ml Of the supernatant liquid obtained in Identification test A, 0.965 to 0.980 g,
Appendix V G.
Heavy metals 1.0 g complies with limit test F for heavy metals, Appendix VII (20 ppm). Use 2 ml of
Assay
For silica To 1 g add 50 ml of toluene, mix well and filter through an ignited silica crucible. Wash
the residue thoroughly with toluene, dry at 105 and ignite to constant weight at 500.
For dimeticone Place 40 mg in a stoppered centrifuge tube, add 20 ml of toluene and shake for 20
minutes. Filter and record the infrared absorption spectrum, Appendix II A, of a 0.5-mm layer of the
filtrate over the range 1330 to 1180 cm-1 (7.52 to 8.47 m). Measure the absorbance of the CH3Si
stretching band at the maximum at 1261 cm-1 (7.93 m). Repeat the operation using dimeticone
EPCRS in place of the preparation being examined.
Action and use Antifoaming agent.
When activated dimethicone is prescribed or demanded, Simeticone for Oral Use shall be dispensed
or supplied.
35-55
Simvastatin
1/01
O
e
H
H
H
OH
H
H
O
O
H
CH3
Me
H
Me
C25H38O5
Me
418.6
79902-63-9
Simvastatin complies with the requirements of the 3rd edition of the European Pharmacopoeia [1563]. These
Action and use Hypolipdaemic.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Simvastatin contains not less than 97.0 per cent and not more than the equivalent of 102.0 per cent
of (1S,3R,7S,8S,8aR)-8-[2-[(2R,4R)-4-hydroxy-6-oxotetrahydro-2H-pyran-2-yl]ethyl]-3,7-dimethyl1,2,3,7,8,8a-hexahydronaphthalen-1-yl 2,2-dimethylbutanoate, calculated with reference to the dried
substance. A suitable antioxidant may be added.
CHARACTERS
A white or almost white, crystalline powder, practically insoluble in water, very soluble in methylene
chloride, freely soluble in alcohol.
IDENTIFICATION
obtained with simvastatin CRS. Examine the substances prepared as discs.
TESTS
2.2.2).
Specific optical rotation (2.2.7). Dissolve 0.125 g in acetonitrile R and dilute to 25.0 ml with the
substance.
Related substances Examine by liquid chromatography (2.2.29) as prescribed under Assay.
Inject 5 l of test solution (a) and continue the chromatography for five times the retention time of
simvastatin. When the chromatograms are recorded under the prescribed conditions the relative
retentions are: impurity A about 0.45, lovastatin (impurity E) and epilovastatin (impurity F) about
0.60, impurity G about 0.80, impurity B about 2.38, impurity C about 2.42 and impurity D about
3.80 (retention time of simvastatin: about 2.6 min). In the chromatogram obtained with test solution
(a): the area of the peak due to lovastatin is not greater than twice the area of the principal peak in
the chromatogram obtained with reference solution (b) (1.0 per cent); the area of any peak apart
from the principal peak and the peak due to lovastatin is not greater than 0.8 times the area of the
the areas of all peaks, apart from the principal peak and the peak due to lovastatin, is not greater than
twice the area of the principal peak in the chromatogram obtained with reference solution (b)
the chromatogram obtained with reference solution (b) (0.05 per cent).
35-56
desiccator under high vacuum at 60C for 3 h.
ASSAY
Solvent mixture. Prepare a mixture of 40 volumes of a solution of a 1.4 g/l solution of potassium
dihydrogen phosphate R, adjusted to pH 4.0 with phosphoric acid R, and 60 volumes of acetonitrile R.
Filter.
Test solution (b). Dissolve 40.0 mg of the substance to be examined in the solvent mixture and dilute
Reference solution (a). Dissolve 1.0 mg of simvastatin CRS and 1.0 mg of lovastatin CRS in the solvent
mixture and dilute to 50.0 ml with the solvent mixture.
Reference solution (b). Dilute 0.5 ml of test solution (a) to 100.0 ml with the solvent mixture.
Reference solution (c). Dissolve 40.0 mg of simvastatin CRS in the solvent mixture and dilute to
a stainless steel column 0.033 m long and 4.6 mm in internal diameter packed with end-capped
Mobile phase A. Mix 50 volumes of acetonitrile R and 50 volumes of a 0.1 per cent V/V solution of
phosphoric acid R,
Mobile phase B. A 0.1 per cent V/V solution of phosphoric acid R in acetonitrile R,
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
0 4.5
100
Isocratic
4.5 4.6
100 95
05
4.6 8.0
95 25
5 75
Linear gradient
Linear gradient
8.0 11.5
25
75
Isocratic
11.5 11.6
25 100
75 0
Linear gradient
11.6 13
100
Re-equilibration

Inject 5 l of reference solution (a). The test and the assay are not valid unless, in the chromatogram obtained, the resolution between the peaks corresponding to lovastatin and simvastatin is at
least 5.0. When the chromatograms are recorded under the prescribed conditions the retention times
are: lovastatin about 1.6 min and simvastatin about 2.6 min. Inject 5 l of reference solution (c).
Adjust the sensitivity of the system so that the height of the principal peak is at least 50 per cent of
the full scale of the recorder. Inject 5 l of test solution (b).
Calculate the content of simvastatin from the peak areas in the chromatograms obtained with test
solution (b) and reference solution (c) and the declared content of simvastatin CRS.
STORAGE
Store under nitrogen, in an airtight container, protected from light.
LABELLING
The label states the name and concentration of any added antioxidant.
35-57
IMPURITIES
Me H
H
H
R=
CH3
Me
Me
H Me
COOH
OH
OH
A. (3R,5R)-7-[(1S,2S,6R,8S,8aR)-8-[(2,2-dimethylbutanoyl)oxy]-2,6-dimethyl-1,2,6,7,8,8ahexahydronaphthalen-1-yl]-3,5-dihydroxyheptanoic acid (hydroxy acid),

O
O
OAc
H
B. (1S,3R,7S,8S,8aR)-8-[2-[(2R,4R)-4-(acetyloxy)-6-oxotetrahydro-2H-pyran-2-yl]ethyl]3,7-dimethyl-1,2,3,7,8,8a-hexahydronaphthalen-1-yl 2,2-dimethylbutanoate (acetate ester),

O
O
C. (1S,3R,7S,8S,8aR)-3,7-dimethyl-8-[2-[(2R)-6-oxo-3,6-dihydro-2H-pyran-2-yl]ethyl]1,2,3,7,8,8a-hexahydronaphthalen-1-yl 2,2-dimethylbutanoate (anhydrosimvastatin),

O
O
H
O
OH
O
OH
H
D. (2R,4R)-2-[[(1S,2S,6R,8S,8aR)-8-[(2,2-dimethylbutanoyl)oxy]-2,6-dimethyl-1,2,6,7,8,8ahexahydronaphthalen-1-yl]ethyl]-6-oxotetrahydro-2H-pyran-4-yl (3R,5R)-7[(1S,2S,6R,8S,8aR)-8-[(2,2-dimethylbutanoyl)oxy]-2,6-dimethyl-1,2,6,7,8,8ahexahydronaphthalen-1-yl]-3,5-dihydroxyheptanoate (dimer),

O
Me H
H
H
H
O
OH
H
O
O
H
R1
H Me
CH3
R2
E. R1 = CH3, R2 = H: (1S,3R,7S,8S,8aR)-8-[2-[(2R,4R)-4-hydroxy-6-oxotetrahydro-2Hpyran-2-yl]ethyl]-3,7-dimethyl-1,2,3,7,8,8a-hexahydronaphthalen-1-yl (2S)-2methylbutanoate (lovastatin),

F. R1 = H, R2 = CH3: (1S,3R,7S,8S,8aR)-8-[2-[(2R,4R)-4-hydroxy-6-oxotetrahydro-2H-pyran2-yl]ethyl]-3,7-dimethyl-1,2,3,7,8,8a-hexahydronaphthalen-1-yl (2R)-2-methylbutanoate
(epilovastatin),
O
e H
H
H
H
O
OH
H
O
O
H
Me
CH2
CH3
Me
G. (1S,7S,8S,8aR)-8-[2-[(2R,4R)-4-hydroxy-6-oxotetrahydro-2H-pyran-2-yl]ethyl]-7-methyl3-methylene-1,2,3,7,8,8a-hexahydronaphthalen-1-yl 2,2-dimethylbutanoate.
__________________________________________________________________________________________________________ Ph Eur
35-58
Soft Soap
Definition Soft Soap is soap made by the interaction of potassium hydroxide or sodium hydroxide
with a suitable vegetable oil or oils or with fatty acids derived therefrom. It yields not less than 44.0%
of fatty acids. It may be coloured with chlorophyll or not more than 0.015% of a suitable green soap
dye.
Characteristics A yellowish white to green or brown, unctuous substance.
Soluble in water and in ethanol (96%).
Chlorides and other ethanol-insoluble substances Dissolve 5 g in 100 ml of hot ethanol (96%)
previously neutralised to phenolphthalein solution R1, filter through a dried and tared filter, wash the
residue thoroughly with hot neutralised ethanol (96%) and dry to constant weight at 105. The
residue weighs not more than 0.15 g.
Free fatty acid or alkali hydroxide Boil 250 ml of ethanol (96%) to remove carbon dioxide, add
0.5 ml of phenolphthalein solution R1, allow to cool to 70 and neutralise, if necessary, with 0.1M
sodium hydroxide VS or 0.05M sulphuric acid VS. To 100 ml of the neutral ethanol add 10 g of the
substance being examined and dissolve it as quickly as possible by heating under a reflux condenser.
Cool to 70 and, if the solution is not pink, titrate at 70 with 0.1M sodium hydroxide VS; not more
than 0.2 ml is required. If the solution is pink add, in a thin stream, 5 ml of hot barium chloride solution previously neutralised to phenolphthalein solution R1, mix thoroughly and titrate with 0.1M hydrochloric acid VS until the pink colour disappears; not more than 1.0 ml is required.
Total free alkali To 100 ml of the neutral ethanol prepared as described in the test for Free fatty
acid or alkali hydroxide add 10 g of the substance being examined and dissolve it as quickly as
possible by heating under a reflux condenser. Add immediately 3 ml of 0.5M sulphuric acid VS and
boil under a reflux condenser on a water bath for at least 10 minutes. If the solution is not pink, cool
to 70 and titrate with 1M sodium hydroxide VS until a pink colour is produced. The volume of 0.5M
sulphuric acid VS neutralised by the substance being examined is not more than 1.0 ml.
Unsaponifiable matter and unsaponified neutral fat Dissolve 5 g in 80 ml of a mixture of 50 ml
of ethanol (96%) and 100 ml of water, without heating more than is necessary, and transfer to a
separating funnel, washing the vessel with the remaining 70 ml of the mixture. Extract with 100 ml of
ether while still slightly warm, run off the ethanolic soap layer into a second separating funnel and
extract with 50 ml of ether. Repeat the extraction with 50 ml of ether and pour the three ether extracts
into a separating funnel containing 20 ml of water. Rotate the separating funnel without violent
shaking and, after allowing the liquids to separate, run off the water. Repeat the washing with water in
the same manner until the separated washings are not more than faintly turbid when acidified. Wash
the ether solution twice by shaking vigorously with 20 ml of 0.5M potassium hydroxide, each washing
with alkali being immediately followed by washing with 20 ml of water, shaking vigorously each time.
Acidify the last alkali washing after separation and, if the liquid becomes turbid, repeat the washing
with 0.5M potassium hydroxide and water until the alkali washing remains clear on acidification. Finally
wash with successive 20-ml quantities of water until the washings do not give a pink colour with
phenolphthalein solution R1. Transfer the ether solution to a tared flask and remove the ether. When
nearly all the ether has evaporated, add 3 ml of acetone. With the aid of a gentle current of air remove
the solvent completely from the flask, which is preferably almost entirely immersed in boiling water,
held obliquely and rotated. Repeat the last operation until the weight of the residue is constant. The
residue weighs not more than 40 mg.
Characteristics of the fatty acids obtained in the Assay
Acid value Not more than 205, Appendix X B, when determined on 2 to 3 g of the fatty acids, using
0.5M potassium hydroxide VS and substituting 2.805 for 5.610 in the formula.
Iodine value Not less than 83 (iodine monochloride method), Appendix X E.
Solidifying point Not above 31, Appendix V B, with the following modifications. Where the
determination is made at 15 to 20 the 1000-ml beaker and cooling liquid need not be used. Where
the room temperature falls outside this range the 1000-ml beaker should contain water maintained at
15 to 20 and the level of this water should not be below the level of the sample in the inner tube.
Transfer about 15 ml of the melted fatty acids to the inner test tube. Before the temperature of the
fatty acids drops to a point 10 above their expected solidifying point, begin agitation in a vertical
manner at a rate of 100 complete up and down motions per minute, the stirrer moving through a
vertical distance of about 38 mm. Continue stirring in this manner until the temperature has
remained constant for 30 seconds or has begun to rise within 30 seconds of ceasing to fall.
Discontinue stirring immediately and lift the stirrer out of the sample. Observe the rise in
temperature; the highest temperature reached after cessation of stirring is the solidifying point of the
fatty acids. When reading the thermometer avoid all undue vibration as this will cause the
temperature to drop before reaching the maximum.
Resin Mix 0.5 ml of the melted fatty acids in a test tube with 2 ml of acetic anhydride, warm, shake
until clear and cool to 15.5. Transfer one drop of this solution to a white porcelain tile, place one
35-59
drop of a cold mixture of equal volumes of sulphuric acid and water adjacent to it and gently bring the
drops together with a glass rod. No transient violet colour is produced.
Assay Dissolve 30 g in 100 ml of water, transfer to a separating funnel, acidify with 1M sulphuric acid
and extract with successive quantities of 50, 40 and 30 ml of ether. Mix the ether solutions in a
separating funnel and wash with water until the washings are free from mineral acid. Transfer the
ether solution to a tared flask, remove the ether and dry the residue of fatty acids to constant weight
at 80.
Preparation
Soap Spirit
35-60
Soda Lime
8006-28-8
Definition Soda Lime is a mixture of sodium hydroxide, or sodium hydroxide and potassium
hydroxide, with calcium hydroxide.
Characteristics White or greyish white granules, or it may be coloured with an indicator to show
when its absorptive capacity is exhausted. It absorbs about 20% of its weight of carbon dioxide.
Partially soluble in water; almost completely soluble in 1M acetic acid.
Identification
A. When moistened with hydrochloric acid and introduced on a platinum wire into a flame, imparts a
yellow colour to the flame.
B. A solution in 1M acetic acid yields reaction C characteristic of calcium salts, Appendix VI.
C. A suspension in water is strongly alkaline to litmus paper.
Hardness of granules Shake 200 g on a sieve no. 2000 for 3 minutes using a mechanical sieve
shaker that reproduces in a uniform manner the circular and tapping motion given to sieves in
manual use and has a frequency of oscillation of 282 to 288 cycles per minute. Place 50 g of the
retained material in a hardness pan 20 cm in diameter having a concave brass bottom, 7.9 mm thick
at the circumference, 3.2 mm thick at the centre and with an inside spherical radius of curvature of
109 cm. Add 15 steel balls, 7.9 mm in diameter, and shake on the mechanical sieve shaker for 30
minutes. Remove the steel balls, transfer the contents of the pan to a sieve no. 2000 and again shake
on the mechanical sieve shaker for 3 minutes. The material retained by the sieve weighs not less than
37.5 g.
Size of granules Shake 500 g on a perforated plate of nominal pore size 6.70 mm; not more than
5 g is retained. Then shake on a sieve no. 4750; not more than 50 g is retained. Shake the unretained
material on a sieve no. 1400; not more than 20 g passes through. Shake the unretained material on a
sieve no. 600; not more than 7.5 g passes through.
Loss on drying When dried to constant weight at 105, loses 14.0 to 21.0% of its weight. Use 1 g.
Moisture absorption Place 10 g in an open glass dish about 50 mm in diameter and 30 mm high in
a desiccator over sulphuric acid (14%) and allow it to remain for 24 hours. The increase in weight is
not more than 7.5%.
Carbon dioxide absorption The activity is not less than 120 minutes when determined by the
following method.
Use a vertically-clamped tube of glass or other suitable transparent material about 25 cm long and
29 to 31 mm internal diameter with closely fitting rubber bungs at each end; the bungs are bored to
receive polythene or glass tubing of about 8 mm external diameter, the tubing being flush with the
inner ends of the bungs. With the lower bung in position, place sufficient nylon mesh support on top
of the bung to produce a bed of mesh about 10 cm deep and press a closely fitting disc of stainless
steel gauze of nominal mesh aperture about 500 m on top of the nylon mesh so that its surface is at
right angles to the axis of the tube. Introduce 59.8 to 60.2 g of the substance being examined onto
the steel gauze in three portions, tamping lightly after the addition of each portion. Place a second
disc of steel gauze on top, followed by a sufficient quantity of nylon mesh such that the soda lime is
kept consolidated by slight pressure when the second bung has been inserted. The exit tube is
connected to a condenser, consisting of two 50-ml separating funnels, leading to a drying tube
packed with anhydrous calcium chloride and then to a carbon dioxide analyser sufficiently sensitive to
detect 0.2% v/v of carbon dioxide. A katharometer, calibrated for carbon dioxide and preferably used
in conjunction with a chart recorder, is suitable.
Using the gas analyser in accordance with the manufacturers instructions, accurately determine
the carbon dioxide content, p, as a percentage v/v, of a nominal 5% carbon dioxide mixture, the
balance gas being oxygen, air or nitrogen as appropriate to the type of gas analyser being used. Suitable compressed gas mixtures are available commercially. Assemble the apparatus described above
and pass the gas mixture downwards into the absorption tube at a rate of 900 cm3 per minute until
such time that the gas analyser shows the content of carbon dioxide in the effluent gas to have risen
to 0.2% v/v. Steps should be taken to vent the effluent gas if an oxygencarbon dioxide mixture is
being used. Record the time taken, t, in minutes. The activity of the soda lime is given, in minutes, by
the expression t p/5.
Storage Soda Lime should be kept in a well-closed container.
Action and use Used to absorb carbon dioxide.
35-61
Sodium Acetate
C2H3NaO2,3H2O
136.1
6131-90-4
Sodium Acetate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0411].
Action and use Used in solutions for dialysis; pharmaceutical aid.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sodium acetate contains not less than 99.0 per cent and not more than the equivalent of 101.0 per
cent of sodium ethanoate, calculated with reference to the dried substance.
CHARACTERS
Colourless crystals, very soluble in water, soluble in alcohol.
IDENTIFICATION
A. 1 ml of solution S (see Tests) gives reaction (b) of acetates (2.3.1).
B. 1 ml of solution S gives reaction (a) of sodium (2.3.1).
TESTS
Reducing substances Dissolve 1.0 g in 100 ml of boiling water R, add 5 ml of dilute sulphuric acid R
and 0.5 ml of 0.002M potassium permanganate, mix and boil gently for 5 min. The pink colour is not
completely discharged.
limit test for sulphates (200 ppm).
Aluminium (2.4.17). If intended for use in the manufacture of dialysis solutions, it complies with
the test for aluminium. Dissolve 20 g in 100 ml of water R and adjust to pH 6.0 by the addition of 1M
hydrochloric acid (about 10 ml). The solution complies with the limit test for aluminium (0.2 ppm).
Use as the reference solution a mixture of 2 ml of aluminium standard solution (2 ppm Al) R, 10 ml of
acetate buffer solution pH 6.0 R and 98 ml of water R. To prepare the blank, use a mixture of 10 ml of
acetate buffer solution pH 6.0 R and 100 ml of water R.
Calcium and magnesium To 200 ml of water R add 10 ml of ammonium chloride buffer solution pH
10.0 R, 0.1 g of mordant black 11 triturate R, 2.0 ml of 0.05M zinc chloride and, dropwise, 0.02M
sodium edetate until the colour changes from violet to blue. Add to the solution 10.0 g of the
substance to be examined and shake to dissolve. Titrate with 0.02M sodium edetate until the blue
colour is restored. Not more than 0.65 ml of 0.02M sodium edetate is required (50 ppm, calculated as
Ca).
at 130C. Introduce the substance to be examined into the oven while the latter is cold.
ASSAY
Dissolve 0.250 g in 50 ml of anhydrous acetic acid R, add 5 ml of acetic anhydride R, mix and allow to
stand for 30 min. Using 0.3 ml of naphtholbenzein solution R as indicator, titrate with 0.1M perchloric
acid until a green colour is obtained.
1 ml of 0.1M perchloric acid is equivalent to 8.20 mg of C2H3NaO2.
STORAGE
35-62
LABELLING
The label states, where applicable, that the substance is suitable for use in the manufacture of dialysis
solutions.
__________________________________________________________________________________________________________ Ph Eur
35-63
Sodium Acid Citrate

Disodium Hydrogen Citrate
C6H6Na2O7,1H2O
263.1
144-33-2
Definition Sodium Acid Citrate contains not less than 98.0% and not more than 104.0% of
C6H6Na2O7, 1H2O.
Characteristics A white powder; odourless or almost odourless.
Freely soluble in water; practically insoluble in ethanol (96%).
Identification Yields the reactions characteristic of sodium salts and of citrates, Appendix VI.
Arsenic 0.50 g dissolved in 25 ml of water complies with the limit test for arsenic, Appendix VII
(2 ppm).
test A for heavy metals, Appendix VII. Use lead standard solution (1 ppm Pb) to prepare the standard
(20 ppm).
Chloride Dissolve 1.0 g in 100 ml of water. 15 ml of the resulting solution complies with the limit test
for chlorides, Appendix VII (330 ppm).
Oxalate Dissolve 1.0 g in 4 ml of water, add 3 ml of hydrochloric acid and 1 g of granulated zinc and
heat on a water bath for 1 minute. Allow to stand for 2 minutes, decant the liquid into a test tube
containing 0.25 ml of a 1% w/v solution of phenylhydrazine hydrochloride and heat to boiling. Cool
rapidly, transfer to a graduated measuring cylinder, add an equal volume of hydrochloric acid and
0.25 ml of potassium hexacyanoferrate(III) solution, shake and allow to stand for 30 minutes. Any red
colour produced is not more intense than that produced by treating in the same manner 4 ml of a
0.005% w/v solution of oxalic acid (150 ppm, calculated as anhydrous oxalic acid).
Sulphate Dissolve 0.50 g in 57 ml of water and add 3 ml of 2M hydrochloric acid. 15 ml of the resulting solution complies with the limit test for sulphates, Appendix VII (0.12%).
Readily carbonisable substances Heat 1.0 g, in powder, with 10 ml of sulphuric acid for 30
minutes in a water bath protected from light. Not more than a pale brown colour is produced.
Assay Heat 2 g until carbonised, cool and boil the residue with 50 ml each of water and 0.5M hydrochloric acid VS. Filter, wash the filter with water and titrate the excess of acid in the filtrate and washings with 0.5M sodium hydroxide VS using methyl orange solution as indicator. Each ml of 0.5M hydrochloric acid VS is equivalent to 65.78 mg of C6H6Na2O7,1H2O.
35-64
Sodium Alendronate
1/01
O
OH
OH
ONa
OH
NH2
C4H12NNaO7P2,3H2O 325.1
121268-17-5
Sodium Alendronate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Inhibition of bone resorption.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sodium alendronate contains not less than 98.0 per cent and not more than the equivalent of
102.0 per cent of (4-amino-1-hydroxybutylidene)bisphosphonic acid monosodium salt trihydrate,
CHARACTERS
A white or almost white, crystalline powder, soluble in water, very slightly soluble in methanol,
practically insoluble in methylene chloride.
IDENTIFICATION
obtained with sodium alendronate CRS. Examine the substances prepared as discs.
TESTS
solution B7 or BY7 (Method II, 2.2.2).
4-aminobutanoic acid Examine by thin-layer chromatography (2.2.27), using a TLC silica gel
plate R.
same solvent.
Reference solution (a). Dissolve 0.10 g of 4-aminobutanoic acid R in water R and dilute to 200 ml with
the same solvent.
Apply to the plate 5 l of the test solution and 5 l of reference solution (b). Allow the plate to dry in
air. Develop over a path of 15 cm using a mixture of 20 volumes of water R, 20 volumes of glacial
acetic acid R and 60 volumes of butanol R. Dry the plate in a current of warm air. Spray with ninhydrin
solution R and heat at 100C to 105C for 15 min. Any spots corresponding to 4-aminobutanoic acid
in the chromatogram obtained with the test solution are not more intense than the spot in the
Phosphate and phosphite Examine the chromatograms obtained in the assay. In the chromatogram obtained with the test solution: the area of any peak corresponding to phosphate is not greater
than that of the peak due to phosphate in the chromatogram obtained with reference solution (d)
(0.5 per cent); the area of any peak corresponding to phosphite is not greater than that of the peak
due to phosphite in the chromatogram obtained with reference solution (d) (0.5 per cent).
Heavy metals (2.4.8).1.0 g complies with limit test F for heavy metals (20 ppm). Prepare the
at 140C to 145C.
35-65
ASSAY
the same solvent.
Reference solution (a). Dissolve 50.0 mg of sodium alendronate CRS in water R and dilute to 25.0 ml
Reference solution (b). Dissolve 3.0 g of phosphoric acid R in water R and dilute to 100.0 ml with the
same solvent. Dilute 1.0 ml of the solution to 100.0 ml with water R.
Reference solution (c). Dissolve 2.5 g of phosphorous acid R in water R and dilute to 100.0 ml with the
a column 0.15 m long and 4.6 mm in internal diameter packed with anion exchange resin R1
(5 m),
as mobile phase at a flow rate of 1.2 ml/min a solution of 0.2 ml of anhydrous formic acid R in
1000 ml of water R, adjusted to pH 3.5 with 2M sodium hydroxide solution,
as detector a refractometer,
a 100 l loop injector,
Inject reference solution (a) six times. The assay is not valid unless the relative standard deviation
of the peak area of sodium alendronate is at most 1.0 per cent. Inject the test solution, reference
solution (a) and reference solution (d). The retention time of sodium alendronate is about 16 min
and the relative retention times are: phosphate about 1.3 and phosphite about 1.6. Record the
chromatograms for twice the retention time of the principal peak in the chromatogram obtained with
the test solution.
Calculate the percentage content of C4H12NNaO7P2 from the peak areas and the declared content
of sodium alendronate CRS.
STORAGE
IMPURITIES
2N
COOH
A. 4-aminobutanoic acid,
B. phosphate,
C. phosphite.
__________________________________________________________________________________________________________ Ph Eur
35-66
Sodium Alginate
9005-38-3
Sodium Alginate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0625].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sodium alginate consists mainly of the sodium salt of alginic acid, which is a mixture of polyuronic
acids [C6H8O6)n] composed of residues of D-mannuronic acid and L-guluronic acid, and is obtained
mainly from algae belonging to the Phaeophyceae.
CHARACTERS
A white or pale yellowish-brown powder, slowly soluble in water forming a viscous, colloidal solution,
practically insoluble in alcohol and in ether.
IDENTIFICATION
A. Dissolve 0.2 g with shaking in 20 ml of water R. To 5 ml of this solution add 1 ml of calcium
chloride solution R. A voluminous gelatinous mass is formed.
B. To 10 ml of the solution prepared in identification test A add 1 ml of dilute sulphuric acid R. A
gelatinous mass is formed.
C. To 5 mg add 5 ml of water R, 1 ml of a freshly prepared 10 g/l solution of 1,3-dihydroxynaphthalene R in alcohol R and 5 ml of hydrochloric acid R. Boil for 3 min, cool, add 5 ml of water R, and shake
with 15 ml of diisopropyl ether R. Carry out a blank test. The upper layer obtained with the substance
to be examined exhibits a deeper bluish-red colour than that obtained with the blank.
D. It complies with the test for sulphated ash. The residue obtained, dissolved in 2 ml of water R,
gives reaction (a) of sodium (2.3.1).
TESTS
Solution S Dissolve 0.10 g in water R, with constant stirring, dilute to 30 ml with the same solvent
and allow to stand for 1 h.
Appearance of solution Dilute 1 ml of solution S to 10 ml with water R. The solution is not more
opalescent than reference suspension II (2.2.1) and not more intensely coloured than intensity 6 of
the range of reference solutions of the most appropriate colour (Method II, 2.2.2).
Chlorides Not more than 1.0 per cent. To 2.50 g add 50 ml of dilute nitric acid R, shake for 1 h and
dilute to 100.0 ml with dilute nitric acid R. Filter. To 50.0 ml of the filtrate add 10.0 ml of 0.1M silver
nitrate and 5 ml of toluene R. Titrate with 0.1M ammonium thiocyanate, using 2 ml of ferric ammonium
sulphate solution R2 as indicator and shaking vigorously towards the end point.
1 ml of 0.1M silver nitrate is equivalent to 3.545 mg of Cl.
Calcium Not more than 1.5 per cent of Ca, determined by atomic absorption spectrometry (Method
II, 2.2.23).
Test solution. Dissolve 0.10 g of the substance to be examined in 50 ml of dilute ammonia R2, heating
on a water-bath. Allow to cool and dilute to 100.0 ml with distilled water R (solution (a)). Dilute
3.0 ml of solution (a) to 100.0 ml with distilled water R.
Reference solutions. Prepare three reference solutions in the same manner as the test solution but add
0.75 ml, 1.0 ml and 1.5 ml respectively of calcium standard solution (100 ppm Ca) R to the 3.0 ml of
solution (a).
Set the zero of the instrument using a mixture of 1.5 volumes of dilute ammonia R2 and 98.5 volumes
of distilled water R. Measure the absorbance at 422.7 nm using a calcium hollow-cathode lamp as
source of radiation and an air-acetylene flame.
Heavy metals (2.4.8). 1.0 g complies with the limit test F for heavy metals (20 ppm). Prepare the
Loss on drying (2.2.32). Not more than 15.0 per cent, determined on 0.1000 g by drying in an
oven at 100C to 105C for 4 h.
Sulphated ash (2.4.14). 30.0 per cent to 36.0 per cent, determined on 0.1000 g and calculated with
per gram, determined by plate-count. It complies with the tests for Escherichia coli and Salmonella
(2.6.13).
__________________________________________________________________________________________________________ Ph Eur
35-67
Sodium Amidotrizoate
corrected 1/01
I
AcHN
COONa
I
NHAc
C11H8I3N2NaO4
636
737-31-5
Sodium Amidotrizoate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Radio-opaque substance used in urography.
Preparation
Sodium Amidotrizoate Injection
When sodium diatrizoate is prescribed or demanded, Sodium Amidotrizoate shall be dispensed or
supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sodium amidotrizoate contains not less than 98.0 per cent and not more than the equivalent of
101.0 per cent of sodium 3,5-bis(acetylamino)-2,4,6-tri-iodobenzoate, calculated with reference to
the anhydrous substance.
CHARACTERS
A white or almost white powder, freely soluble in water, slightly soluble in alcohol, practically
insoluble in acetone.
IDENTIFICATION
obtained with sodium amidotrizoate CRS. Dry both the substance to be examined and the reference
substance at 100C to 105C for 3 h.
C. Heat 50 mg gently in a small porcelain dish over a naked flame. Violet vapour is evolved.
D. It gives reaction (a) of sodium (2.3.1).
TESTS
Solution S Dissolve 10 g in carbon dioxide-free water R and dilute to 20 ml with the same solvent.
plate R.
Prepare the solutions in subdued light and develop the chromatograms protected from light.
Test solution (a). Dissolve 0.50 g of the substance to be examined in a 3 per cent V/V solution of
ammonia R in methanol R and dilute to 10 ml with the same solution.
Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with a 3 per cent V/V solution of ammonia R
in methanol R.
Reference solution (a). Dilute 1 ml of test solution (b) to 50 ml with a 3 per cent V/V solution of
ammonia R in methanol R.
Reference solution (b). Dissolve 50 mg of sodium amidotrizoate CRS in a 3 per cent V/V solution of
35-68
ammonia R in methanol R and dilute to 10 ml with the same solution.
20 volumes of anhydrous formic acid R, 25 volumes of methyl ethyl ketone R and 60 volumes of
toluene R. Allow the plate to dry until the solvents have evaporated and examine in ultraviolet light at
is not more intense than the spot in the chromatogram obtained with reference solution (a) (0.2 per
cent).
Free aromatic amines Maintain the solutions and reagents in iced water, protected from light. To 0.50 g
in a 50 ml volumetric flask add 15 ml of water R. Shake and add 1 ml of dilute sodium hydroxide
solution R. Cool in iced water, add 5 ml of a freshly prepared 5 g/l solution of sodium nitrite R and
12 ml of dilute hydrochloric acid R. Shake gently and allow to stand for exactly 2 min after adding the
hydrochloric acid. Add 10 ml of a 20 g/l solution of ammonium sulphamate R. Allow to stand for
5 min, shaking frequently, and add 0.15 ml of a 100 g/l solution of -naphthol R in alcohol R. Shake
and allow to stand for 5 min. Add 3.5 ml of buffer solution pH 10.9 R, mix and dilute to 50.0 ml with
water R. The absorbance (2.2.25), measured within 20 min at 485 nm using as the compensation
liquid a solution prepared at the same time and in the same manner but omitting the substance to be
examined, is not greater than 0.30.
Free iodine and iodides Not more than 50 ppm. Dissolve 1.0 g in distilled water R and dilute to
10 ml with the same solvent. Add dropwise dilute nitric acid R until the precipitation is complete, then
add 3 ml of dilute nitric acid R. Filter and wash the precipitate with 5 ml of water R. Collect the filtrate
and washings. Add 1 ml of strong hydrogen peroxide solution R and 1 ml of methylene chloride R. Shake.
The lower layer is not more intensely coloured than a reference solution prepared simultaneously and
in the same manner, using a mixture of 5 ml of iodide standard solution (10 ppm I) R, 3 ml of dilute
nitric acid R and 15 ml of water R.
Heavy metals (2.4.8). Dilute 4 ml of solution S to 20 ml with water R. 12 ml of this solution
ASSAY
To 0.150 g in a 250 ml round-bottomed flask add 5 ml of strong sodium hydroxide solution R, 20 ml of
water R, 1 g of zinc powder R and a few glass beads. Boil under a reflux condenser for 30 min. Allow
to cool and rinse the condenser with 20 ml of water R, adding the rinsings to the flask. Filter through
a sintered-glass filter and wash the filter with several quantities of water R. Collect the filtrate and
washings. Add 40 ml of dilute sulphuric acid R and titrate immediately with 0.1M silver nitrate. Determine the end-point potentiometrically (2.2.20), using a suitable electrode system such as silvermercurous sulphate.
1 ml of 0.1M silver nitrate is equivalent to 21.20 mg of C11H8I3N2NaO4.
STORAGE
IMPURITIES
COOH
I
R2
NHAc
R1
I
A. R1 = NH2, R2 = I: 3-acetylamino-5-amino-2,4,6-tri-iodobenzoic acid,

B. R1 = NHCOCH3, R2 = H: 3,5-bis(acetylamino)-2,4-di-iodobenzoic acid.
__________________________________________________________________________________________________________ Ph Eur
35-69
Sodium Aurothiomalate
39377-38-3
Definition Sodium Aurothiomalate consists mainly of the disodium salt of (aurothio)succinic acid. It
contains not less than 44.5% and not more than 46.0% of Au and not less than 10.8% and not more
than 11.5% of Na, both calculated with reference to the dried substance.
Characteristics A fine, pale yellow powder; odour, slight; hygroscopic.
Very soluble in water.
Identification
A. To 2 ml of solution A add 2 ml of hydrogen peroxide solution (20 vol) and 1 ml of 5M sodium
hydroxide. A precipitate is produced which appears brownish black by reflected light and bluish green
by transmitted light.
B. Solution A yields a black precipitate with hydrogen sulphide which is insoluble in 2M hydrochloric
acid but soluble in ammonium polysulphide solution.
C. Extract a portion of the residue obtained in test A with 10 ml of 2M hydrochloric acid. The solution, after neutralisation if necessary, yields the reactions characteristic of sodium salts and the reaction
characteristic of sulphates, Appendix VI.
Acidity or alkalinity pH of a 10% w/v solution, 6.0 to 7.0, Appendix V L.
Stability Dissolve 1.0 g in 10 ml of water, filter, seal in an ampoule, heat at 100 for 1 hour, cool and
add sufficient water to produce 100 ml. The solution remains bright and is not more intensely
coloured than a 0.010% w/v solution of potassium hexacyanoferrate(III).
Assay For Au Heat 0.2 g with 10 ml of sulphuric acid and continue to boil gently until a clear, pale
yellow liquid is produced. Cool, add about 1 ml of nitric acid dropwise and boil again for 1 hour.
Cool, dilute with 70 ml of water, boil for 5 minutes, filter, wash the residue of gold with hot water,
dry and ignite for 3 hours at a temperature not lower than 600.
For Na Evaporate to dryness the filtrate and washings obtained in the Assay for Au, moisten with
sulphuric acid and ignite for 3 hours at 600. Each g of residue is equivalent to 0.3237 g of Na.
Storage Sodium Aurothiomalate should be kept in a well-closed container and protected from light.
Action and use Used in treatment of rheumatoid arthritis.
Preparation
Sodium Aurothiomalate Injection
35-70
Sodium Benzoate
COONa
C7H5NaO2
144.1
532-32-1
Sodium Benzoate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0123].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sodium benzoate contains not less than 99.0 per cent and not more than the equivalent of 100.5 per
cent of sodium benzenecarboxylate, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline or granular powder or flakes, slightly hygroscopic, freely soluble in water, sparingly
soluble in alcohol (90 per cent V/V).
IDENTIFICATION
A. It gives reactions (b) and (c) of benzoates (2.3.1).
B. It gives the reactions of sodium (2.3.1).
TESTS
Acidity or alkalinity To 10 ml of solution S add 10 ml of carbon dioxide-free water R and 0.2 ml of
phenolphthalein solution R. Not more than 0.2 ml of 0.1M sodium hydroxide or 0.1M hydrochloric acid is
Halogenated compounds. All glassware used must be chloride-free and may be prepared by soaking
overnight in a 500 g/l solution of nitric acid R, rinsed with water R and stored full of water R. It is
recommended that glassware be reserved exclusively for this test.
To 20.0 ml of solution S add 5 ml of water R and dilute to 50.0 ml with alcohol R (test solution).
Determination of ionised chlorine. In three 25 ml volumetric flasks, prepare the following solutions.
Solution (a). To 4.0 ml of the test solution add 3 ml of dilute sodium hydroxide solution R and 3 ml of
alcohol R. This solution is used to prepare solution A.
Solution (b). To 3 ml of dilute sodium hydroxide solution R add 2 ml of water R and 5 ml of alcohol R.
This solution is used to prepare solution B.
Solution (c). To 4.0 ml of chloride standard solution (8 ppm Cl) R add 6.0 ml of water R. This solution
is used to prepare solution C.
In a fourth 25 ml volumetric flask, place 10 ml of water R. To each flask add 5 ml of ferric
ammonium sulphate solution R5, mix and add dropwise and with swirling 2 ml of nitric acid R and 5 ml
of mercuric thiocyanate solution R. Shake. Dilute the contents of each flask to 25.0 ml with water R and
allow the solutions to stand in a water-bath at 20C for 15 min. Measure at 460 nm in a 2 cm cell the
absorbance (2.2.25) of solution A using solution B as the compensation liquid, and the absorbance of
solution C using the solution obtained with 10 ml of water R as the compensation liquid. The absorbance of solution A is not greater than that of solution C (200 ppm).
Determination of total chlorine
Solution (a). To 10.0 ml of the test solution add 7.5 ml of dilute sodium hydroxide solution R and
0.125 g of nickel-aluminium alloy R and heat on a water-bath for 10 min. Allow to cool to room
temperature, filter into a 25 ml volumetric flask and wash the filter with three quantities, each of 2 ml,
of alcohol R (a slight precipitate may form that disappears on acidification). Dilute the filtrate and
washings to 25.0 ml with water R. This solution is used to prepare solution A.
Solution (b). In the same manner, prepare a similar solution replacing the test solution by a mixture of
5 ml of alcohol R and 5 ml of water R. This solution is used to prepare solution B.
Solution (c). To 6.0 ml of chloride standard solution (8 ppm Cl) R add 4.0 ml of water R. This solution is
used to prepare solution C.
In four 25 ml volumetric flasks, place separately 10 ml of solution (a), 10 ml of solution (b), 10 ml of
35-71
solution (c) and 10 ml of water R. To each flask add 5 ml of ferric ammonium sulphate solution R5, mix
and add dropwise and with swirling 2 ml of nitric acid R and 5 ml of mercuric thiocyanate solution R.
Shake. Dilute the contents of each flask to 25.0 ml with water R and allow the solutions to stand in a
water-bath at 20C for 15 min. Measure at 460 nm in a 2 cm cell the absorbance (2.2.25) of solution
A using solution B as the compensation liquid, and the absorbance of solution C using the solution
obtained with 10 ml of water R as the compensation liquid. The absorbance of solution A is not
greater than that of solution C (300 ppm).
100C to 105C.
ASSAY
Dissolve 0.250 g in 20 ml of anhydrous acetic acid R, heating to 50C if necessary. Cool. Using
0.05 ml of naphtholbenzein solution R as indicator, titrate with 0.1M perchloric acid until a green colour
is obtained.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
36-1
Sodium Bicarbonate
1/01
NaHCO3
84.0
144-55-8
Sodium Bicarbonate complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Sodium Hydrogen Carbonate [0195]. These requirements are reproduced after the heading Definition
below.
Action and use Antacid; used in treatment of electrolyte deficiency.
Preparations
Sodium Bicarbonate Ear Drops
Sodium Bicarbonate Eye Lotion
Sodium Bicarbonate Intravenous Infusion
Compound Sodium Bicarbonate Tablets
Ph Eur _______________________________________________________________________________________________________________
DEFINITION
Sodium hydrogen carbonate contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of NaHCO3.
CHARACTERS
A white, crystalline powder, soluble in water, practically insoluble in alcohol. When heated in the dry
state or in solution, it gradually changes into sodium carbonate.
IDENTIFICATION
A. To 5 ml of solution S (see Tests) add 0.1 ml of phenolphthalein solution R. A pale pink colour is
produced. Heat; gas is evolved and the solution becomes red.
B. It gives the reaction of carbonates and bicarbonates (2.3.1).
C. Solution S gives reaction (a) of sodium (2.3.1).
TESTS
Solution S Dissolve 5.0 g in 90 ml of carbon dioxide-free water R and dilute to 100.0 ml with the
same solvent.
Carbonates The pH (2.2.3) of freshly prepared solution S is not more than 8.6.
Chlorides (2.4.4). To 7 ml of solution S add 2 ml of nitric acid R and dilute to 15 ml with water R.
The solution complies with the limit test for chlorides (150 ppm).
Sulphates (2.4.13). To a suspension of 1.0 g in 10 ml of distilled water R add hydrochloric acid R until
neutral and about 1 ml in excess. Dilute to 15 ml with distilled water R. The solution complies with
the limit test for sulphates (150 ppm).
Ammonium (2.4.1). 10 ml of solution S diluted to 15 ml with water R complies with the limit test
for ammonium (20 ppm). Prepare the standard using a mixture of 5 ml of water R and 10 ml of
ammonium standard solution (1 ppm NH4) R.
Calcium (2.4.3). To a suspension of 1.0 g in 10 ml of distilled water R add hydrochloric acid R until
neutral and dilute to 15 ml with distilled water R. The solution complies with the limit test for calcium
(100 ppm).
Iron (2.4.9). Dissolve 0.5 g in 5 ml of dilute hydrochloric acid R and dilute to 10 ml with water R. The
Heavy metals (2.4.8). Dissolve 2.0 g in a mixture of 2 ml of hydrochloric acid R and 18 ml of
water R. 12 ml of the solution complies with limit test A for heavy metals (10 ppm). Prepare the
ASSAY
Dissolve 1.500 g in 50 ml of carbon dioxide-free water R. Titrate with 1M hydrochloric acid, using 0.2 ml
of methyl orange solution R as indicator.
1 ml of 1M hydrochloric acid is equivalent to 84.0 mg of NaHCO3.
__________________________________________________________________________________________________________ Ph Eur
36-2
Sodium Bromide
NaBr
102.9
7647-15-6
Sodium Bromide complies with the requirements of the 3rd edition of the European Pharmacopoeia [0190].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sodium bromide contains not less than 98.0 per cent and not more than the equivalent of 100.5 per
cent of NaBr, calculated with reference to the dried substance.
CHARACTERS
A white, granular powder or small, colourless, transparent or opaque crystals, slightly hygroscopic,
freely soluble in water, soluble in alcohol.
IDENTIFICATION
A. It gives the reactions of bromides (2.3.1).
B. Solution S (see Tests) gives the reactions of sodium (2.3.1).
TESTS
the indicator.
Bromates To 10 ml of solution S add 1 ml of starch solution R, 0.1 ml of a 100 g/l solution of
potassium iodide R and 0.25 ml of 0.5M sulphuric acid and allow to stand protected from light for
5 min. No blue or violet colour develops.
Chlorides In a conical flask, dissolve 1.000 g in 20 ml of dilute nitric acid R. Add 5 ml of strong
hydrogen peroxide solution R and heat on a water-bath until the solution is completely decolourised.
Wash down the sides of the flask with a little water R and heat on a water-bath for 15 min. Allow to
cool, dilute to 50 ml with water R and add 5.0 ml of 0.1M silver nitrate and 1 ml of dibutyl phthalate R.
Shake and titrate with 0.1M ammonium thiocyanate, using 5 ml of ferric ammonium sulphate solution R2
as indicator. Not more than 1.7 ml of 0.1M silver nitrate is used (0.6 per cent). Note the volume of
0.1M silver nitrate used (see Assay).
Iodides To 5 ml of solution S add 0.15 ml of ferric chloride solution R1 and 2 ml of chloroform R.
Shake and allow to separate. The chloroform layer is colourless (Method I, 2.2.2).
(20 ppm).
100C to 105C for 3 h.
ASSAY
solution R2 as indicator and shaking vigorously towards the end-point. Correct for the amount of
chloride present, as determined in the test for chlorides.
1 ml of 0.1M silver nitrate is equivalent to 10.29 mg of NaBr.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
36-3
Sodium Butyl Hydroxybenzoate

Sodium Butylparaben
COOBun
NaO
C11H13NaO3
216.2
36457-20-2
Definition Sodium Butyl Hydroxybenzoate is the sodium salt of butyl 4-hydroxybenzoate. It

contains not less than 99.0% and not more than 102.0% of C11H13NaO3, calculated with reference
Characteristics A white powder; odourless or almost odourless; hygroscopic.
Freely soluble in water and in ethanol (96%).
Identification
A. Dissolve 0.5 g in 5 ml of water and acidify to litmus paper with hydrochloric acid. A white precipitate
is produced. Wash the precipitate with water and dry. The infrared absorption spectrum of the precipitate, Appendix II A, is concordant with the reference spectrum of butyl hydroxybenzoate (RS 036).
B. The residue on ignition yields the reactions characteristic of sodium salts, Appendix VI.
Alkalinity pH of a 0.1% w/v solution, 9.5 to 10.5, Appendix V L.
Clarity of solution Dissolve 1.0 g in 10 ml of water. The solution is clear, Appendix IV A.
Chloride Dissolve 1.0 g in 100 ml of water, add 1 ml of nitric acid and filter. 15 ml of the filtrate
complies with the limit test for chlorides, Appendix VII (330 ppm).
Sulphate Dissolve 0.50 g in 40 ml of water, add 3.5 ml of 2M hydrochloric acid, dilute to 60 ml with
water and filter. 15 ml of the filtrate complies with the limit test for sulphates, Appendix VII (0.12%).
plate precoated with silica gel F254 the surface of which has been modified with chemically-bonded
octadecylsilyl groups (Whatman KC18F plates are suitable) and a mixture of 70 volumes of methanol,
30 volumes of water and 1 volume of glacial acetic acid as the mobile phase. Apply separately to the
plate 2 l of each of the following solutions. For solution (1) dilute a 2.0% w/v solution of the
substance being examined in water with an equal volume of acetone. For solution (2) dilute 1 volume
of solution (1) to 25 volumes with a mixture of equal volumes of acetone and water. After removal of
the plate, allow it to dry in air and examine under ultraviolet light (254 nm). Any secondary spot in the
Assay Gently boil 0.1 g under a reflux condenser with 25 ml of 1.25M sodium hydroxide for 30
minutes. Allow to cool, add 25 ml of 0.0333M potassium bromate VS, 5 ml of a 12.5% w/v solution of
potassium bromide and 10 ml of hydrochloric acid and immediately stopper the flask. Shake for 15
minutes and allow to stand for 15 minutes. Add 25 ml of dilute potassium iodide solution and shake
vigorously. Titrate the liberated iodine with 0.1M sodium thiosulphate VS using starch mucilage, added
towards the end of the titration, as indicator. Repeat the operation without the substance being
examined. The difference between the titrations represents the amount of potassium bromate
required. The volume of 0.0333M potassium bromate VS used is equivalent to half of the volume of
0.1M sodium thiosulphate VS required for the titration. Each ml of 0.0333M potassium bromate VS is
equivalent to 7.207 mg of C11H13NaO3.
Storage Sodium Butyl Hydroxybenzoate should be kept in a well-closed container.
36-4
Sodium Calcium Edetate

NaOOCCH2
CH2COONa
Ca
O
C10H12CaN2Na2O8,xH2O
374.3 (anhydrous)
62-33-9 (anhydrous)
Sodium Calcium Edetate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Chelating substance used in treatment of lead poisoning.
Preparation
Sodium Calcium Edetate Intravenous Infusion
When sodium calciumedetate is prescribed or demanded, Sodium Calcium Edetate shall be
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sodium calcium edetate contains not less than 98.0 per cent and not more than the equivalent of
102.0 per cent of disodium [(ethylenedinitrilo)tetra-acetato]calciate(2), calculated with reference to
the anhydrous substance. It contains a variable amount of water of crystallisation.
CHARACTERS
A white or almost white powder, hygroscopic, freely soluble in water, practically insoluble in alcohol
and in ether.
IDENTIFICATION
obtained with sodium calcium edetate CRS. Examine the substances prepared as discs.
B. Dissolve 2 g in 10 ml of water R, add 6 ml of lead nitrate solution R, shake and add 3 ml of
potassium iodide solution R. No yellow precipitate is formed. Make alkaline to red litmus paper R by the
addition of dilute ammonia R2 and add 3 ml of ammonium oxalate solution R. A white precipitate is
formed.
C. Dissolve 0.5 g in 10 ml of water R. Make alkaline to red litmus paper R by the addition of dilute
ammonia R2 and add 3 ml of ammonium oxalate solution R. At most, a slight precipitate is formed.
D. Ignite. The residue gives the reactions of calcium (2.3.1).
E. The residue obtained in Identification test D gives the reactions of sodium (2.3.1).
TESTS
Disodium edetate Dissolve 5.0 g in 250 ml of water R. Add 10 ml of ammonium chloride buffer
solution pH 10.0 R and about 50 mg of mordant black 11 triturate R. Not more than 1.5 ml of 0.1M
magnesium chloride is required to change the colour of the indicator to violet (1.0 per cent).
Chlorides (2.4.4). To 20 ml of solution S add 30 ml of dilute nitric acid R, allow to stand for 30 min
and filter. 2.5 ml of the filtrate diluted to 15 ml with water R complies with the limit test for chlorides
(0.1 per cent).
Iron (2.4.9). 2.5 ml of solution S diluted to 10 ml with water R complies with the limit test for iron
(80 ppm). Add 0.25 g of calcium chloride R to each solution before the addition of the thioglycollic
acid R.
Water (2.5.12). 5.0 per cent to 13.0 per cent, determined on 0.100 g by the semi-micro determination of water (Method B).
36-5
ASSAY
Dissolve 0.500 g in water R and dilute to 300 ml with the same solvent. Add 2 g of hexamethylenetetramine R and 2 ml of dilute hydrochloric acid R. Titrate with 0.1M lead nitrate, using about
50 mg of xylenol orange triturate R as indicator.
1 ml of 0.1M lead nitrate is equivalent to 37.43 mg of C10H12CaN2Na2O8.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
36-6
Sodium Caprylate
corrected 1/01
Sodium Octanoate
COONa
3C
C8H15NaO2
166.2
1984-06-1
Sodium Caprylate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1471].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sodium caprylate contains not less than 99.0 per cent and not more than the equivalent of 101.0 per
cent of sodium octanoate, calculated with reference to the anhydrous substance.
CHARACTERS
A white, crystalline powder, very soluble or freely soluble in water, freely soluble in acetic acid,
sparingly soluble in alcohol, practically insoluble in acetone.
IDENTIFICATION
A. Examine the chromatograms obtained in the test for related substances. The retention time and
same as those of the principal peak in the chromatogram obtained with reference solution (a).
B. To 0.2 ml of solution S (see Tests) add 0.3 ml of water R. The solution gives reaction (b) of
sodium (2.3.1).
TESTS
Test solution. Dissolve 0.116 g in water R and dilute to 5 ml with the same solvent. Add 1 ml of a
2.8 per cent V/V solution of sulphuric acid R and shake with 10 ml of ethyl acetate R. Separate the
organic layer and dry over anhydrous sodium sulphate R.
Reference solution (a). Dissolve 0.10 g of caprylic acid CRS in ethyl acetate R and dilute to 10 ml with
the same solvent.
Reference solution (b). Dilute 1 ml of the test solution to 100 ml with ethyl acetate R. Dilute 5 ml of the
solution to 50 ml with ethyl acetate R.
a fused-silica column 30 m long and 0.25 mm in internal diameter coated with macrogol 20,000 2nitroterephthalate R (film thickness 0.25 m),
Column
Time
(min)
Temperature
(C)
Rate
(C/min)
Comment
01
1 25
25 35
100
isothermal
linear gradient
isothermal
100 220
220
Injection port
250
Detector
250
Inject 1 l of reference solution (b). The test is not valid unless in the chromatogram obtained the
principal peak has a signal-to-noise ratio of at least 5.
Inject 1 l of the test solution and 1 l of reference solution (a). Calculate the percentage of related
substances from the areas of the peaks in the chromatogram obtained with the test solution by the
normalisation procedure, disregarding any peaks with an area less than 0.5 times the area of the peak
in the chromatogram obtained with reference solution (b) and any peak due to the solvent. The
36-7
content of any related substance is not greater than 0.3 per cent and the sum of the related
substances is not greater than 0.5 per cent.
Heavy metals (2.4.8). Dissolve 2.0 g in glacial acetic acid R and dilute to 10 ml with the same acid.
Add 10 ml of alcohol R. 12 ml of the solution complies with limit test B for heavy metals (10 ppm).
Prepare the standard using 1 ml of lead standard solution (10 ppm Pb) R and 9 ml of a mixture of
equal volumes of glacial acetic acid R and alcohol R.
ASSAY
IMPURITIES
COOH
3C
A. hexanoic acid,
H3C
COOH
B. heptanoic acid,
3C
COOH
C. nonanoic acid,
COOH
H3C
D. decanoic acid,
3C
CH3
COOH
E. valproic acid,
COOR
H3C
F. R = CH3: methyl octanoate,

G. R = C2H5: ethyl octanoate,
COOMe
H3C
H. methyl decanoate,
O
H3C
CH3
I. undecan-2-one,
O
H3C
O
and enantiomer
J. (RS)-5-butyltetrahydrofuran-2-one (-hydroxyoctanoic acid lactone).

__________________________________________________________________________________________________________ Ph Eur
36-8
Anhydrous Sodium Carbonate

Na2CO3
106.0
497-19-8
Anhydrous Sodium Carbonate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0773]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Anhydrous sodium carbonate contains not less than 99.5 per cent and not more than the equivalent
of 100.5 per cent of Na2CO3, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, slightly granular powder, hygroscopic, freely soluble in water, practically
insoluble in alcohol.
IDENTIFICATION
A. Dissolve 1 g in water R and dilute to 10 ml with the same solvent. The solution is strongly alkaline
(2.2.4).
B. The solution prepared for Identification test A gives the reaction of carbonates (2.3.1).
C. The solution prepared for Identification test A gives reaction (a) of sodium (2.3.1).
D. It complies with the test for loss on drying (see Tests).
TESTS
Solution S Dissolve 2.0 g in portions in a mixture of 5 ml of hydrochloric acid R and 25 ml of distilled
water R. Heat the solution to boiling and cool. Add dilute sodium hydroxide solution R until the solution
is neutral and dilute to 50 ml with distilled water R.
more intensely coloured than reference solution Y6 (Method I, 2.2.2).
Alkali hydroxides and bicarbonates Dissolve 0.4 g in 20 ml of water R. Add 20 ml of barium
chloride solution R1 and filter. To 10 ml of the filtrate add 0.1 ml of phenolphthalein solution R. The
solution does not become red. Boil the remainder of the filtrate for 2 min. The solution remains clear
(2.2.1).
Chlorides (2.4.4). Dissolve 0.4 g in water R, add 4 ml of dilute nitric acid R and dilute to 15 ml with
Loss on drying (2.2.32). Not more than 1.0 per cent, determined on 1.000 g by drying at 300C.
ASSAY
Dissolve 1.000 g in 25 ml of water R. Add 0.2 ml of methyl orange solution R as indicator. Titrate with
1M hydrochloric acid until the colour changes from yellow to red.
1 ml of 1M hydrochloric acid is equivalent to 52.99 mg of Na2CO3.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
36-9
Sodium Carbonate Decahydrate

Na2CO3,10H2O
286.1
497-19-8
Sodium Carbonate Decahydrate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0191]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sodium carbonate decahydrate contains not less than 36.7 per cent and not more than the equivalent
of 40.0 per cent of Na2CO3.
CHARACTERS
A white, crystalline powder or colourless, transparent crystals, efflorescent, freely soluble in water,
practically insoluble in alcohol.
IDENTIFICATION
(2.2.4).
C. The solution prepared for Identification test A gives the reactions of sodium (2.3.1).
TESTS
Alkali hydroxides and bicarbonates Dissolve 1.0 g in 20 ml of water R, add 20 ml of barium
solution does not become red. Heat the remainder of the filtrate to boiling for 2 min. The solution
remains clear (2.2.1).
(20 ppm).
ASSAY
Dissolve 2.000 g in 25 ml of water R. Titrate with 1M hydrochloric acid, using 0.2 ml of methyl orange
STORAGE
__________________________________________________________________________________________________________ Ph Eur
36-10
Sodium Carbonate Monohydrate

Na2CO3,H2O
124.0
5698-11-6
Sodium Carbonate Monohydrate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0192]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sodium carbonate monohydrate contains not less than 83.0 per cent and not more than the equivalent of 87.5 per cent of Na2CO3.
CHARACTERS
alcohol.
IDENTIFICATION
(2.2.4).
C. The solution prepared for Identification test A gives the reactions of sodium (2.3.1).
TESTS
Alkali hydroxides and bicarbonates Dissolve 0.4 g in 20 ml of water R, add 20 ml of barium
solution does not become red. Heat the remainder of the filtrate to boiling for 2 min. The solution
remains clear (2.2.1).
(50 ppm).
ASSAY
Dissolve 1.000 g in 25 ml of water R. Titrate with 1M hydrochloric acid, using 0.2 ml of methyl orange
STORAGE
__________________________________________________________________________________________________________ Ph Eur
36-11
Sodium Cetostearyl Sulphate

Sodium Cetostearyl Sulphate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0847]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sodium cetostearyl sulphate is a mixture of sodium cetyl sulphate (C16H33NaO4S; Mr 344.5) and
sodium stearyl sulphate (C18H37NaO4S; Mr 372.5). It contains not less than 90.0 per cent of sodium
cetostearyl sulphate and not less than 40.0 per cent of sodium cetyl sulphate, both contents calculated with reference to the anhydrous substance. A suitable buffer may be added.
CHARACTERS
A white or pale yellow, amorphous or crystalline powder, soluble in hot water giving an opalescent
solution, practically insoluble in cold water, partly soluble in alcohol.
IDENTIFICATION
First identification: B, D, F.
A. Examine by thin-layer chromatography (2.2.27), using a TLC silanised silica gel plate R.
Test solution. Dissolve 50 mg of the substance to be examined in 10 ml of alcohol (70 per cent V/V) R,
heating on a water-bath.
Reference solution. Dissolve 50 mg of sodium cetostearyl sulphate CRS in 10 ml of alcohol (70 per cent
V/V) R, heating on a water-bath.
of water R, 40 volumes of acetone R and 40 volumes of methanol R. Allow the plate to dry in air and
spray with a 50 g/l solution of phosphomolybdic acid R in alcohol R. Heat at 120C until the appearance
of the spots (about 3 h). The principal spots in the chromatogram obtained with the test solution are
similar in position and colour to the principal spots in the chromatogram obtained with the reference
solution.
B. Examine the chromatograms obtained in the assay. The retention times of the two principal peaks
in the chromatogram obtained with test solution (c) are similar to those of the two principal peaks in
the chromatogram obtained with the reference solution.
C. Dissolve 0.1 g in 10 ml of water R and shake. A foam is formed.
D. It gives a yellow colour to a non-luminous flame.
E. To 0.1 ml of the solution prepared for identification test C add 0.1 ml of a 1 g/l solution of
methylene blue R and 2 ml of dilute sulphuric acid R. Add 2 ml of methylene chloride R and shake. The
methylene chloride layer has an intense blue colour.
F. Mix about 10 mg with 10 ml of ethanol R. Heat to boiling on a water-bath, shaking frequently.
Filter immediately and evaporate to dryness. Dissolve the residue in 7 ml of water R, add 3 ml of
dilute hydrochloric acid R and evaporate the solution to half its volume. Allow to cool. Filter. To the
filtrate add 1 ml of barium chloride solution R1. A white, crystalline precipitate is formed.
TESTS
Acidity or alkalinity Dissolve 0.5 g with heating in a mixture of 10 ml of water R and 15 ml of
alcohol (90 per cent V/V) R. Add 0.1 ml of phenolphthalein solution R1. The solution is colourless. Add
0.1 ml of 0.1M sodium hydroxide. The solution is red.
Sodium chloride and sodium sulphate Not more than a total of 8.0 per cent.
Sodium chloride. Dissolve 5.00 g in 50 ml of water R, add dilute nitric acid R dropwise until the
solution is neutral to blue litmus paper R. Add 2 ml of potassium chromate solution R and titrate with
0.1M silver nitrate.
1 ml of 0.1M silver nitrate is equivalent to 5.844 mg of NaCl.
Sodium sulphate. Dissolve 0.500 g in 20 ml of water R, warming gently if necessary, and add 1 ml of a
0.5 g/l solution of dithizone R in acetone R. If the solution is red, add 1M nitric acid, dropwise, until a
bluish-green colour is obtained. Add 2.0 ml of dichloroacetic acid solution R and 80 ml of acetone R.
Titrate with 0.01M lead nitrate until a persistent orange-red colour is obtained.
1 ml of 0.01M lead nitrate is equivalent to 1.420 mg of Na2SO4.
Free cetostearyl alcohol Not more than 4.0 per cent. Examine the chromatogram obtained with
test solution (a) in the assay.
Calculate the percentage content of free cetostearyl alcohol in the substance to be examined, using
the expression:
36-12
S
100 mH
S Ha(corr) m
S = sum of the areas of the peaks corresponding to cetyl alcohol and stearyl alcohol in the
chromatogram obtained with test solution (a),
mH = mass of the internal standard added in the preparation of test solution (a), in milligrams,
SHa(corr) = corrected area of the peak corresponding to the internal standard in the chromatogram
obtained with test solution (a),
m = mass of sodium cetostearyl sulphate used for the preparation of test solution (a), in
milligrams.
of water.
ASSAY
Internal standard solution. Dissolve 0.20 g of heptadecanol CRS in ethanol R and dilute to 50 ml with
the same solvent.
Test solution (a). Dissolve 0.300 g of the substance to be examined in 50 ml of ethanol R and add 2 ml
of the internal standard solution and 48 ml of water R. Shake with four quantities, each of 25 ml, of
pentane R, adding sodium chloride R, if necessary, to facilitate the separation of the layers. Combine
the organic layers. Reserve the hydro-alcoholic layer for the preparation of test solutions (c) and (d).
Wash the organic layer with two quantities, each of 30 ml, of water R. Dry over anhydrous sodium
sulphate R and filter.
Test solution (b). Dissolve 0.300 g of the substance to be examined in 50 ml of ethanol R and add
50 ml of water R. Shake with four quantities, each of 25 ml, of pentane R, adding sodium chloride R, if
necessary, to facilitate the separation of the layers. Combine the organic layers, wash with two
quantities, each of 30 ml, of water R. Dry over anhydrous sodium sulphate R and filter.
Test solution (c). Transfer 25 ml of the hydro-alcoholic solution obtained in the preparation of test
solution (a) to a 200 ml flask that can be fitted with a reflux condenser. Add 20 ml of hydrochloric
acid R and 10 ml of the internal standard solution. Boil under a reflux condenser for 2 h. Allow to
cool and shake with four quantities, each of 20 ml, of pentane R. Combine the organic layers and
wash with two quantities, each of 20 ml, of water R. Dry over anhydrous sodium sulphate R and filter.
Test solution (d). Transfer 25 ml of the hydro-alcoholic solution obtained in the preparation of test
solution (a) to a 200 ml flask that can be fitted with a reflux condenser. Add 20 ml of hydrochloric
acid R and 10 ml of ethanol R. Boil under a reflux condenser for 2 h. Allow to cool and shake with
four quantities, each of 20 ml, of pentane R. Combine the organic layers and wash with two quantities, each of 20 ml, of water R. Dry over anhydrous sodium sulphate R and filter.
Reference solution. Dissolve 50 mg of cetyl alcohol CRS and 50 mg of stearyl alcohol CRS in ethanol R
a fused-silica column 25 m long and 0.25 mm in internal diameter coated with poly(dimethyl)siloxane R or another suitable polar phase,
Time
(min)
Temperature
(C)
Rate
(C per min)
Comment
Column 020
150250
linear
gradient
Injection
port
Detector
250
250
The substances are eluted in the following order: cetyl alcohol, heptadecanol (internal standard) and
stearyl alcohol.
Correction for interference. Inject 1 l of test solution (a) and 1 l of test solution (b). If the chromatogram obtained with test solution (b) shows a peak with the same retention time as the peak corresponding to the internal standard in the chromatogram obtained with test solution (a), calculate the
ratio:
r=
Sci
Si
36-13
Sci = area of the peak corresponding to cetyl alcohol in the chromatogram obtained with test
solution (b),
Si = area of the peak with the same retention time as the peak corresponding to the internal
standard in the chromatogram obtained with test solution (a).
If r is less than 300, calculate the corrected area SHa(corr) of the peak corresponding to the internal
standard in the chromatogram obtained with test solution (a):
S Ha(corr) = S Ha
Si S c
S ci
SHa = area of the peak corresponding to the internal standard in the chromatogram obtained
with test solution (a),
Sc = area of the peak corresponding to cetyl alcohol in the chromatogram obtained with test
solution (a).
Inject 1 l of test solution (c) and 1 l of test solution (d). Carry out the correction for interference
in the same manner as for test solution (a) and calculate the corrected area SHc(corr) of the peak
corresponding to the internal standard in the chromatogram obtained with test solution (c).
Inject equal volumes of the reference solution, test solution (c) and test solution (d). Identify the
peaks in the chromatograms obtained with the test solutions by comparison of their retention times
with those of the peaks in the chromatogram obtained with the reference solution. Determine the
area of each peak.
Calculate the percentage content of sodium cetyl sulphate in the substance to be examined, using
the expression:
( A 1.421) mH 100
SHc(corr) m
A = area of the peak corresponding to cetyl alcohol in the chromatogram obtained with test
solution (c),
mH = mass of the internal standard added in the preparation of test solution (c), in milligrams,
SHc(corr) = corrected area of the peak corresponding to the internal standard in the chromatogram
obtained with test solution (c),
m = mass of the substance to be examined in test solution (c), in milligrams.
Calculate the percentage content of sodium stearyl sulphate in the substance to be examined, using
the expression:
( B 1.377) mH 100
SHc(corr) m
B = area of the peak corresponding to stearyl alcohol in the chromatogram obtained with test
solution (c).
The percentage content of sodium cetostearyl sulphate corresponds to the sum of the percentage
content of sodium cetyl sulphate and the percentage content of sodium stearyl sulphate.
LABELLING
The label states, where appropriate, the name and concentration of any added buffer.
__________________________________________________________________________________________________________ Ph Eur
36-14
Sodium Chloride
NaCl
58.44
7647-14-5
Sodium Chloride complies with the requirements of the 3rd edition of the European Pharmacopoeia [0193].
Action and use Used in treatment of electrolyte deficiency.
Preparations
Oral Rehydration Salts
Potassium Chloride and Sodium Chloride Intravenous Infusion
Potassium Chloride, Sodium Chloride and Glucose Intravenous Infusion
Sodium Chloride Eye Drops
Sodium Chloride Eye Lotion
Sodium Chloride Intravenous Infusion
Sodium Chloride and Glucose Intravenous Infusion
Sodium Chloride Irrigation Solution
Compound Sodium Chloride Mouthwash
Sodium Chloride Solution
Sodium Chloride Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sodium chloride contains not less than 99.0 per cent and not more than the equivalent of 100.5 per
cent of NaCl, calculated with reference to the dried substance.
CHARACTERS
ethanol.
IDENTIFICATION
TESTS
Solution S. Dissolve 20.0 g in carbon dioxide-free water R prepared from distilled water R and dilute to
Appearance of solution. Solution S is clear (2.2.1) and colourless (Method II, 2.2.2).
Acidity or alkalinity. To 20 ml of solution S add 0.1 ml of bromothymol blue solution R1. Not more
the indicator.
Bromides. To 1.0 ml of solution S add 4.0 ml of water R, 2.0 ml of phenol red solution R2 and 1.0 ml
of a 0.1 g/l solution of chloramine R and mix immediately. After exactly 2 min, add 0.15 ml of 0.1M
sodium thiosulphate, mix and dilute to 10.0 ml with water R. The absorbance (2.2.25) of the solution
measured at 590 nm, using water R as the compensation liquid, is not greater than that of a standard
prepared at the same time and in the same manner, using 5.0 ml of a 3.0 mg/l solution of potassium
bromide R (50 ppm).
Ferrocyanides Dissolve 2.0 g in 6 ml of water R. Add 0.5 ml of a mixture of 5 ml of a 10 g/l solution of ferric ammonium sulphate R in a 2.5 g/l solution of sulphuric acid R and 95 ml of a 10 g/l solution of ferrous sulphate R. No blue colour develops within 10 min.
Iodides Moisten 5 g by the dropwise addition of a freshly prepared mixture of 0.15 ml of sodium
nitrite solution R, 2 ml of 0.5M sulphuric acid, 25 ml of iodide-free starch solution R and 25 ml of water R.
After 5 min, examine in daylight. The substance shows no blue colour.
Nitrites To 10 ml of solution S add 10 ml of water R. Measure the absorbance (2.2.25) of the solution at 354 nm. The absorbance is not greater than 0.01.
Phosphates (2.4.11). Dilute 2 ml of solution S to 100 ml with water R. The solution complies with
the limit test for phosphates (25 ppm).
haemodialysis solutions or haemofiltration solutions, it complies with the test for aluminium.
Dissolve 20.0 g in 100 ml of water R and add 10 ml of acetate buffer solution pH 6.0 R. The solution
complies with the limit test for aluminium (0.2 ppm). Use as the reference solution a mixture of 2 ml
36-15
of aluminium standard solution (2 ppm Al) R, 10 ml of acetate buffer solution pH 6.0 R and 98 ml of
water R. To prepare the blank, use a mixture of 10 ml of acetate buffer solution pH 6.0 R and 100 ml of
water R.
Barium. To 5 ml of solution S add 5 ml of distilled water R and 2 ml of dilute sulphuric acid R. After
2 h, any opalescence in the solution is not more intense than that in a mixture of 5 ml of solution S
and 7 ml of distilled water R.
using a mixture of 4 ml of iron standard solution (1 ppm Fe) R and 6 ml of water R.
Potassium. If intended for use in the manufacture of parenteral dosage forms or haemodialysis,
haemofiltration or peritoneal dialysis solutions, it contains not more than 500 ppm of K, determined
by atomic emission spectrometry (Method I, 2.2.22).
the same solvent.
Reference solutions. Dissolve in water R 1.144 g of potassium chloride R, previously dried at 100C to
105C for 3 h, and dilute to 1000.0 ml with the same solvent (600 g of K per millilitre). Dilute as
required.
100C to 105C for 2 h.
without a further appropriate procedure for the removal of bacterial endotoxins, it contains not more
than 5 I.U. of endotoxin per gram.
ASSAY
Dissolve 1.000 g in water R and dilute to 100 ml with the same solvent. To 10.0 ml of the solution
solution R2 as indicator and shaking vigorously towards the end-point.
LABELLING
The label states:
forms,
where applicable, that the substance is free from bacterial endotoxins,
solutions, haemodialysis solutions or haemofiltration solutions.
__________________________________________________________________________________________________________ Ph Eur
36-16
Sodium Citrate
Trisodium Citrate
CH2COONa
HO
COONa
CH2COONa
C6H5Na3O7,2H2O
294.1
6132-04-3
Sodium Citrate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0412].
Action and use Systemic alkalinising substance.
Preparations
Sodium Citrate Eye Drops
Sodium Citrate Irrigation Solution
Sodium Citrate Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sodium citrate contains not less than 99.0 per cent and not more than the equivalent of 101.0 per
cent of trisodium 2-hydroxypropane-1,2,3-tricarboxylate, calculated with reference to the anhydrous
substance.
CHARACTERS
A white, crystalline powder or white, granular crystals, slightly deliquescent in moist air, freely soluble
in water, practically insoluble in alcohol.
IDENTIFICATION
A. To 1 ml of solution S (see Tests add 4 ml of water R. The solution gives the reaction of citrates
(2.3.1).
B. 1 ml of solution S gives reaction (a) of sodium (2.3.1).
TESTS
indicator.
solution is not more intense than that in a standard prepared at the same time in the same manner
using 4 ml of a 50 mg/l solution of oxalic acid R (300 ppm).
Water (2.5.12). 11.0 per cent to 13.0 per cent, determined on 0.300 g by the semi-micro determination of water. After adding the substance to be examined, stir for 15 min before titrating.
Pyrogens (2.6.8). If intended for use in large-volume preparations for parenteral use, the competent
authority may require that it comply with the test for pyrogens. Inject per kilogram of the rabbits
36-17
mass 10 ml of a freshly prepared solution in water for injections R containing per millilitre 10.0 mg of
the substance to be examined and 7.5 mg of pyrogen-free calcium chloride R.
ASSAY
Dissolve 0.150 g in 20 ml of anhydrous acetic acid R, heating to about 50C. Allow to cool. Using
0.25 ml of naphtholbenzein solution R as indicator, titrate with 0.1M perchloric acid until a green colour
is obtained.
1 ml of 0.1M perchloric acid is equivalent to 8.602 mg of C6H5Na3O7.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
36-18
Sodium Cromoglicate
NaOOC
H
O
C23H14Na2O11
COONa
OH
O
512.3
O
15826-37-6
Sodium Cromoglicate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Prophylaxis of allergic conditions.
Preparations
Sodium Cromoglicate Eye Drops
Sodium Cromoglicate Powder for Inhalation
When sodium cromoglycate is prescribed or demanded, Sodium Cromoglicate shall be dispensed or
supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sodium cromoglicate contains not less than 98.0 per cent and not more than the equivalent of
101.0 per cent of disodium 5,5-[(2-hydroxy-1,3-propanediyl)dioxy]bis(4-oxo-4H-chromene-2carboxylate), calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, hygroscopic, soluble in water, practically insoluble in
IDENTIFICATION
A. Dissolve 10.0 mg in phosphate buffer solution pH 7.4 R and dilute to 100.0 ml with the same
solvent. Dilute 10.0 ml of this solution to 100.0 ml with the same solvent. Examined between
The ratio of the absorbance at the maximum at 327 nm to that at the maximum at 239 nm is 0.25 to
0.30.
obtained with sodium cromoglicate CRS. Examine the substances prepared as discs.
C. Dissolve about 5 mg in 0.5 ml of methanol R. Add 3 ml of a solution in methanol R containing 5 g/l
of aminopyrazolone R and 1 per cent V/V of hydrochloric acid R. Allow to stand for 5 min. The solution
shows an intense yellow colour.
TESTS
not more intensely coloured than reference solution BY5 (Method II, 2.2.2).
colourless. Add 0.2 ml of 0.01M sodium hydroxide. The solution is pink. Add 0.4 ml of 0.01M hydrochloric acid. The solution is colourless. Add 0.25 ml of methyl red solution R. The solution is red.
coating substance.
Test solution. Dissolve 0.2 g of the substance to be examined in a mixture of 1 volume of acetone R, 4
volumes of tetrahydrofuran R and 6 volumes of water R and dilute to 10 ml with the same mixture of
solvents.
Reference solution. Dissolve 10 mg of 1,3-bis(2-acetyl-3-hydroxyphenoxy)-2-propanol CRS in chloroform R and dilute to 100 ml with the same solvent.
volumes of glacial acetic acid R, 50 volumes of ethyl acetate R and 50 volumes of toluene R. Allow the
36-19
plate to dry in air and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained
with the test solution, apart from the principal spot (which remains at the starting point), is not more
Oxalate Dissolve 0.10 g in 20 ml of water R, add 5.0 ml of iron salicylate solution R and dilute to
50.0 ml with water R. Determine the absorbance (2.2.25) at 480 nm. The absorbance is not less than
that of a standard prepared in the same manner using 0.35 mg of oxalic acid R instead of the
substance to be examined.
diphosphorus pentoxide R at 100C to 105C and at a pressure of 300 Pa to 600 Pa.
ASSAY
Dissolve 0.200 g with heating in a mixture of 5 ml of 2-propanol R and 25 ml of ethylene glycol R. Cool
and add 30 ml of dioxan R. Titrate with 0.1M perchloric acid, determining the end-point potentiometrically (2.2.20).
1 ml of 0.1M perchloric acid is equivalent to 25.62 mg of C23H14Na2O11.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
36-20
Sodium Cyclamate
NHSO3
Na+
C6H12NNaO3S
201.2
139-05-9
Sodium Cyclamate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0774].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sodium cyclamate contains not less than 98.5 per cent and not more than the equivalent of 101.0 per
cent of sodium N-cyclohexylsulphamate, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder or colourless crystals, freely soluble in water, slightly soluble in alcohol.
IDENTIFICATION
obtained with sodium cyclamate CRS.
B. Examine the chromatograms obtained in the test for sulphamic acid. The principal spot in the
C. To 1 ml of solution S (see Tests), add 1 ml of water R and 2 ml of silver nitrate solution R1 and
shake. A white, crystalline precipitate is formed.
D. To 1 ml of solution S add 5 ml of water R, 2 ml of dilute hydrochloric acid R and 4 ml of barium
chloride solution R1 and mix. The solution is clear. Add 2 ml of sodium nitrite solution R. A voluminous
white precipitate is formed and gas is given off.
E. A mixture of 1 ml of solution S and 1 ml of water R gives reaction (a) of sodium (2.3.1).
TESTS
Solution S Dissolve 5 g in carbon dioxide-free water R prepared from distilled water R and dilute to
Absorbance (2.2.25). The absorbance of solution S, measured at 270 nm, is not greater than 0.10.
Sulphamic acid Examine by thin-layer chromatography (2.2.27), using a TLC silica gel G plate R.
Test solution (a). Use solution S.
Reference solution (a). Dissolve 0.10 g of sodium cyclamate CRS in water R and dilute to 10 ml with the
same solvent.
Reference solution (b). Dissolve 10 mg of sulphamic acid R in water R and dilute to 100 ml with the
same solvent.
of concentrated ammonia R, 10 volumes of water R, 20 volumes of ethyl acetate R and 70 volumes of
propanol R. Dry the plate in a current of warm air, heat at 105C for 5 min and spray the hot plate
with strong sodium hypochlorite solution R diluted to a concentration of 5 g/l of active chlorine. Place
the plate in a current of cold air until an area of coating below the points of application gives at most
a faint blue colour with a drop of potassium iodide and starch solution R; avoid prolonged exposure to
cold air. Spray with potassium iodide and starch solution R and examine the chromatograms within
5 min. Any spot corresponding to sulphamic acid in the chromatogram obtained with test solution
(a) is not more intense than the spot in the chromatogram obtained with reference solution (b)
(0.1 per cent).
Aniline, cyclohexylamine and dicyclohexylamine Not more than 1 ppm of aniline, not more
than 10 ppm of cyclohexylamine and not more than 1 ppm of dicyclohexylamine determined by gas
36-21
chromatography (2.2.28) using tetradecane R as the internal standard.
Internal standard solution. Dissolve 2 l of tetradecane R in methylene chloride R and dilute to 100 ml
Test solution. Dissolve 2.00 g of the substance to be examined in 20 ml of water R and add 0.5 ml of
strong sodium hydroxide solution R and shake with 30 ml of toluene R. Shake 20 ml of the upper layer
with 4 ml of a mixture of equal volumes of dilute acetic acid R and water R. Separate the lower layer
and add 0.5 ml of concentrated sodium hydroxide solution R and 0.5 ml of the internal standard solution. Shake and use the lower layer for chromatography immediately after separation.
Reference solution. Dissolve 10.0 mg (about 12 l) of cyclohexylamine R, 1.0 mg (about 1.1 l) of
dicyclohexylamine R and 1.0 mg (about 1 l) of aniline R in water R and dilute to 1000 ml with the
same solvent. Dilute 10.0 ml of this solution to 100.0 ml with water R (solution A). To 20.0 ml of
solution A add 0.5 ml of strong sodium hydroxide solution R and extract with 30 ml of toluene R. Shake
20 ml of the upper layer with 4 ml of a mixture of equal volumes of dilute acetic acid R and water R.
Separate the lower layer and add 0.5 ml of concentrated sodium hydroxide solution R and 0.5 ml of the
internal standard solution. Shake and use the lower layer for chromatography immediately after
separation.
a fused silica column 25 m long and 0.32 mm in internal diameter coated with poly(dimethyl)(diphenyl)siloxane R (0.51 m),
a split vent at a flow rate of 20 ml/min,
Column
Time
(min)
Temperature
(C)
01
19
85
85 150
9 13 150
Injection port
Detector
Rate
(C/min)
8
Comment
isothermal
linear gradient
isothermal
250
270
Inject 1.5 l of each solution. When the chromatograms are recorded in the conditions prescribed,
the retention times relative to cyclohexylamine (about 2.3 min) are the following: tetradecane about
1.4, dicyclohexylamine about 4.3 and aniline about 4.5.
with the limit test for sulphates (0.1 per cent).
Loss on drying (2.2.32). Not more than 1.0 per cent, determined on 1.000 g in an oven at 100C to
105C for 4 h.
ASSAY
Dissolve without heating 0.150 g in 60 ml of anhydrous acetic acid R. Titrate with 0.1M perchloric acid,
1 ml of 0.1M perchloric acid is equivalent to 20.12 mg of C6H12NNaO3S.
IMPURITIES
2N
SO3H
A. sulphamic acid,
NH2
B. aniline (phenylamine),
NH2
C. cyclohexanamine,
H
N
D. N-cyclohexylcyclohexanamine.
__________________________________________________________________________________________________________ Ph Eur
36-22
Anhydrous Sodium Dihydrogen Phosphate

NaH2PO4
120.0
7558-80-7
Definition Anhydrous Sodium Dihydrogen Phosphate contains not less than 98.0% and not more
than 100.5% of NaH2PO4, calculated with reference to the dried substance.
Characteristics White, slightly deliquescent crystals or granules.
Very soluble in water; very slightly soluble in ethanol (96%).
Dissolve 10.0 g in sufficient carbon dioxide-free water prepared from distilled water to produce 100 ml
(solution S).
Identification
A. Solution S is faintly acid, Appendix V K.
B. Solution S yields the reactions characteristic of phosphates, Appendix VI.
C. Solution S neutralised with a 10% w/v solution of potassium hydroxide yields reaction A
characteristic of sodium salts, Appendix VI.
D. Complies with the test for Loss on drying.
Acidity pH of a mixture of 5 ml of solution S and 5 ml of carbon dioxide-free water, 4.2 to 4.5,
Appendix V L.
Clarity and colour of solution Solution S is clear, Appendix IV A, and colourless, Appendix IV B,
Method II.
Heavy metals 12 ml of solution S complies with limit test A for heavy metals, Appendix VII. Use lead
standard solution (1 ppm Pb) to prepare the standard (10 ppm).
Iron 10 ml of solution S complies with the limit test for iron, Appendix VII (10 ppm).
Chloride Dilute 2.5 ml of solution S to 15 ml with water. The resulting solution complies with the
limit test for chlorides, Appendix VII (200 ppm).
Sulphate Dilute 5 ml of solution S to 15 ml with distilled water. The resulting solution complies with
the limit test for sulphates, Appendix VII (300 ppm).
Reducing substances To 5 ml of solution S add 0.25 ml of 0.02M potassium permanganate and 5 ml
of 1M sulphuric acid and heat in a water bath for 5 minutes. The solution retains a slight red colour.
1 g.
Assay Dissolve 2 g in 50 ml of water and titrate with carbonate-free 1M sodium hydroxide VS determining the end point potentiometrically. Each ml of 1M sodium hydroxide VS is equivalent to 0.120 g
of NaH2PO4.
Storage Anhydrous Sodium Dihydrogen Phosphate should be kept in a well-closed container.
36-23
Sodium Dihydrogen Phosphate Monohydrate

NaH2PO4,H2O
138.0
10049-21-5
Definition Sodium Dihydrogen Phosphate Monohydrate contains not less than 98.0% and not more
than 100.5% of NaH2PO4, calculated with reference to the dried substance.
Characteristics Colourless crystals or a white powder.
Very soluble in water; very slightly soluble in ethanol (96%).
Dissolve 10.0 g in sufficient carbon dioxide-free water prepared from distilled water to produce 100 ml
(solution S).
Identification
A. Solution S is faintly acid, Appendix V K.
B. Solution S yields the reactions characteristic of phosphates, Appendix VI.
C. Solution S neutralised with a 10% w/v solution of potassium hydroxide yields reaction A
characteristic of sodium salts, Appendix VI.
D. Complies with the test for Loss on drying.
Acidity pH of a mixture of 5 ml of solution S and 5 ml of carbon dioxide-free water, 4.2 to 4.5,
Appendix V L.
Clarity and colour of solution Solution S is clear, Appendix IV A, and colourless, Appendix IV B,
Method II.
Heavy metals 12 ml of solution S complies with limit test A for heavy metals, Appendix VII. Use lead
Iron 10 ml of solution S complies with the limit test for iron, Appendix VII (10 ppm).
Chloride Dilute 2.5 ml of solution S to 15 ml with water. The resulting solution complies with the
limit test for chlorides, Appendix VII (200 ppm).
Sulphate Dilute 5 ml of solution S to 15 ml with distilled water. The resulting solution complies with
the limit test for sulphates, Appendix VII (300 ppm).
of 1M sulphuric acid and heat in a water bath for 5 minutes. The solution retains a slight red colour.
Loss on drying When dried to constant weight at 130, loses 11.5 to 14.5% of its weight. Use 0.5 g.
Assay Dissolve 2.2 g in 50 ml of water and titrate with carbonate-free 1M sodium hydroxide VS
determining the end point potentiometrically. Each ml of 1M sodium hydroxide VS is equivalent to
0.120 g of NaH2PO4.
Storage Sodium Dihydrogen Phosphate Monohydrate should be kept in a well-closed container.
36-24
Sodium Dihydrogen Phosphate Dihydrate

Sodium Acid Phosphate
156.0
13472-35-0
NaH2PO4,2H2O
Sodium Dihydrogen Phosphate Dihydrate complies with the requirements of the 3rd edition of the European
Preparation
Phosphates Enema
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sodium dihydrogen phosphate dihydrate contains not less than 98.0 per cent and not more than the
equivalent of 100.5 per cent of NaH2PO4, calculated with reference to the dried substance.
CHARACTERS
A white powder or colourless crystals, very soluble in water, very slightly soluble in alcohol.
IDENTIFICATION
A. Solution S (see Tests) is faintly acid (2.2.4).
B. Solution S gives the reactions of phosphates (2.3.1).
C. Solution S previously neutralised using a 100 g/l solution of potassium hydroxide R gives reaction
(a) of sodium (2.3.1).
TESTS
pH (2.2.3). To 5 ml of solution S add 5 ml of carbon dioxide-free water R. The pH of the solution is
4.2 to 4.5.
of dilute sulphuric acid R and heat in a water-bath for 5 min. The solution retains a slight red colour.
Sulphates (2.4.13). To 5 ml of solution S add 0.5 ml of hydrochloric acid R and dilute to 15 ml with
at 130C.
ASSAY
Dissolve 2.500 g in 40 ml of water R. Titrate with carbonate-free 1M sodium hydroxide, determining
1 ml of 1M sodium hydroxide is equivalent to 0.120 g of NaH2PO4.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
36-25
Sodium Feredetate
_
OOC
_
OOC
COO
COO
C10H12N2O8FeNa,H2O 385.1
_
Na+ Fe3+
15708-41-5 (anhydrous)
Definition Sodium Feredetate is iron(III) sodium ethylenediaminetetra-acetate monohydrate. It

contains not less than 98.0% and not more than 102.0% of C10H12O8N2FeNa, calculated with
Characteristics A yellow or yellowish brown, crystalline powder; hygroscopic.
Identification
A. The infrared absorption spectrum, Appendix II A, is concordant with the reference spectrum of sodium
feredetate (RS 378).
B. Ignite 0.5 g and allow to cool. Dissolve the residue in 2 ml of hydrochloric acid, add sufficient water
to produce 20 ml and filter. The filtrate yields reaction C characteristic of iron salts, Appendix VI.
Dilute 1 volume of the filtrate to 10 volumes with water; the solution yields reaction B characteristic
of iron salts, Appendix VI.
C. Dissolve 2 g in 30 ml of water, slowly add 6.5 ml of a 20% w/v solution of potassium hydroxide,
shake and filter the resulting suspension. Evaporate 8 ml of the filtrate to dryness, ignite and dissolve
the residue in 4 ml of water. The solution yields reaction B characteristic of sodium salts, Appendix VI.
Free iron Not more than 500 ppm when determined by the following method. Dissolve 0.2 g in
sufficient water to produce 20 ml and filter. Label three tubes A, B and C. Place 5 ml of the filtrate
into each of tubes A and B and 4 ml of water and 1 ml of an iron standard solution prepared in the
following manner in tube C. For the iron standard solution dilute 25 volumes of a 0.1726% w/v
solution of ammonium iron(III) sulphate in 0.05M sulphuric acid to 200 volumes with water (25 ppm of
Fe(III)). Add 1 ml of a 1.0% w/v solution of disodium catechol-3,5-disulphonate into each of tubes A
and C and 1 ml of water to tube B. Measure the absorbance of solution A at 670 nm, Appendix II B,
using solution B in the reference cell and of solution C using water in the reference cell. The absorbance of solution A is not greater than that of solution C.
Heavy metals 1 g complies with limit test D for heavy metals, Appendix VII (20 ppm). Use 2 ml of
lead standard solution (10 ppm Pb) to prepare the standard solution.
Free sodium edetate To 4 ml of a 1.0% w/v solution add 2 ml of ferric iron standard solution
(50 ppm) and 1 ml of a 1.0% w/v solution of disodium catechol-3,5-disulphonate and mix. Prepare a
standard in the same manner using 4 ml of a 0.010% w/v solution of disodium edetate in place of the
solution of the substance being examined. Measure the absorbance of the solutions at 670 nm,
Appendix II B, using water in the reference cell. The absorbance of the solution of the substance
being examined is not less than that of the standard solution (1%).
Sulphates Dissolve 2 g in 40 ml of water, add 5 ml of a 20% w/v solution of sodium hydroxide and
sufficient water to produce 50 ml, shake and filter. Evaporate 6.2 ml of this solution to dryness and
ignite until no trace of carbon remains. Cool and dissolve the residue in 10 ml of distilled water.
Neutralise the solution with 2M hydrochloric acid using litmus paper as external indicator and add 2 ml
in excess. Boil the solution, cool, dilute to 15 ml with distilled water and filter. The filtrate complies
with the limit test for sulphates, Appendix VII (600 ppm).
Loss on drying When dried over silica gel at 120 at a pressure not exceeding 2 kPa for 5 hours,
loses 4.0 to 13.5% of its weight. Use 1 g.
Assay Dissolve 1 g in 40 ml of water in an iodine flask, add 20 ml of hydrochloric acid and 3 g of
potassium iodide, close the flask and allow to stand for 5 minutes. Titrate the liberated iodine with
0.1M sodium thiosulphate VS using starch mucilage as indicator. Repeat the procedure without the
substance being examined. The difference between the titrations represents the amount of sodium
thiosulphate required. Each ml of 0.1M sodium thiosulphate VS is equivalent to 36.71 mg of
C10H12O8N2FeNa.
Storage Sodium Feredetate should be kept in an airtight container.
Action and use Used in prevention and treatment of iron deficiency.
Preparation
Sodium Feredetate Oral Solution
36-26
Sodium Fluoride
NaF
41.99
7681-49-4
Sodium Fluoride complies with the requirements of the 3rd edition of the European Pharmacopoeia [0514].
Action and use Used in prevention of dental caries.
Preparations
Sodium Fluoride Mouthwash
Sodium Fluoride Oral Drops
Sodium Fluoride Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sodium fluoride contains not less than 98.5 per cent and not more than the equivalent of 100.5 per
cent of NaF, calculated with reference to the dried substance.
CHARACTERS
A white powder or colourless crystals, soluble in water, practically insoluble in alcohol.
IDENTIFICATION
A. To 2 ml of solution S (see Tests) add 0.5 ml of calcium chloride solution R. A gelatinous white
precipitate is formed which dissolves on adding 5 ml of ferric chloride solution R1.
B. To about 4 mg add a mixture of 0.1 ml of alizarin S solution R and 0.1 ml of zirconyl nitrate solution R and mix. The colour changes from red to yellow.
TESTS
Solution S Dissolve 2.5 g in carbon dioxide-free water R without heating and dilute to 100 ml with the
same solvent.
Acidity or alkalinity Dissolve 2.5 g of potassium nitrate R in 40 ml of solution S and dilute to 50 ml
with carbon dioxide-free water R. Cool to 0C and add 0.2 ml of phenolphthalein solution R. If the
solution is colourless, not more than 1.0 ml of 0.1M sodium hydroxide is required to produce a red
colour that persists for at least 15 s. If the solution is red, not more than 0.25 ml of 0.1M hydrochloric
acid is required to change the colour of the indicator.
Fluorosilicates Heat to boiling the neutralised solution obtained in the test for acidity or alkalinity
and titrate whilst hot. Not more than 0.75 ml of 0.1M sodium hydroxide is required to change the
colour of the indicator to red.
Sulphates (2.4.13). Dissolve 0.25 g in 10 ml of a saturated solution of boric acid R in distilled water R.
Add 5 ml of distilled water R and 0.6 ml of hydrochloric acid R1. The solution complies with the limit
test for sulphates (200 ppm). Prepare the standard by mixing 0.6 ml of hydrochloric acid R1, 5 ml of
sulphate standard solution (10 ppm SO4) R and 10 ml of a saturated solution of boric acid R in distilled
water R.
130C for 3 h.
ASSAY
To 80.0 mg add a mixture of 5 ml of acetic anhydride R and 20 ml of anhydrous acetic acid R and heat
to dissolve. Allow to cool and add 20 ml of dioxan R. Using 0.1 ml of crystal violet solution R as
indicator, titrate with 0.1M perchloric acid until a green colour is obtained. Carry out a blank titration.
1 ml of 0.1M perchloric acid is equivalent to 4.199 mg of NaF.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
36-27
Sodium Fusidate
H
Me
HO
COONa
OAc
Me
H
Me H
Me
H
H
Me
HO
H
Me
C31H47NaO6
538.7
751-94-0
Sodium Fusidate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0848].
Preparation
Sodium Fusidate Ointment
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sodium fusidate is sodium ent-16-acetoxy-3,11-dihydroxy-4,8,14-trimethyl-18-nor-5,10cholesta-(17Z)-17(20),24-dien-21-oate, an antimicrobial substance produced by the growth of
certain strains of Fusidium coccineum or by any other means. It contains not less than 97.5 per cent
and not more than the equivalent of 101.0 per cent of C31H47NaO6, calculated with reference to the
CHARACTERS
A white or almost white, crystalline powder, slightly hygroscopic, freely soluble in water and in
alcohol.
IDENTIFICATION
A. Examine by infrared absorption spectrophotometry (2.2.24). Dissolve 0.1 g in 5 ml of water R,
add 5 ml of chloroform R and 0.1 ml of dilute phosphoric acid R. Shake vigorously for 1 min, allow to
separate and filter the lower layer through absorbent cotton covered with anhydrous sodium
sulphate R. Repeat the extraction with two quantities, each of 5 ml, of chloroform R and evaporate the
combined extracts to dryness under reduced pressure. Dry the residue over diphosphorus pentoxide R
under reduced pressure for 2 h, dissolve in 1 ml of chloroform R. Compare the spectrum obtained
with the Ph. Eur. reference spectrum of fusidic acid.
B. Examine by thin-layer chromatography (2.2.27), using silica gel HF254 R as the coating substance.
the same solvent.
Reference solution. Dissolve 24 mg of diethanolamine fusidate CRS in methanol R and dilute to 10 ml
2.5 volumes of methanol R, 10 volumes of glacial acetic acid R, 10 volumes of cyclohexane R and 80
volumes of chloroform R. Dry the plate in a current of hot air. Examine in ultraviolet light at 254 nm.
The principal spot in the chromatogram obtained with the test solution is similar in position and size
to the principal spot in the chromatogram obtained with the reference solution.
C. Ignite 1 g. The residue gives reaction (a) of sodium (2.3.1).
TESTS
Appearance of solution Dissolve 1.5 g in 10 ml of water R. The solution is not more intensely
coloured than reference solution B6 (Method II, 2.2.2).
9.0.
36-28
Reference solution (a). Dissolve 5 mg of 3-ketofusidic acid CRS in 5 ml of the mobile phase. To 1.0 ml
of this solution add 0.20 ml of the test solution and dilute to 20.0 ml with the mobile phase.
Reference solution (b). Dilute 20 l of the test solution to 100.0 ml with the mobile phase.
a steel column 0.125 m to 0.15 m long and 4 mm to 5 mm in internal diameter packed with
as mobile phase at a flow rate of 2 ml per minute a mixture of 10 volumes of methanol R, 20
volumes of a 10 g/l solution of phosphoric acid R, 20 volumes of water R and 50 volumes of
acetonitrile R,
Continue the chromatography for at least 3.5 times the retention time of the principal peak. In the
chromatogram obtained with the test solution, the sum of the areas of the peaks, apart from the
principal peak and the solvent peak, is not greater than twice the area of the peak corresponding to
sodium fusidate in the chromatogram obtained with reference solution (a) (2.0 per cent). Disregard
any peak with an area less than that of the principal peak in the chromatogram obtained with reference solution (b). The test is not valid unless: the resolution between the peaks corresponding to
3-ketofusidic acid and sodium fusidate in the chromatogram obtained with reference solution (a) is at
least 2.5; the principal peak in the chromatogram obtained with reference solution (b) has a signalto-noise ratio of at least 3.
of water.
ASSAY
Dissolve 0.500 g in 30 ml of water R and add 40 ml of alcohol R. Titrate with 0.1M hydrochloric acid,
1 ml of 0.1M hydrochloric acid is equivalent to 53.87 mg of C31H47NaO6.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
36-29
Sodium Hyaluronate
corrected 1/01
COONa
O
CH2OH
O
OH
OH
H
HO
OH
NHAc
n
(C14H20NNaO11)n
9067-32-7
Sodium Hyaluronate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use High viscosity mucopolysaccharide.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sodium hyaluronate is the sodium salt of hyaluronic acid, a glycosaminoglycan consisting of
D-glucuronic acid and N-acetyl-D-glucosamine disaccharide units. It contains not less than 95.0 per
cent and not more than the equivalent of 105.0 per cent of sodium hyaluronate, calculated with
reference to the dried substance. It has an intrinsic viscosity of not less than 90 per cent and not more
than 120 per cent of the value stated on the label.
PRODUCTION
Sodium hyaluronate is extracted from cocks combs or obtained by fermentation from Streptococci,
Lancefield Groups A and C. It is produced by methods of manufacture designed to minimise or
eliminate infectious agents. When produced by fermentation of gram-positive bacteria, the process
must be shown to reduce or eliminate pyrogenic or inflammatory components of the cell wall.
CHARACTERS
A white or almost white, very hygroscopic powder or a fibrous aggregate, sparingly soluble to soluble
in water, practically insoluble in acetone, in ethanol and in ether.
IDENTIFICATION
spectrum of sodium hyaluronate.
TESTS
Solution S Weigh a quantity of the substance to be examined equivalent to 0.10 g of the dried
substance and add 30.0 ml of a 9 g/l solution of sodium chloride R. Mix gently on a shaker until
dissolved (about 12 h).
pH (2.2.3). Dissolve the substance to be examined in carbon dioxide-free water R to obtain a solution
containing a quantity equivalent to 5 mg of the dried substance per millilitre. The pH of the solution
is 5.0 to 8.5.
Intrinsic viscosity Sodium hyaluronate is very hygroscopic and must be protected from moisture during
weighing.
Buffer solution (0.15M sodium chloride in 0.01M phosphate buffer solution pH 7.0). Dissolve 0.78 g of
sodium dihydrogen phosphate R and 4.50 g of sodium chloride R in water R and dilute to 500.0 ml with
the same solvent (solution A). Dissolve 1.79 g of disodium hydrogen phosphate R and 4.50 g of sodium
chloride R in water R and dilute to 500.0 ml with the same solvent (solution B). Mix solutions A and
B until a pH of 7.0 is reached. Filter through a sintered-glass filter (4).
Test solution (a) (concentration c1 of sodium hyaluronate). Weigh 0.200 g (m0p) (NOTE: this value is only
indicative and should be adjusted after an initial measurement of the viscosity of test solution (a)) of the
substance to be examined and dilute with 50.0 g (m0s) of buffer solution at 4C. Mix the solution by
shaking at 4C during 24 h. Weigh 5.00 g (m1p) of this solution and dilute with 100.0 g (m1s) of
buffer solution at 25C. Mix this solution by shaking for 20 min. Filter the solution through a
sintered-glass filter (100), and discard the first 10 ml.
36-30
Test solution (b) (concentration c2 of sodium hyaluronate). Weigh 30.0 g (m2p) of test solution (a) and
dilute with 10.0 g (m2s) of buffer solution at 25C. Mix this solution by shaking for 20 min. Filter the
solution through a sintered-glass filter (100) and discard the first 10 ml.
Test solution (c) (concentration c3 of sodium hyaluronate). Weigh 20.0 g (m3p) of test solution (a) and
Test solution (d) (concentration c4 of sodium hyaluronate). Weigh 10.0 g (m4p) of test solution (a) and
Determine the flow-time for the buffer solution (t0) and the flow times for the four test solutions (t1,
t2, t3 and t4), at 25.00 0.03C (2.2.9). Use an appropriate suspended level viscometer (specifications: viscometer constant about 0.005 mm2/s2, kinematic viscosity range 1 to 5 mm2/s2, internal
diameter of tube R 0.53 mm, volume of bulb C 5.6 ml, internal diameter of tube N 2.8 mm to
3.2 mm) with a funnel-shaped lower capillary end. Use the same viscometer for all measurements;
measure all outflow times in triplicate. The test is not valid unless the results do not differ by more
than 0.35 per cent from the mean and if the flow time t1 is not less than 1.6 and not more than 1.8
times t0. If this is not the case, adjust the value of m0p and repeat the procedure.
Calculation of the relative viscosities
Since the densities of the sodium hyaluronate solutions and of the solvent are almost equal, the
relative viscosities ri (being r1, r2, r3 and r4) can be calculated from the ratio of the flow times
for the respective solutions ti (being t1, t2, t3 and t4) to the flow time of the solvent t0, but taking into
account the kinetic energy correction factor for the capillary (B = 30 800 s3), as shown below:
ti
ri
t0
B
t12
B
t02
Calculation of the concentrations

Calculation of the concentration c1 (expressed in kg/m3) of sodium hyaluronate in test solution (a)
c1 = m0 p
m1p
1
x 100 h
100
100
m0p + m0s m1p + m1s
25
x = percentage content of sodium hyaluronate as determined under Assay,

h = loss on drying as a percentage,
25 = 1005 kg/m3 (density of the test solution at 25C).
Calculation of the other concentrations
c2 = c1
c3 = c1
c4 = c1
m2 p
m2s + m2 p
m3p
m3s + m3p
m4p
m4s + m4p
Calculation of the intrinsic viscosity

The intrinsic viscosity [] is calculated by linear least-squares regression analysis using the Martin
equation:
log
1
= log [ ]+ k[ ] c
c
The decimal antilogarithm of the intercept is the intrinsic viscosity expressed in m3/kg.
Sulphated glycosaminoglycans If the product is extracted from cocks combs, it complies with the
following requirement. Appropriate safety precautions are to be taken when handling perchloric acid at
elevated temperature.
Test solution. Introduce a quantity of the substance to be examined equivalent to 50.0 mg of the dried
substance into a test-tube 150 mm long and 16 mm in internal diameter and dissolve in 1.0 ml of
perchloric acid R.
36-31
Reference solution. Dissolve 0.149 g of sodium sulphate R in water R and dilute to 100.0 ml with the
same solvent. Dilute 10.0 ml to 100.0 ml with water R. Evaporate 1.0 ml in a test-tube 150 mm long
and 16 mm in internal diameter in a heating block at 90C to 95C, and dissolve the residue in
1.0 ml of perchloric acid R.
Plug each test-tube with a piece of glass wool. Place the test-tubes in a heating block or a silicone oil
bath maintained at 180C and heat until clear, colourless solutions are obtained (about
12 h). Remove the test-tubes and cool to room temperature. Add to each test-tube 3.0 ml of a
33.3 g/l solution of barium chloride R, cap and shake vigorously. Allow the test-tubes to stand for
30 min. Shake each test-tube once again, and determine the absorbance (2.2.25) at 660 nm, using
water R as a blank.
The absorbance obtained with the test solution is not greater than the absorbance obtained with
the reference solution (1 per cent).
Nucleic acids The absorbance (2.2.25) of solution S at 260 nm is not greater than 0.5.
Protein Not more than 0.3 per cent. If intended for use in the manufacture of parenteral dosage
forms, not more than 0.1 per cent.
Test solution (a). Dissolve the substance to be examined in water R to obtain a solution containing a
quantity equivalent to about 10 mg of the dried substance per millilitre.
Test solution (b). Mix equal volumes of test solution (a) and water R.
Reference solutions. Prepare a 0.5 mg/ml stock solution of bovine albumin R in water R. Prepare five
dilutions of the stock solution containing between 5 g/ml and 50 g/ml of bovine albumin R.
Add 2.0 ml of freshly prepared cupri-tartaric solution R3 to test-tubes containing 2.0 ml of water R
(blank), 2.0 ml of the test solutions (a) or (b) or 2.0 ml of the reference solutions. Mix after each
addition. After about 10 min, add to each test-tube 0.50 ml of phosphomolybdotungstic reagent R
prepared immediately before use. Mix after each addition. After 30 min, measure the absorbance
(2.2.25) of each solution at 750 nm against the blank. From the calibration curve obtained with the
five reference solutions determine the content of protein in the test solutions.
Chlorides (2.4.4). Dissolve 67 mg in 100 ml of water R. 15 ml of the solution complies with the
limit test for chlorides (0.5 per cent).
Iron Not more than 80 ppm of Fe, determined by atomic absorption spectrometry (Method II,
2.2.23).
Test solution. Dissolve a quantity of the substance to be examined equivalent to 0.25 g of the dried
substance in 1 ml of nitric acid R by heating on a water-bath. Cool and dilute to 10.0 ml with water R.
Reference solutions. Prepare two reference solutions in the same manner as the test solution, adding
1.0 ml and 2.0 ml respectively of iron standard solution (10 ppm Fe) R to the dissolved substance to be
examined.
Measure the absorbance at 248.3 nm, using an iron hollow-cathode lamp as source of radiation, a
transmission band of 0.2 nm and an air-acetylene flame.
to 110C over diphosphorus pentoxide R for 6 h.
Microbial contamination Total aerobic viable count (2.6.12) not more than 102 micro-organisms
per gram. Use 1 g of the substance to be examined.
Sterility (2.6.1). If intended for use in the manufacture of sterile dosage forms without a further
0.5 I.U. of endotoxin per milligram. If intended for use in the manufacture of intra-ocular preparations or intra-articular preparations without a further appropriate procedure for the removal of
bacterial endotoxins, not more than 0.05 I.U. of endotoxin per milligram.
ASSAY
Determine the glucuronic acid content by reaction with carbazole as described below.
Reagent A. Dissolve 0.95 g of disodium tetraborate R in 100.0 ml of sulphuric acid R.
Reagent B. Dissolve 0.125 g of carbazole R in 100.0 ml of ethanol R.
Test solution. Prepare this solution in triplicate. Dissolve 0.170 g of the substance to be examined in
water R and dilute to 100.0 g with the same solvent. Dilute 10.0 g of this solution to 200.0 g with
water R.
Reference stock solution. Dissolve 0.100 g of D-glucuronic acid R, previously dried to constant mass in
vacuum over diphosphorus pentoxide R (2.2.32), in water R and dilute to 100.0 g with the same solvent.
Reference solutions. Prepare five dilutions of the reference stock solution containing between 6.5 g/g
and 65 g/g of D-glucuronic acid R.
36-32
Place 25 test-tubes, numbered 1 to 25, in iced water. Add 1.0 ml of the five reference solutions in
triplicate to the test-tubes 1 to 15 (reference tubes), 1.0 ml of the three test solutions in triplicate to
the test-tubes 16 to 24 (sample tubes), and 1.0 ml of water R to test-tube 25 (blank). Add 5.0 ml of
freshly prepared reagent A to each test-tube. Tightly close the test-tubes with plastic caps, shake the
contents, and place on a water bath for exactly 15 min. Cool in iced water, and add to each test tube
0.20 ml of reagent B. Recap the tubes, shake, and put them again on a water-bath for exactly 15 min.
Cool to room temperature and measure the absorbance (2.2.25) of the solutions at 530 nm, against
the blank.
From the calibration curve obtained with the mean absorbances read for each reference solution,
determine the mean concentrations of D-glucuronic acid in the test solutions.
Calculate the percentage content of sodium hyaluronate from the expression:
cg
cs
100
401.3
100 h 194.1
cg = mean of concentrations of D-glucuronic acid in the test solutions, in milligrams per gram,
cs = mean of concentrations of the substance to be examined in the test solutions, in
milligrams per gram,
Z = determined percentage content of C6H10O7 in D-glucuronic acid R,
h = loss on drying as a percentage,
401.3 = relative molecular mass of the disaccharide fragment,
194.1 = relative molecular mass of glucuronic acid.
STORAGE
Store in an airtight container, protected from light and humidity. If the substance is sterile, store in a
sterile, airtight, tamper-proof container.
LABELLING
The label states:
the intrinsic viscosity,
the origin of the substance,
the intended use of the substance,
where applicable, that the substance is suitable for parenteral administration other than intraarticular administration,
where applicable, that the substance is suitable for parenteral administration, including intraarticular administration,
where applicable that the material is suitable for intra-ocular use.
__________________________________________________________________________________________________________ Ph Eur
36-33
Sodium Hydroxide
Caustic Soda
NaOH
40.00
1310-73-2
Sodium Hydroxide complies with the requirements of the 3rd edition of the European Pharmacopoeia [0677].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sodium hydroxide contains not less than 97.0 per cent and not more than the equivalent of 100.5 per
cent of total alkali, calculated as NaOH.
CHARACTERS
White, crystalline masses, supplied as pellets, sticks or slabs, deliquescent, readily absorbing carbon
dioxide, very soluble in water, freely soluble in alcohol.
IDENTIFICATION
A. Dissolve 0.1 g in 10 ml of water R. Dilute 1 ml of the solution to 100 ml with water R. The pH
(2.2.3) of the final solution is not less than 11.0.
B. 2 ml of solution S (see Tests) gives reaction (a) of sodium (2.3.1).
TESTS
Solution S Carry out the procedure described below with caution. Dissolve 5.0 g in 12 ml of distilled
water R. Add 17 ml of hydrochloric acid R1, adjust to pH 7 with 1M hydrochloric acid and dilute to
50 ml with distilled water R.
Appearance of solution Dissolve 1.0 g in 10 ml of water R. The solution is clear (2.2.1) and colourless (Method II, 2.2.2).
Carbonates Not more than 2.0 per cent, calculated as Na2CO3, as determined in the Assay.
Chlorides (2.4.4). Dissolve 1.0 g in 5 ml of water R, acidify the solution with about 4 ml of nitric
acid R and dilute to 15 ml with water R. The solution, without addition of dilute nitric acid R,
Sulphates (2.4.13). Dissolve 3.0 g in 6 ml of distilled water R, adjust to pH 7 with hydrochloric acid R
(about 7.5 ml) and dilute to 15 ml with distilled water R. The solution complies with the limit test for
sulphates (50 ppm).
ASSAY
Dissolve 2.000 g in about 80 ml of carbon dioxide-free water R. Add 0.3 ml of phenolphthalein solution R
and titrate with 1M hydrochloric acid. Add 0.3 ml of methyl orange solution R and continue the titration
with 1M hydrochloric acid.
1 ml of 1M hydrochloric acid used in the second part of the titration is equivalent to 0.1060 g of
Na2CO3.
1 ml of 1M hydrochloric acid used in the combined titrations is equivalent to 40.00 mg of total alkali,
calculated as NaOH.
STORAGE
Store in an airtight, non-metallic container.
__________________________________________________________________________________________________________ Ph Eur
36-34
Sodium Iodide
NaI
149.9
7681-82-5
Sodium Iodide complies with the requirements of the 3rd edition of the European Pharmacopoeia [0196].
Preparation
Sodium Iodide Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sodium iodide contains not less than 99.0 per cent and not more than the equivalent of 100.5 per
cent of NaI, calculated with reference to the dried substance.
CHARACTERS
alcohol.
IDENTIFICATION
A. Solution S (see Tests) gives the reactions of iodides (2.3.1).
B. Solution S gives the reactions of sodium (2.3.1).
TESTS
Alkalinity To 12.5 ml of solution S add 0.1 ml of bromothymol blue solution R1. Not more than
0.7 ml of 0.01M hydrochloric acid is required to change the colour of the indicator.
Iodates To 10 ml of solution S add 0.25 ml of iodide-free starch solution R and 0.2 ml of dilute
sulphuric acid R and allow to stand protected from light for 2 min. No blue colour develops.
Thiosulphates To 10 ml of solution S add 0.1 ml of starch solution R and 0.1 ml of 0.005M iodine. A
blue colour is produced.
(20 ppm).
100C to 105C for 3 h.
ASSAY
add 40 ml of hydrochloric acid R and titrate with 0.05M potassium iodate until the colour changes from
red to yellow. Add 5 ml of chloroform R and continue the titration, shaking vigorously, until the
chloroform layer is decolorised.
1 ml of 0.05M potassium iodate is equivalent to 14.99 mg of NaI.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
36-35
Sodium Lactate Solution

Sodium Lactate Solution complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Systemic alkalinising agent.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sodium lactate consists of a mixture of enantiomers. It is usually the racemate but the (+)-(S) isomer
may predominate. Sodium lactate solution contains not less than 50.0 per cent m/m of sodium
2-hydroxypropionate (C3H5NaO3; Mr 112.1) and not less than 96.0 per cent and not more than
104.0 per cent of the declared content of sodium lactate (stated on the label).
CHARACTERS
A clear, colourless, slightly syrupy liquid, miscible with water and with alcohol.
IDENTIFICATION
A. To 0.1 ml add 10 ml of water R. 5 ml of the solution gives the reaction of lactate (2.3.1).
TESTS
Solution S Dilute a quantity of the substance to be examined corresponding to 40.0 g of sodium
lactate to 200 ml with distilled water R.
Appearance of solution The substance to be examined is clear (2.2.1) and not more intensely
coloured than reference solution BY7 (Method II, 2.2.2).
pH (2.2.3). The pH of the substance to be examined is 6.5 to 9.0.
Reducing sugars and sucrose To 5 ml of the substance to be examined add 2 ml of dilute sodium
hydroxide solution R and 0.2 ml of copper sulphate solution R. The solution is clear and blue and
remains so on boiling. Add to the hot solution 4 ml of hydrochloric acid R. Boil for 1 min. Add 6 ml of
strong sodium hydroxide solution R and heat to boiling again. The solution is clear and blue.
Residual solvents (2.4.24). Examine by head-space gas chromatography (2.2.28).
Solvent solution. Dissolve 0.5 g of methanol R and 1.0 g of ethanol R in water R and dilute to 1000.0 ml
with the same solvent. Dilute 5.0 ml of the solution to 100.0 ml with water R.
Sample solution. Dissolve a quantity of the substance to be examined equivalent to 0.500 g of sodium
lactate in water R and dilute to 10.0 ml with the same solvent.
Test solution. Add 2.0 ml of the sample solution and 1.0 ml of water R to an injection vial.
Reference solution. Add 2.0 ml of the sample solution and 1.0 ml of the solvent solution to an injection
vial.
Cool the vials in an ice-bath and add to each 3.0 ml of a 500 g/l solution of sodium hydroxide R.
Inject 1 ml of the gaseous phase of the test solution and 1 ml of the gaseous phase of the reference
solution. The areas of the peaks due to methanol and ethanol in the chromatogram obtained with the
test solution are not greater than half of the areas of the corresponding peaks in the chromatogram
obtained with the reference solution (250 ppm of methanol and 500 ppm of ethanol, calculated as
sodium lactate).
If intended for use in the manufacture of parenteral dosage forms, dialysis, haemodialysis or
haemofiltration solutions, not more than 50 ppm of methanol, calculated as sodium lactate.
Chloride (2.4.4). Dilute 5 ml of solution S to 15 ml with water R. The solution complies with the
limit test for chlorides (50 ppm calculated as sodium lactate).
Oxalates and phosphates To 1 ml of the substance to be examined add 15 ml of alcohol R and 2 ml
of calcium chloride solution R. Heat at 75C for 5 min. Any opalescence in the solution is not more
intense than that of a standard prepared at the same time and in the same manner using a mixture of
1 ml of the substance to be examined, 15 ml of alcohol R and 2 ml of water R.
Sulphates (2.4.13). Dilute 7.5 ml of solution S to 15.0 ml with distilled water R. The solution
complies with the limit test for sulphates (100 ppm calculated as sodium lactate).
Aluminium If intended for the use in the manufacture of parenteral dosage forms, dialysis,
haemodialysis or haemofiltration solutions, it contains not more than 0.1 ppm of Al, determined by
atomic absorption spectrometry (Method I, 2.2.23). For the preparation of the solutions, use
equipment that is aluminium-free or that will not release aluminium under the conditions of use
(glass, polyethylene, etc).
Modifier solution. Dissolve 100.0 g of ammonium nitrate R in a mixture of 50 ml of water R and 4 ml of
nitric acid R and dilute to 200 ml with water R.
36-36
Blank solution. Dilute to 2.0 ml of the modifier solution to 25.0 ml with water R.
Test solution. To 1.25 g add 2.0 ml of the modifier solution and dilute to 25.0 ml with water R.
Reference solutions. Prepare the reference solutions immediately before use (0.010 ppm to 0.050 ppm
of aluminium) using aluminium standard solution (200 ppm Al) R.
Measure the absorbance at 309.3 nm using an aluminium hollow-cathode lamp as the source of
radiation, a graphite furnace as atomisation device, and argon R as the carrier gas. The device is
equipped with a non-specific absorption correction system. Heat the oven to 120C for as many
seconds as there are microlitres of solution introduced into the apparatus, then heat at 1000C for
30 s and finally at 2700C for 6 s.
Barium To 10 ml of solution S add 10 ml of calcium sulphate solution R. Allow to stand for 30 min.
Any opalescence (2.2.1) in the solution is not more intense than that of a standard prepared at the
same time and in the same manner using a mixture of 10 ml of solution S and 10 ml of distilled
water R.
Heavy metals (2.4.8). 12 ml of solution S complies with limit test A for heavy metals (10 ppm
calculated as sodium lactate). Prepare the standard using lead standard solution (2 ppm Pb) R.
test for iron (10 ppm calculated as sodium lactate).
of endotoxin per gram.
ASSAY
Dissolve a quantity of the substance to be examined corresponding to 75.0 mg of sodium lactate in a
mixture of 10 ml of anhydrous acetic acid R and 20 ml of acetic anhydride R. Allow to stand for 15 min.
Add 1 ml of naphtholbenzein solution R and titrate with 0.1M perchloric acid.
STORAGE
LABELLING
The label states:
where applicable, that the substance is suitable for use in the manufacture of dialysis,
haemodialysis and haemofiltration solutions,
forms,
the declared content of sodium lactate.
__________________________________________________________________________________________________________ Ph Eur
36-37
Sodium Lauryl Sulphate

Sodium Lauryl Sulphate complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Sodium Laurilsulfate [0098]. These requirements are reproduced after the heading Definition below.
Action and use Anionic emulsifying agent.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sodium laurilsulfate is a mixture of sodium alkyl sulphates consisting chiefly of sodium dodecyl
sulphate, C12H25NaO4S (Mr 288.4). It contains not less than 85.0 per cent of sodium alkyl sulphates,
calculated as C12H25NaO4S.
CHARACTERS
A white or pale yellow powder or crystals, freely soluble in water giving an opalescent solution, partly
soluble in alcohol.
IDENTIFICATION
A. Dissolve 0.1 g in 10 ml of water R and shake. A copious foam is formed.
B. To 0.1 ml of the solution prepared for Identification test A, add 0.1 ml of a 1 g/l solution of
methylene blue R and 2 ml of dilute sulphuric acid R. Add 2 ml of methylene chloride R and shake. An
intense blue colour develops in the methylene chloride layer.
C. Mix about 10 mg with 10 ml of ethanol R. Heat to boiling on a water-bath, shaking frequently.
Filter immediately and evaporate the ethanol. Dissolve the residue in 8 ml of water R, add 3 ml of
dilute hydrochloric acid R, evaporate the solution to half its volume and allow to cool. Separate the
congealed fatty alcohols by filtration. To the filtrate add 1 ml of barium chloride solution R1. A white,
crystalline precipitate is formed.
D. Ignite 0.5 g. The residue gives reaction (a) of sodium (2.3.1).
TESTS
Alkalinity Dissolve 1.0 g in 100 ml of carbon dioxide-free water R and add 0.1 ml of phenol red solution R. Not more than 0.5 ml of 0.1M hydrochloric acid is required to change the colour of the
indicator.
Non-esterified alcohols Dissolve 10 g in 100 ml of water R, add 100 ml of alcohol R and shake the
solution with three quantities, each of 50 ml, of pentane R, adding sodium chloride R, if necessary, to
promote separation of the two layers. Wash the combined organic layers with three quantities, each
of 50 ml, of water R, dry over anhydrous sodium sulphate R, filter and evaporate on a water-bath until
the odour of the solvent is no longer perceptible. Heat the residue at 105C for 15 min and cool. The
residue weighs not more than 0.4 g (4 per cent).
Sodium chloride and sodium sulphate Not more than a total of 8.0 per cent of NaCl and
Na2SO4.
Sodium chloride. Dissolve 5.00 g in 50 ml of water R, add dilute nitric acid R dropwise until the solution is neutral to blue litmus paper R, add 2 ml of potassium chromate solution R and titrate with 0.1M
silver nitrate.
Sodium sulphate. Dissolve 0.500 g in 20 ml of water R, warming gently if necessary, and add 1 ml of a
0.5 g/l solution of dithizone R1 in acetone R. If the solution is red, add 1M nitric acid, dropwise, until
the solution becomes bluish-green. Add 2.0 ml of dichloroacetic acid solution R and 80 ml of acetone R.
Titrate with 0.01M lead nitrate until a persistent violet-red or orange-red colour is obtained. Carry out
a blank titration.
1 ml of 0.01M lead nitrate is equivalent to 1.420 mg of Na2SO4.
ASSAY
Dissolve 1.15 g in water R, warming if necessary, and dilute to 1000.0 ml with the same solvent. To
20.0 ml of the solution add 15 ml of chloroform R and 10 ml of dimidium bromidesulphan blue mixed
solution R. Titrate with 0.004M benzethonium chloride, shaking vigorously and allowing the layers to
separate before each addition, until the pink colour of the chloroform layer is completely discharged
and a greyish-blue colour is obtained.
1 ml of 0.004M benzethonium chloride is equivalent to 1.154 mg of sodium alkyl sulphates, calculated as C12H25NaO4S.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
36-38
Sodium Metabisulphite
Sodium Pyrosulphite
Na2S2O5
190.1
7681-57-4
Sodium Metabisulphite complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sodium metabisulphite (sodium disulphite) contains not less than 95.0 per cent and not more than
the equivalent of 100.5 per cent of Na2S2O5.
CHARACTERS
A white or almost white, crystalline powder or colourless crystals, freely soluble in water, slightly
soluble in alcohol.
IDENTIFICATION
A. Solution S (see Tests) complies with the test for pH (see Tests).
B. To 0.4 ml of iodinated potassium iodide solution R add 8 ml of distilled water R and 1 ml of a 1 to 10
dilution of solution S in distilled water R. The solution is colourless and gives reaction (a) of sulphates
(2.3.1).
TESTS
Thiosulphates To 5 ml of solution S add 5 ml of dilute hydrochloric acid R. The solution remains
clear (2.2.1) for not less than 15 min.
Arsenic (2.4.2). Mix 0.20 g with 2 ml of water R in a dish. Add, drop by drop, 1.5 ml of nitric
acid R. Evaporate the mixture to dryness on a water-bath. Heat over a flame until no more vapour is
evolved. Take up the residue in 25 ml of water R. The solution complies with limit test A for arsenic
(5 ppm).
Heavy metals (2.4.8). To 40 ml of solution S in a silica crucible, add 10 ml of hydrochloric acid R
and evaporate to dryness. Dissolve the residue in 19 ml of water R and add 1 ml of a 40 g/l solution
of sodium fluoride R. 12 ml of the solution complies with limit test A for heavy metals (20 ppm).
ASSAY
Dissolve 0.200 g in 50.0 ml of 0.05M iodine. Add 5 ml of dilute hydrochloric acid R. Titrate with 0.1M
sodium thiosulphate using 1 ml of starch solution R, added towards the end of the titration, as indicator.
1 ml of 0.05M iodine is equivalent to 4.753 mg of Na2S2O5.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
36-39
Sodium Methyl Hydroxybenzoate

Sodium Methylparaben
COOMe
NaO
C8H7NaO3
174.1
5026-62-0
Sodium Methyl Hydroxybenzoate complies with the requirements of the 3rd edition of the European Pharmacopoeia for Sodium Methyl Parahydroxybenzoate [1262]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sodium methyl parahydroxybenzoate contains not less than 99.0 per cent and not more than the
equivalent of 102.0 per cent of sodium 4-(methoxycarbonyl)phenolate, calculated with reference to
CHARACTERS
A white, crystalline powder, freely soluble in water, sparingly soluble in alcohol, practically insoluble
IDENTIFICATION
A. Dissolve 0.5 g in 50 ml of water R. Immediately add 5 ml of hydrochloric acid R1. Filter and wash
the precipitate with water R. Dry under vacuum at 80C for 2 h. The precipitate obtained melts
(2.2.14) at 125C to 128C.
B. Examine the precipitate obtained in identification test A by infrared absorption spectrophotometry
(2.2.24), comparing with the spectrum obtained with methyl parahydroxybenzoate CRS.
the chromatogram obtained with reference solution (c).
D. To about 10 mg in a test-tube add 1 ml of sodium carbonate solution R, boil for 30 s and cool. Add
5 ml of aminopyrazolone solution R and 1 ml of potassium ferricyanide solution R and mix. An orange to
red colour develops.
E. To 1 ml of solution S (see Tests) add 1 ml of water R. The solution gives reaction (a) of sodium
(2.3.1).
TESTS
Appearance of solution Solution S examined immediately after preparation is clear (2.2.1) and not
more intensely coloured than reference solution BY6 (Method I, 2.2.2).
Test solution (a). Dissolve 0.100 g in 10 ml of water R. Immediately add 2 ml of hydrochloric acid R
and shake with 50 ml of ether R. Evaporate the upper layer to dryness and dissolve the residue to
10 ml with acetone R.
Reference solution (a). Dissolve 34.3 mg of 4-hydroxybenzoic acid R in acetone R and dilute to 100 ml
Reference solution (b). Dilute 0.5 ml of test solution (a) to 100 ml with acetone R.
Reference solution (c). Dissolve 10 mg of methyl parahydroxybenzoate CRS in acetone R and dilute to
36-40
Reference solution (d). Dissolve 10 mg of ethyl parahydroxybenzoate CRS in 1 ml of test solution (a)
volume of glacial acetic acid R, 30 volumes of water R and 70 volumes of methanol R. Allow the plate
to dry in air and examine in ultraviolet light at 254 nm. In the chromatogram obtained with test
solution (a): any spot due to 4-hydroxybenzoic acid is not more intense than the spot in the chromatogram obtained with reference solution (a) (4 per cent) and any spot, apart from the principal spot
and the spot due to 4-hydroxybenzoic acid, is not more intense than the spot in the chromatogram
obtained with reference solution (b) (0.5 per cent). The test is not valid unless the chromatogram
obtained with reference solution (d) shows two clearly separated principal spots.
Chlorides (2.4.4). To 10 ml of solution S, add 30 ml of water R and 1 ml of nitric acid R and dilute
to 50 ml with water R. Shake and filter. Dilute 10 ml of the filtrate to 15 ml with water R. The
solution complies with the limit test for chlorides (350 ppm). Prepare the standard using 14 ml of
chloride standard solution (5 ppm Cl) R to which 1 ml of water R has been added.
Sulphates (2.4.13). To 25 ml of solution S, add 5 ml of distilled water R and 10 ml of hydrochloric
acid R and dilute to 50 ml with distilled water R. Shake and filter. Dilute 10 ml of the filtrate to 15 ml
with distilled water R. The solution complies with the limit test for sulphates (300 ppm).
ASSAY
STORAGE
IMPURITIES
O
OR
HO
B. R = CH2 - CH3: ethyl 4-hydroxybenzoate,
C. R = CH2 - CH2 - CH3: propyl 4-hydroxybenzoate,
D. R = CH2 - CH2 - CH2 - CH3: butyl 4-hydroxybenzoate.
__________________________________________________________________________________________________________ Ph Eur
36-41
Sodium Molybdate Dihydrate

1/01
Na2MoO4,2H2O
241.9
10102-40-6
Sodium Molybdate Dihydrate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1565]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sodium molybdate dihydrate contains not less than 98.0 per cent and not more than the equivalent
of 100.5 per cent of Na2MoO4, calculated with reference to the dried substance.
CHARACTERS
A white powder or colourless crystals, freely soluble in water.
IDENTIFICATION
A. It complies with the test for loss on drying (see Tests).
B. Dissolve 0.2 g in 5 ml of a mixture of equal volumes of nitric acid R and water R and add 0.1 g of
ammonium chloride R. Add 0.3 ml of disodium hydrogen phosphate solution R and heat slowly at 50C to
60C. A yellow precipitate is formed.
C. Dissolve 0.15 g in 2 ml of water R, the solution gives reaction (a) of sodium (2.3.1).
TESTS
Chlorides To 10 ml of a mixture of equal volumes of nitric acid R and water R add 10 ml of solution
S with shaking. Add 1 ml of 0.1M silver nitrate. Any opalescence in the solution is not more intense
after 5 min than that of a standard solution prepared at the same time in the same manner with 2 ml
of chloride standard solution (50 ppm Cl) R (50 ppm).
Phosphates Dissolve 2.0 g by heating in 13 ml of water R. In the still hot solution, dissolve 8.0 g of
ammonium nitrate R1. Add this solution to 27 ml of a mixture of equal volumes of nitric acid R and
water R. Any yellow colour or opalescence in the solution is not more intense within 3 h than that in a
standard solution prepared at the same time as follows: dissolve 1.0 g in 12 ml of water R and add
1 ml of phosphate standard solution (200 ppm PO4) R (200 ppm).
standard using 1 ml of ammonium standard solution (1 ppm NH4) R.
Heavy metals To 10 ml of solution S, add 2 ml of water R, 6 ml of 4M sodium hydroxide and 2 ml of
concentrated ammonia R (solution A). To 0.5 ml of thioacetamide reagent R add a mixture of 15 ml of
solution A and 5 ml of water R. Any coloration of the solution is not more intense after 2 min than
that of a standard solution prepared at the same time as follows: to 0.5 ml of thioacetamide reagent R
add a mixture of 5 ml of solution A, 1 ml of lead standard solution (10 ppm Pb) R and 14 ml of water R
(10 ppm).
at 140C for 3 h.
ASSAY
Dissolve 0.100 g in 30 ml of water R, add 0.5 g of hexamethylenetetramine R and 0.1 ml of a 250 g/l
solution of nitric acid R. Heat to 60C. Titrate with 0.05M lead nitrate using 4-(2-pyridylazo)resorcinol
monosodium salt R as indicator.
1 ml of 0.05M lead nitrate is equivalent to 10.30 mg of Na2MoO4.
__________________________________________________________________________________________________________ Ph Eur
36-42
Sodium Nitroprusside
Na2Fe(CN)5NO,2H2O
298.0
13755-38-9
Sodium Nitroprusside complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Sodium Nitroprusside Intravenous Infusion
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sodium nitroprusside contains not less than 99.0 per cent and not more than the equivalent of
100.5 per cent of sodium pentacyanonitrosylferrate(III), calculated with reference to the anhydrous
substance.
CHARACTERS
Reddish-brown powder or crystals, freely soluble in water, slightly soluble in alcohol.
IDENTIFICATION
A. Dissolve 0.700 g in water R and dilute to 100.0 ml with the same solvent. Examined between
350 nm and 600 nm (2.2.25) immediately after preparation, the solution shows an absorption maximum at 395 nm, a shoulder at about 510 nm and a minimum at 370 nm. The specific absorbance at
the maximum is 0.65 to 0.80.
B. Dissolve about 20 mg in 2 ml of water R and add 0.1 ml of sodium sulphide solution R. A deep
violet-red colour is produced.
C. Dissolve 50 mg in 1 ml of water R and acidify the solution by the addition of hydrochloric acid R.
Place a drop of the solution in an oxidising flame. A persistent yellow colour is produced.
TESTS
Insoluble matter Dissolve 10 g without heating in 50 ml of water R. Allow to stand for 30 min and
filter through a sintered-glass filter (16). Wash the filter with cold water R until the filtrate is colourless. Dry the residue on the filter at 105C. The residue weighs not more than 1 mg (100 ppm).
Chlorides (2.4.4). In a metallic crucible (nickel) mix 1.0 g of the substance to be examined with
8 ml of a 200 g/l solution of sodium hydroxide R. Heat slowly and evaporate carefully to dryness over a
small flame, then heat to a dull red colour for 30 min. Allow to cool and transfer the solid residue
with three successive portions, each of 8 ml of dilute sulphuric acid R. Filter the sulphuric acid extracts
on a filter paper and collect the filtrates. Render the filtrate acid to litmus paper R by adding, if
necessary, a few drops of dilute sulphuric acid R. Wash the crucible and the filter paper with three
successive portions of 10 ml of water R, add the washings to the main sulphuric acid solution and
dilute to 60 ml with water R. Mix. 15 ml of the solution complies with the limit test for chlorides
(200 ppm).
Ferricyanides Dissolve 1.25 g in acetate buffer solution pH 4.6 R and dilute to 50.0 ml with the same
buffer solution. Use three 50 ml volumetric flasks (A, B, C). To flask B add 1.0 ml of ferricyanide
standard solution (50 ppm Fe(CN)6) R. To flasks A and B add 1 ml of a 5 g/l solution of ferrous
ammonium sulphate R. To the three flasks add 10.0 ml of the solution of the substance to be examined. Dilute the contents of each flask to 50.0 ml with water R. Allow to stand for 30 min. The
absorbance (2.2.25) of the solution in flask A measured at 720 nm using the solution in flask C as the
compensation liquid is not greater than the absorbance of the solution in flask B measured at 720 nm
using the solution in flask A as the compensation liquid (200 ppm).
Ferrocyanides Dissolve 4.0 g in water R and dilute to 100.0 ml with the same solvent. Use three
50 ml volumetric flasks (A, B, C). To flask B add 2.0 ml of ferrocyanide standard solution (100 ppm
Fe(CN)6) R. To flasks A and B add 1 ml of ferric chloride solution R2. To the three flasks add 25.0 ml
of the solution of the substance to be examined. Dilute the contents of each flask to 50.0 ml with
water R. Allow to stand for 30 min. The absorbance (2.2.25) of the solution in flask A measured at
695 nm using the solution in flask C as the compensation liquid is not greater than the absorbance of
the solution in flask B measured at 695 nm using the solution in flask A as the compensation liquid
(200 ppm).
Sulphates For the preparation and dilution of the solutions use distilled water R.
Test solution. Dissolve 3.6 g in 120 ml of water, add with mixing 4 ml of sulphate standard solution
(10 ppm SO4) R and 20 ml of a 250 g/l solution of cupric chloride R and dilute to 150.0 ml with water.
Allow to stand for 16 h and filter or centrifuge until a clear light-blue solution is obtained.
36-43
Reference solution. To 40 ml of sulphate standard solution (10 ppm SO4) R add 80 ml of water and
12 ml to 13 ml of a 250 g/l solution of cupric chloride R. Dilute to 150.0 ml with water. The volume of
cupric chloride solution added is such that the colour of the final solution matches that of the test
solution.
Allow the solutions to stand. Filter both solutions, discarding the first 25 ml of filtrate. To 100 ml of
each filtrate, add 0.5 ml of acetic acid R. Mix and add 2 ml of a 250 g/l solution of barium chloride R
and mix again. The test solution is not more opalescent than the reference solution (100 ppm).
ASSAY
Dissolve 0.250 g in 100 ml of water R and add 0.1 ml of dilute sulphuric acid R. Titrate with 0.1M
silver nitrate, determining the end-point potentiometrically (2.2.20) with a silver-mercurous sulphate
electrode system.
1 ml of 0.1M silver nitrate is equivalent to 13.10 mg of Na2[Fe(CN)5NO].
STORAGE
__________________________________________________________________________________________________________ Ph Eur
36-44
Sodium Perborate
NaBO2,H2O2,3H2O
153.9
7632-04-4
Definition Sodium Perborate contains not less than 96.0% and not more than 103.0% of
NaBO2,H2O2, 3H2O.
Characteristics Colourless, prismatic crystals or a white powder, stable in the crystalline form;
Sparingly soluble in water, with some decomposition.
Identification
A. Mix 1 ml of a saturated solution with 1 ml of 1M sulphuric acid and 0.2 ml of dilute potassium
dichromate solution, shake with 2 ml of ether and allow to stand. A blue colour is produced in the ether
layer.
B. The mixture obtained on treating with sulphuric acid and methanol burns with a greenish flame
when ignited.
C. Yields the reactions characteristic of sodium salts, Appendix VI.
D. An aqueous solution is alkaline to litmus solution.
Arsenic Heat 0.38 g in a porcelain dish with 2 ml of sulphuric acid and 2 ml of water until white
fumes are evolved. Cool, add 2 ml of water and heat again until white fumes are evolved. Cool, add
50 ml of water and 5 ml of stannated hydrochloric acid and dilute to 75 ml with water. 25 ml of the
resulting solution complies with the limit test for arsenic, Appendix VII (8 ppm).
Heavy metals Dissolve 2.0 g in 10 ml of 2M hydrochloric acid with the aid of heat, evaporate to
dryness, with stirring, and dissolve the residue in 20 ml of hot water. 12 ml of the resulting solution
complies with limit test A for heavy metals, Appendix VII. Use lead standard solution (1 ppm Pb) to
prepare the standard (10 ppm).
Iron 6 ml of the solution obtained in the test for Heavy metals diluted to 10 ml with water complies
with the limit test for iron, Appendix VII (80 ppm).
Chloride 0.15 g complies with the limit test for chlorides, Appendix VII (330 ppm).
Sulphate 15 ml of a solution prepared by dissolving 0.13 g in 150 ml of water complies with the limit
test for sulphates, Appendix VII (1.2%).
Assay Dissolve 0.3 g in 50 ml of water, add 10 ml of 1M sulphuric acid and titrate with 0.02M
potassium permanganate VS.
Each ml of 0.02M potassium permanganate VS is equivalent to 7.693 mg of NaBO2,H2O2,3H2O.
Storage Sodium Perborate should be kept in an airtight container.
36-45
Sodium Picosulfate
O
N
ONa
S
ONa
S
O
C18H13NNa2O8S2,H2O
499.4
10040-45-6 (anhydrous)
Sodium Picosulfate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1031].
Preparation
Sodium Picosulfate Oral Powder
When sodium picosulphate is prescribed or demanded, Sodium Picosulfate shall be dispensed or
supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sodium picosulfate contains not less than 98.5 per cent and not more than the equivalent of
100.5 per cent of 4,4-(pyridin-2-ylmethylene)bisphenyl bis(sodium sulphate), calculated with reference to the anhydrous substance.
CHARACTERS
A white or almost white, crystalline powder, freely soluble in water, slightly soluble in alcohol,
IDENTIFICATION
obtained with sodium picosulfate CRS. Examine the substances prepared as discs.
B. Examine the chromatograms obtained in the test for related substances in ultraviolet light at
C. To 5 ml of solution S (see Tests) add 1 ml of dilute hydrochloric acid R and heat to boiling. Add
1 ml of barium chloride solution R1. A white precipitate is formed.
D. To about 10 mg add 3 ml of sulphuric acid R and 0.1 ml of potassium dichromate solution R1. A
violet colour develops.
E. The solution S gives reaction (a) of sodium (2.3.1).
TESTS
Acidity or alkalinity To 10 ml of solution S add 0.05 ml of phenolphthalein solution R. The solution
is colourless. Not more than 0.25 ml of 0.01M sodium hydroxide is required to change the colour of
the indicator to pink.
coating substance.
36-46
Reference solution (a). Dissolve 20 mg of sodium picosulfate CRS in methanol R and dilute to 5 ml with
the same solvent.
Reference solution (c). Dissolve 0.20 g of the substance to be examined in 2 ml of a 103 g/l solution of
hydrochloric acid R. Heat rapidly to boiling and maintain boiling for 10 s. Cool in iced water and
2.5 volumes of anhydrous formic acid R, 12.5 volumes of water R, 25 volumes of methanol R and 60
volumes of ethyl acetate R. Dry the plate in a current of hot air for 15 min and examine in ultraviolet
light at 254 nm. Spray with a 200 g/l solution of hydrochloric acid R in methanol R and heat at 110C
for 10 min. Spray the hot plate with a freshly prepared solution containing 50 g/l of ferric chloride R
and 1 g/l of potassium ferricyanide R. Examine the wet plate. Any spot in the chromatogram obtained
with test solution (a), apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (b) (0.2 per cent). The test is not valid unless the chromatogram obtained with reference solution (c) shows three clearly separated spots. A fourth spot may
be present on the starting-line.
ASSAY
Dissolve 0.400 g in 80 ml of methanol R. Titrate with 0.1M perchloric acid, determining the end-point
1 ml of 0.1M perchloric acid is equivalent to 48.14 mg of C18H13NNa2O8S2.
STORAGE
IMPURITIES
OSO 3 Na
OH
A. 4-[(pyridin-2-yl)(4-hydroxyphenyl)methyl]phenyl sodium sulphate,

OH
OH
B. 4,4-[(pyridin-2-yl)methylene]bisphenol.
__________________________________________________________________________________________________________ Ph Eur
36-47
Sodium Polystyrene Sulphonate

Definition Sodium Polystyrene Sulphonate is a cation-exchange resin prepared in the sodium form
containing not less than 9.4% w/w and not more than 11.0% w/w of sodium, calculated with reference to the dried substance. Each g exchanges not less than 2.8 mEq and not more than 3.4 mEq of
potassium, calculated with reference to the dried substance.
Characteristics A cream to light brown, fine powder.
Practically insoluble in water and in ethanol (96%).
Identification
A. The infrared absorption spectrum, Appendix II A, is concordant with the reference spectrum of sodium
polystyrene sulphonate (RS 318).
B. Yields the reactions characteristic of sodium salts, Appendix VI.
Particle size Not more than 1% w/w is retained on a 150 m sieve, Appendix XVII B. Use 20 g and
sieve for 5 minutes.
Potassium Not more than 0.1% of K when determined by atomic emission spectrophotometry,
Appendix II D, measuring at 766.5 nm and using a solution prepared in the following manner. To
1.1 g of the substance being examined add 5 ml of hydrochloric acid, heat to boiling, cool and add
10 ml of water. Filter, wash the filter and residue with water and dilute the filtrate and washings to
25 ml with water. Use potassium solution (100 ppm K), suitably diluted with water, to prepare the
standard solutions.
Calcium Not more than 0.1% of Ca when determined by atomic emission spectrophotometry, Appendix
II D, measuring at 422.7 nm and using a solution prepared in the following manner. To 1.1 g of the
substance being examined add 5 ml of hydrochloric acid, heat to boiling, cool and add 10 ml of water.
Filter, wash the filter and residue with water and dilute the filtrate and washings to 25 ml with water.
Use calcium standard solution (400 ppm Ca), suitably diluted with water, to prepare the standard
solutions.
Arsenic 1 g complies with the limit test for arsenic, Appendix VII (1 ppm).
Heavy metals Not more than 10 ppm when determined by the following method. Heat 4 g until
charred, cool, add 4 ml of lead-free nitric acid and 0.5 ml of sulphuric acid dropwise, heat cautiously
until white fumes are no longer evolved. Ignite in a muffle furnace at 500 to 600 until a white
residue is obtained. Cool, add 4 ml of hydrochloric acid and dilute to 20 ml. The resulting solution
complies with limit test A for heavy metals, Appendix VII. Use 2 ml of lead standard solution (10 ppm
Pb) to prepare the standard.
Styrene Not more than 1 ppm when determined by liquid chromatography, Appendix III D, using the
following solutions. For solution (1) shake 10 g of the substance being examined with 10 ml of
acetone for 30 minutes, centrifuge and use the supernatant liquid. Solution (2) contains 0.0001% w/v
of styrene in acetone.
4 mm) packed with stationary phase C (Bondapak C18 is suitable), (b) a mixture of equal volumes of
acetonitrile and water as the mobile phase with a flow rate of 2 ml per minute and (c) a detection
Inject separately 20 l of each solution. The area of any peak corresponding to styrene in the
chromatogram obtained with solution (1) is not greater than the area of the principal peak in the
Potassium exchange capacity Prepare a solution containing 0.9533% w/v of potassium chloride in
water (solution A). To 1.6 g of the substance being examined in a dry 250-ml ground-glass-stoppered
flask add 100 ml of solution A, stopper and shake for 15 minutes. Filter, discard the first 20 ml of
filtrate and dilute 4 ml of the filtrate to 1000 ml with water. Determine the concentration of
potassium in this solution by atomic emission spectrophotometry, Appendix II D, measuring at 766.5 nm
and using a calibration curve prepared from the following solutions. Into each of five 1000-ml
graduated flasks place 0, 1, 2, 3 and 4 ml of solution A respectively and 4, 3, 2, 1 and 0 ml of a
0.763% w/v solution of sodium chloride and dilute the contents of each flask to 1000 ml with water.
Calculate the potassium exchange capacity of the substance being examined in milliequivalents per g
taking the concentration of potassium in solution A as 128 milliequivalents of K per litre.
Microbial contamination Carry out a quantitative evaluation for Enterobacteria and certain other
Gram-negative bacteria, Appendix XVI B1. 0.01 g of the substance being examined gives a negative
result, Table I (most probable number of bacteria per gram fewer than 102).
Assay For sodium Moisten 0.9 g in a platinum crucible with sulphuric acid, ignite very gently and
allow to cool. Moisten with sulphuric acid again, ignite at 800 until a carbon-free ash is obtained and
allow to cool. Add 20 ml of water to the crucible, warm gently on a water bath until solution is
36-48
effected, cool, transfer quantitatively to a 100-ml graduated flask and add sufficient water to produce
100 ml. Dilute 5 ml of this solution to 1000 ml with water (solution B). Carry out the method for
atomic emission spectrophotometry, Appendix II D, measuring at 589 nm using solution B as the test
solution and sodium standard solution (200 ppm Na) suitably diluted with water to prepare the
standard solutions.
Storage Sodium Polystyrene Sulphonate should be kept in an airtight container.
Action and use Used in the treatment of hyperkalaemia.
36-49
Sodium Propionate
H3C
C3H5NaO2
COONa
96.06
137-40-6
Definition Sodium Propionate contains not less than 99.0% and not more than 101.0% of
C3H5NaO2, calculated with reference to the anhydrous substance.
Characteristics Colourless deliquescent crystals or a white granular powder; odourless or with a
slight characteristic odour.
Freely soluble in water; soluble in ethanol (96%).
Identification
A. Yields the reactions characteristic of sodium salts, Appendix VI.
B. Warm with 1M sulphuric acid. Propionic acid, recognisable by its odour, is produced.
Acidity or alkalinity Dissolve 2.0 g in 20 ml of carbon dioxide-free water and add 0.1 ml of phenolphthalein solution R1. Not more than 0.6 ml of either 0.1M hydrochloric acid VS or 0.1M sodium
hydroxide VS is required to change the colour of the solution.
Heavy metals 12 ml of a 10% w/v solution complies with limit test A for heavy metals, Appendix VII.
Use 10 ml of lead standard solution (1 ppm Pb) to prepare the standard (10 ppm).
C3H5NaO2.
Storage Sodium Propionate should be kept in a well-closed container.
36-50
Sodium Propyl Hydroxybenzoate

Sodium Propylparaben
COOPrn
NaO
C10H11NaO3
202.2
35285-69-9
Sodium Propyl Hydroxybenzoate complies with the requirements of the 3rd edition of the European Pharmacopoeia for Sodium Propyl Parahydroxybenzoate [1263]. These requirements are reproduced after the heading
Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sodium propyl parahydroxybenzoate contains not less than 99.0 per cent and not more than the
equivalent of 104.0 per cent of sodium 4-(propoxycarbonyl)phenolate, calculated with reference to
CHARACTERS
A white, crystalline powder, freely soluble in water, sparingly soluble in alcohol, practically insoluble
IDENTIFICATION
A. Dissolve 0.5 g in 50 ml of water R. Immediately add 5 ml of hydrochloric acid R1. Filter, and wash
the precipitate with water R. Dry at 80C in vacuo for 2 h. The melting point (2.2.14) of the precipitate is 96C to 99C.
B. Examine the precipitate obtained in identification test A by infrared absorption spectrophotometry
(2.2.24), comparing with the spectrum obtained with propyl parahydroxybenzoate CRS.
the chromatogram obtained with reference solution (c).
D. To about 10 mg in a test-tube add 1 ml of sodium carbonate solution R, boil for 30 s and cool. Add
5 ml of aminopyrazolone solution R and 1 ml of potassium ferricyanide solution R and mix. An orange to
red colour develops.
E. To 1 ml of solution S (see Tests) add 1 ml of water R. The solution gives reaction (a) of sodium
(2.3.1).
TESTS
Solution S Dissolve 5.0 g in carbon dioxide-free water R prepared from distilled water R, and dilute to
Appearance of solution Solution S, examined immediately after preparation, is clear (2.2.1) and
not more intensely coloured than reference solution BY6 (Method II, 2.2.2).
Test solution (a). Dissolve 0.100 g of the substance to be examined in 10 ml of water R. Immediately
add 2 ml of hydrochloric acid R and shake with 50 ml of ether R. Evaporate the upper layer to dryness
and take up the residue with 10 ml of acetone R.
Reference solution (a). Dissolve 34.3 mg of 4-hydroxybenzoic acid R in acetone R and dilute to 100 ml
Reference solution (b). Dilute 0.5 ml of test solution (a) to 100 ml with acetone R.
Reference solution (c). Dissolve 10 mg of propyl parahydroxybenzoate CRS in acetone R and dilute to
Reference solution (d). Dissolve 10 mg of ethyl parahydroxybenzoate CRS in 1 ml of test solution (a)
36-51
volume of glacial acetic acid R, 30 volumes of water R and 70 volumes of methanol R. Allow the plate
to dry in air and examine in ultraviolet light at 254 nm. In the chromatogram obtained with test
solution (a): any spot due to 4-hydroxybenzoic acid is not more intense than the spot in the chromatogram obtained with reference solution (a) (4 per cent); and any spot, apart from the principal spot
and the spot due to 4-hydroxybenzoic acid, is not more intense than the spot in the chromatogram
obtained with reference solution (d) shows two clearly separated principal spots.
Chlorides (2.4.4). To 10 ml of solution S add 30 ml of water R and 1 ml of nitric acid R and dilute
to 50 ml with water R. Shake and filter. Dilute 10 ml of the filtrate to 15 ml with water R. The
solution complies with the limit test for chlorides (350 ppm). Prepare the standard using 14 ml of
chloride standard solution (5 ppm Cl) R to which 1 ml of water R has been added.
Sulphates (2.4.13). To 25 ml of solution S add 5 ml of distilled water R and 10 ml of hydrochloric
acid R and dilute to 50 ml with distilled water R. Shake and filter. Dilute 10 ml of the filtrate to 15 ml
with distilled water R. The solution complies with the limit test for sulphates (300 ppm).
ASSAY
STORAGE
IMPURITIES
O
OR
HO
B. R = CH3: methyl 4-hydroxybenzoate,
C. R = CH2 - CH3: ethyl 4-hydroxybenzoate,
D. R= CH2 - CH2 - CH2 - CH3: butyl 4-hydroxybenzoate.
__________________________________________________________________________________________________________ Ph Eur
36-52
Sodium Salicylate
COONa
OH
C7H5NaO3
160.1
54-21-7
Sodium Salicylate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0413].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sodium salicylate contains not less than 99.0 per cent and not more than the equivalent of 101.0 per
cent of sodium 2-hydroxybenzenecarboxylate, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder or small, colourless crystals or shiny flakes, freely soluble in water, sparingly soluble in alcohol and practically insoluble in ether.
IDENTIFICATION
obtained with sodium salicylate CRS.
B. Solution S (see Tests) gives the reactions of salicylates (2.3.1).
C. It gives reaction (b) of sodium (2.3.1).
TESTS
Acidity To 20 ml of solution S add 0.1 ml of phenol red solution R. The solution is yellow. Not more
than 2.0 ml of 0.01M sodium hydroxide is required to change the colour of the indicator to reddishviolet.
Chlorides (2.4.4). To 5 ml of solution S add 5 ml of water R and 10 ml of dilute nitric acid R and
filter. 10 ml of the filtrate diluted to 15 ml with water R complies with the limit test for chlorides
(200 ppm).
Heavy metals (2.4.8). Dissolve 1.6 g in 16 ml of a mixture of 5 volumes of water R and 10 volumes
of alcohol R. 12 ml of the solution complies with limit test B for heavy metals (20 ppm). Prepare the
standard using lead standard solution (2 ppm Pb) prepared by diluting lead standard solution (100 ppm
Pb) R with a mixture of 5 volumes of water R and 10 volumes of alcohol R.
100C to 105C.
ASSAY
STORAGE
__________________________________________________________________________________________________________ Ph Eur
36-53
Sodium Starch Glycollate (Type A)

Sodium Starch Glycollate (Type A) complies with the requirements of the 3rd edition of the European
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sodium starch glycollate (type A) is the sodium salt of a cross-linked partly O-carboxymethylated
potato starch. It contains not less than 2.8 per cent and not more than 4.2 per cent of Na (Ar 22.99),
calculated with reference to the substance washed with alcohol (80 per cent V/V) and dried.
CHARACTERS
A white or almost white, fine, free-flowing powder, very hygroscopic, practically insoluble in
methylene chloride. It gives a translucent suspension in water.
Examined under a microscope it is seen to consist of: granules, irregularly shaped, ovoid or pear-shaped,
30 m to 100 m in size, or rounded, 10 m to 35 m in size; compound granules consisting of two
to four components occur occasionally; the granules have an eccentric hilum and clearly visible
concentric striations; between crossed nicol prisms, the granules show a distinct black cross intersecting at the hilum; small crystals are visible at the surface of the granules. The granules show
considerable swelling in contact with water.
IDENTIFICATION
B. Prepare with shaking and without heating a mixture of 4.0 g and 20 ml of carbon dioxide-free
water R. The mixture has the appearance of a gel. Add 100 ml of carbon dioxide-free water R and
shake. A suspension forms that settles after standing.
C. To 5 ml of the suspension obtained in Identification test B add 0.05 ml of iodine solution R1. A
dark blue colour is produced.
D. Solution S2 (see Tests) gives reaction (a) of sodium (2.3.1).
TESTS
Solution S1 Centrifuge the suspension obtained in Identification test B at 2500 g for 10 min. Collect
carefully the supernatant liquid.
Solution S2 Place 2.5 g in a silica or platinum crucible and add 2 ml of a 500 g/l solution of sulphuric
acid R. Heat on a water-bath, then cautiously over a naked flame, raising the temperature
progressively, and then incinerate in a muffle furnace at 600 25C. Continue heating until all black
particles have disappeared. Allow to cool, add a few drops of dilute sulphuric acid R and heat and
incinerate as above. Allow to cool, add a few drops of ammonium carbonate solution R, evaporate to
dryness and incinerate cautiously. Allow to cool and dissolve the residue in 50 ml of water R.
Appearance of solution S1 Solution S1 is clear (2.2.1) and colourless (Method II, 2.2.2).
pH (2.2.3). The pH of solution S1 is 5.5 to 7.5.
Sodium glycollate Carry out the test protected from light.
Test solution. Place 0.20 g of the substance to be examined in a beaker. Add 5 ml of acetic acid R and
5 ml of water R. Stir until dissolution is complete (about 10 min). Add 50 ml of acetone R and 1 g of
sodium chloride R. Filter through a fast filter paper impregnated with acetone R, rinse the beaker and
filter with acetone R. Combine the filtrate and washings and dilute to 100.0 ml with acetone R. Allow
to stand for 24 h without shaking. Use the clear supernatant liquid.
Reference solution. Dissolve 0.310 g of glycollic acid R, previously dried in vacuo over diphosphorus
pentoxide R, in water R and dilute to 500.0 ml with the same solvent. To 5.0 ml of this solution, add
5 ml of acetic acid R and allow to stand for about 30 min. Add 50 ml of acetone R and 1 g of sodium
chloride R and dilute to 100.0 ml with acetone R.
Heat 2.0 ml of the test solution on a water-bath for 20 min. Cool to room temperature and add
20.0 ml of 2,7-dihydroxynaphthalene solution R. Shake and heat in a water-bath for 20 min. Cool
under running water, transfer to a volumetric flask and dilute to 25.0 ml with sulphuric acid R,
maintaining the flask under running water. Within 10 min, measure the absorbance at 540 nm
(2.2.25) using water R as the compensation liquid. The absorbance of the solution prepared with the
test solution is not greater than that of a solution prepared at the same time and in the same manner
with 2.0 ml of the reference solution (2.0 per cent).
Sodium chloride Not more than 7.0 per cent. Shake 1.00 g with 20 ml of alcohol (80 per cent
36-54
V/V) R for 10 min and filter. Repeat the operation four times. Dry the residue to constant mass at
100C and set aside for the Assay. Combine the filtrates. Evaporate to dryness, take up the residue
with water R and dilute to 25.0 ml with the same solvent. To 10.0 ml of the solution add 30 ml of
water R and 5 ml of dilute nitric acid R. Titrate with 0.1M silver nitrate, determining the end-point
potentiometrically (2.2.20), using a silver indicator electrode.
Iron (2.4.9). 10 ml of solution S2 complies with the limit test for iron (20 ppm).
Microbial contamination It complies with the test for Escherichia coli and Salmonella (2.6.13).
ASSAY
To 0.500 g of the dried and crushed residue obtained in the test for sodium chloride add 80 ml of
anhydrous acetic acid R and heat under a reflux condenser for 2 h. Cool the solution to room temperature. Titrate with 0.1M perchloric acid, determining the end-point potentiometrically (2.2.20). Carry
out a blank test.
1 ml of 0.1M perchloric acid is equivalent to 2.299 mg of Na.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
36-55
Sodium Starch Glycollate (Type B)

Sodium Starch Glycollate (Type B) complies with the requirements of the 3rd edition of the European
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sodium starch glycollate (type B) is the sodium salt of a cross-linked partly O-carboxymethylated
potato starch. It contains not less than 2.0 per cent and not more than 3.4 per cent of Na (Ar 22.99),
calculated with reference to the substance washed with alcohol (80 per cent V/V) and dried.
CHARACTERS
A white or almost white, fine, free-flowing powder, very hygroscopic, practically insoluble in
methylene chloride. It gives a translucent suspension in water.
Examined under a microscope it is seen to consist of: granules, irregularly shaped, ovoid or pear shaped,
IDENTIFICATION
B. Prepare with shaking and without heating a mixture of 4.0 g and 20 ml of carbon dioxide-free
water R. The mixture has the appearance of a gel. Add 100 ml of carbon dioxide-free water R and
shake. A suspension forms that settles after standing.
C. To 5 ml of the suspension obtained in Identification test B add 0.05 ml of iodine solution R1. A
dark blue colour is produced.
D. Solution S2 (see Tests) gives reaction (a) of sodium (2.3.1).
TESTS
Solution S1 Centrifuge the suspension obtained in Identification test B at 2500 g for 10 min. Collect
carefully the supernatant liquid.
Solution S2 Place 2.5 g in a silica or platinum crucible and add 2 ml of a 500 g/l solution of sulphuric
particles have disappeared. Allow to cool, add a few drops of dilute sulphuric acid R and heat and
incinerate as above. Allow to cool, add a few drops of ammonium carbonate solution R, evaporate to
Appearance of solution S1 Solution S1 is clear (2.2.1) and colourless (Method II, 2.2.2).
pH (2.2.3). The pH of solution S1 is 3.0 to 5.0.
Sodium glycollate Carry out the test protected from light.
20.0 ml of 2,7-dihydroxynaphthalene solution R. Shake and heat in a water-bath for 20 min. Cool
under running water, transfer quantitatively to a volumetric flask and dilute to 25.0 ml with sulphuric
acid R, maintaining the flasks under running water. Within 10 min, measure the absorbance at
540 nm (2.2.25) using water R as the compensation liquid. The absorbance of the solution prepared
with the test solution is not greater than that of a solution prepared at the same time and in the same
manner with 2.0 ml of the reference solution (2.0 per cent).
Sodium chloride Not more than 7.0 per cent. Shake 1.00 g with 20 ml of alcohol (80 per cent
V/V) R for 10 min and filter. Repeat the operation four times. Dry the residue to constant mass at
36-56
100C and set aside for the Assay. Combine the filtrates. Evaporate to dryness, take up the residue
with water R and dilute to 25.0 ml with the same solvent. To 10.0 ml of the solution add 30 ml of
water R and 5 ml of dilute nitric acid R. Titrate with 0.1M silver nitrate, determining the end-point
potentiometrically (2.2.20) using a silver indicator electrode.
ASSAY
anhydrous acetic acid R and heat under a reflux condenser for 2 h. Cool the solution to room temperature. Titrate with 0.1M perchloric acid, determining the end-point potentiometrically (2.2.20).
Carry out a blank test.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
36-57
Sodium Starch Glycollate (Type C)

1/01
Sodium Starch Glycolate (Type C) complies with the requirements of the 3rd edition of the European
Pharmacopoeia for Sodium Starch Glycolate (Type C) [1566]. These requirements are reproduced after the
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sodium starch glycolate (type C) is the sodium salt of a cross-linked by physical dehydration, partly
O-carboxymethylated starch. It contains not less than 2.8 per cent and not more than 5.0 per cent of
Na (Ar 22.99), calculated with reference to the substance washed with alcohol (80 per cent V/V) and
dried.
CHARACTERS
A white or almost white, fine, free-flowing powder, very hygroscopic, soluble in water, practically
insoluble in methylene chloride. It gives a translucent gel-like product in water.
Examined under a microscope it is seen to consist of granules, irregularly shaped, ovoid or pear-shaped,
IDENTIFICATION
B. Mix with shaking and without heating 4.0 g and 20 ml of carbon dioxide-free water R. The mixture
has the appearance of a gel. Add 100 ml of carbon dioxide-free water R and shake: the gel remains
stable (difference from types A and B). Keep the gel for the tests for appearance of gel and pH.
C. To 5 ml of the gel obtained in identification test B add 0.05 ml of iodine solution R1. A dark blue
colour is produced.
D. Solution S (see Tests) gives reaction (a) of sodium (2.3.1).
TESTS
Solution S Place 2.5 g in a silica or platinum crucible and add 2 ml of a 500 g/l solution of sulphuric
particles have disappeared. Allow to cool, add a few drops of sulphuric acid R and heat and incinerate
as described above. Allow to cool, add a few drops of ammonium carbonate solution R, evaporate to
Appearance of gel The gel prepared under identification test B is colourless (Method II, 2.2.2).
pH (2.2.3). The pH of the gel prepared under identification test B is 5.5 to 7.5.
Sodium glycolate Carry out the test protected from light.
20.0 ml of 2,7-dihydroxynaphthalene solution R. Shake and heat on a water-bath for 20 min. Cool
under running water, transfer to a volumetric flask and dilute to 25.0 ml with sulphuric acid R,
maintaining the flask under running water. Within 10 min, measure the absorbance at 540 nm
(2.2.25) using water R as the compensation liquid. The absorbance of the solution prepared with the
test solution is not greater than that of a solution prepared at the same time and in the same manner
with 2.0 ml of the reference solution (2.0 per cent).
Sodium chloride Not more than 1 per cent. Shake 1.00 g with 20 ml of alcohol (80 per cent V/V) R
36-58
for 10 min and filter. Repeat the operation four times. Dry the residue to constant mass at 100C and
set aside for the assay. Combine the filtrates. Evaporate to dryness, take up the residue with water R
and dilute to 25.0 ml with the same solvent. To 10.0 ml of the solution add 30 ml of water R and
5 ml of dilute nitric acid R. Titrate with 0.1M silver nitrate, determining the end-point potentiometrically (2.2.20), using a silver indicator electrode.
Iron (2.4.9).10 ml of solution S complies with the limit test for iron (20 ppm).
Heavy metals (2.4.8).1.0 g complies with limit test D for heavy metals (20 ppm). Prepare the
100C to 105C for 4 h.
ASSAY
anhydrous acetic acid R and heat under a reflux condenser for 2 h. Cool the solution to room temperature. Titrate with 0.1M perchloric acid, determining the end-point potentiometrically (2.2.20). Carry
out a blank test.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
36-59
Sodium Stearyl Fumarate

1/01
H3C
COONa
O
C22H39NaO4
390.5
Sodium Stearyl Fumarate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sodium stearyl fumarate contains not less than 99.0 per cent and not more than 101.5 per cent of
sodium octadecyl (E)-but-2-enedioate, calculated with reference to the anhydrous substance.
CHARACTERS
A fine, white or almost white powder with agglomerates of flat circular shaped particles, practically
insoluble in water, slightly soluble in methanol, practically insoluble in acetone and in ethanol.
IDENTIFICATION
with sodium stearyl fumarate CRS.
TESTS
Silylation solution. To 2 ml of N,O-bis(trimethylsilyl)trifluoroacetamide R add 0.02 ml of
trimethylchlorosilan R and mix.
Test solution. Introduce 15.0 mg of the substance to be examined in a vial with a screw cap and add
1 ml of the silylation solution. Seal the vial and heat at about 70C for 1 h. After the reaction a
precipitate remains in the vial; filter the solution through a nylon filter (pore size 0.45 m).
Reference solution. Introduce 1.0 mg of sodium stearyl maleate CRS and 1.0 mg of sodium stearyl
fumarate CRS into a vial with a screw cap and add 1 ml of the silylation solution. Seal the vial and
heat at about 70C for 1 h.
a fused-silica column 15 m long and 0.53 mm in internal diameter coated with poly(diphenyl)(dimethyl)siloxane R (film thickness 0.15 m),
Column
Time
(min)
Temperature
(C)
Rate (C
per min)
01
1 21
180
180 320
21 26 320
Injection port
250
Detector
320
Comment
isothermal
linear gradient
isothermal
Inject 2 l of each solution. The test is not valid unless, in the chromatogram obtained with the
reference solution, the resolution between the peaks is not less than 1.5. Calculate the percentage
content of the known impurities from the areas of the peaks in the chromatogram obtained with the
test solution by the normalisation procedure, identifying the peaks by their relative retention (see
Table 1567-1). The content of any impurity is not greater than 0.5 per cent and the sum of the
contents is not greater than 5.0 per cent.
36-60
Table 15671
Impurity
retention
Stearyl alcohol
Stearyl alcohol TMS-ether
(trimethylsilyl-ether)
Monopalmityl fumarate TMS-ether
Monoheptadecyl fumarate TMS-ether
Monostearyl maleate TMS-ether
Monostearyl fumarate TMS-ether
0.30
0.35
Monononadecyl fumarate TMS-ether

Monoeicosenyl fumarate TMS-ether
Distearyl fumarate
1.05
1.15
2.25
0.80
0.85
0.90
1 (Rt 9.3 min)
ASSAY
Dissolve 0.250 g, accurately weighed, in 10 ml of methylene chloride R and add 30 ml of anhydrous
acetic acid R. Titrate with 0.1M perchloric acid determining the end-point potentiometrically (2.2.20).
__________________________________________________________________________________________________________ Ph Eur
36-61
Sodium Stibogluconate
COONa
COONa
OCH
HCO
O
CH
OH
HCO
Sb
HCOH
CH2OH
HO
O
Sb
HC
OCH
HOCH
CH2OH
16037-91-5
Definition Sodium Stibogluconate is mainly the disodium salt of oxy-bis[gluconato(3)O2,O3,O4hydroxo-antimony]. It contains not less than 30.0% and not more than 34.0% of antimony(V),
calculated with reference to the dried and methanol-free substance.
Production The method of manufacture is such as to ensure consistently controlled reaction
stoichiometry in order to yield sodium stibogluconate that is satisfactory with regard to intrinsic
toxicity.
Characteristics A colourless, mostly amorphous powder; odourless or almost odourless.
Very soluble in water; practically insoluble in ethanol (96%) and in ether.
Identification
A. A solution is dextrorotatory.
B. Pass hydrogen sulphide into a 5% w/v solution for several minutes. An orange precipitate is
produced.
C. When heated, it chars without melting leaving a residue which yields the reactions characteristic of
antimony compounds and the reactions characteristic of sodium salts, Appendix VI.
Stability and acidity of solution Heat a solution containing the equivalent of 10% w/v of
antimony(V) in an autoclave at 115.5 and at a pressure of 170 kPa for 30 minutes. The resulting
solution is colourless or almost colourless and has a pH of 5.0 to 5.6, Appendix V L.
Antimony(III) Dissolve 2 g in 30 ml of water, add 15 ml of hydrochloric acid and titrate with
0.00833M potassium bromate VS using methyl orange solution as indicator. Not more than 1.3 ml of
0.00833M potassium bromate VS is required.
Chloride Dissolve 2.5 g in 50 ml of water and add 2 ml of 2M nitric acid and 75 ml of acetate buffer
pH 5.0. Titrate with 0.1M silver nitrate VS determining the end point potentiometrically. Not more
than 3.0 ml of 0.1M silver nitrate VS is required.
Methanol Not more than 2.0% w/w when determined by the following method. Carry out the
method for gas chromatography, Appendix III B, using the following solutions. For solution (1) add
1 ml of a 1.0% v/v solution of methanol to 5 ml of a 0.2% v/v solution of absolute ethanol (internal
standard). For solution (2) add 5 ml of water to 0.5 g of the substance being examined and mix with
the aid of ultrasound until solution is complete. For solution (3) add 5 ml of a 0.2% v/v solution of
the internal standard to 0.5 g of the substance being examined and mix with the aid of ultrasound
until solution is complete.
with porous polymer beads (80 to 100 mesh) (Porapak Q and Chromosorb 101 are suitable) and
maintained at 130.
Calculate the percentage w/w of methanol taking 0.792 g as its weight per ml at 20.
Assay Dissolve 0.16 g in 30 ml of hydrochloric acid, add 70 ml of orthophosphoric acid and stir carefully
until completely mixed. Titrate with 0.05M ammonium iron(II) sulphate VS prepared using sulphuric
acid (1%) and determining the end point potentiometrically using a platinum electrode and a silver
silver chloride reference electrode. Each ml of 0.05M ammonium iron(II) sulphate VS is equivalent to
3.044 mg of antimony(V).
Storage Sodium Stibogluconate should be kept in a well-closed container.
Action and use Antiprotozoal.
Preparation
Sodium Stibogluconate Injection
36-62
Anhydrous Sodium Sulphate

Na2SO4
142.0
7757-82-6
Anhydrous Sodium Sulphate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0099]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Anhydrous sodium sulphate contains not less than 99.0 per cent and not more than the equivalent of
100.5 per cent of Na2SO4, calculated with reference to the dried substance.
CHARACTERS
A white powder, hygroscopic, freely soluble in water.
IDENTIFICATION
TESTS
the indicator.
Calcium (2.4.3). 10 ml of solution S diluted to 15 ml with distilled water R complies with the limit
test for calcium (450 ppm).
(90 ppm).
Magnesium To 10 ml of solution S add 1 ml of glycerol (85 per cent) R, 0.15 ml of titan yellow solution R, 0.25 ml of ammonium oxalate solution R and 5 ml of dilute sodium hydroxide solution R and
shake. Any pink colour in the test solution is not more intense than that in a standard prepared at the
same time in the same manner using a mixture of 5 ml of magnesium standard solution (10 ppm Mg) R
and 5 ml of water R (200 ppm).
130C.
ASSAY
Dissolve 1.30 g in 50 ml of water R. Pass through a column of strongly acidic ion-exchange resin R at a
flow rate of about 4 ml per minute. Wash with water R (about 300 ml) until 50 ml requires not more
than 0.05 ml of 0.1M sodium hydroxide for neutralisation. Titrate the eluate with 1M sodium hydroxide,
using 0.1 ml of methyl orange solution R as indicator.
1 ml of 1M sodium hydroxide is equivalent to 71.0 mg of Na2SO4.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
36-63
Sodium Sulphate
Glaubers Salt
Na2SO4,10H2O
322.2
7727-73-3
Sodium Sulphate complies with the requirements of the 3rd edition of the European Pharmacopoeia for Sodium
Sulphate Decahydrate [0100]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sodium sulphate decahydrate contains not less than 99.0 per cent and not more than the equivalent of
100.5 per cent of Na2SO4, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder or colourless, transparent crystals, freely soluble in water, practically
insoluble in alcohol. It partly dissolves in its own water of crystallisation at about 33C.
IDENTIFICATION
TESTS
than 0.5 ml of 0.01M hydrochloric acid or 0.01M sodium hydroxide is required to change the colour of the
indicator.
Calcium (2.4.3). 10 ml of solution S diluted to 15 ml with distilled water R complies with the limit test
for calcium (200 ppm).
(40 ppm).
Magnesium To 10 ml of solution S add 1 ml of glycerol (85 per cent) R, 0.15 ml of titan yellow solution R, 0.25 ml of ammonium oxalate solution R and 5 ml of dilute sodium hydroxide solution R and shake.
Any pink colour in the test solution is not more intense than that in a standard prepared at the same
time in the same manner using a mixture of 5 ml of magnesium standard solution (10 ppm Mg) R and
5 ml of water R (100 ppm).
Loss on drying (2.2.32). 52.0 per cent to 57.0 per cent, determined on 1.00 g by drying at 30C for
1 h, then at 130C.
ASSAY
Dissolve 3.00 g in 50 ml of water R. Pass through a column of strongly acidic ion-exchange resin R at a
flow rate of about 4 ml per minute. Wash with water R (about 300 ml) until 50 ml requires not more
than 0.05 ml of 0.1M sodium hydroxide for neutralisation. Titrate the eluate with 1M sodium hydroxide,
using 0.1 ml of methyl orange solution R as indicator.
1 ml of 1M sodium hydroxide is equivalent to 71.0 mg of Na2SO4.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
36-64
Anhydrous Sodium Sulphite

Na2SO3
126.0
7757-83-7
Anhydrous Sodium Sulphite complies with the requirements of the 3rd edition of the European Pharmacopoeia [0775]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Anhydrous sodium sulphite contains not less than 95.0 per cent and not more than the equivalent of
100.5 per cent of Na2SO3.
CHARACTERS
A white powder, freely soluble in water, very slightly soluble in alcohol.
IDENTIFICATION
A. Solution S (see Tests) is slightly alkaline (2.2.4).
B. To 5 ml of solution S, add 0.5 ml of 0.05M iodine. The solution is colourless and gives reaction (a)
of sulphates (2.3.1).
D. It complies with the limits of the Assay.
TESTS
Solution S Dissolve 5 g in water R and dilute to 100 ml with the same solvent.
Solution S1 To 10.0 g add 25 ml of water R. Shake until mostly dissolved and carefully and
progressively add 15 ml of hydrochloric acid R. Heat to boiling. Cool and dilute to 100.0 ml with
water R.
Appearance of solution Solution S is clear (2.2.1) and colourless (Method I, 2.2.2).
Thiosulphates To 2.00 g add 100 ml of water R. Shake and add 10 ml of formaldehyde solution R
and 10 ml of acetic acid R. Allow to stand for 5 min. Add 0.5 ml of starch solution R and titrate with
0.05M iodine. Carry out a blank titration. The difference between the volumes used in the titrations is
not more than 0.15 ml (0.1 per cent).
Selenium To 3.0 g add 10 ml of formaldehyde solution R and carefully and progressively add 2 ml of
hydrochloric acid R. Heat on a water-bath for 20 min. Any pink colour in the solution is not more
intense than that of a standard prepared at the same time and in the same manner using 1.0 g of the
substance to be examined to which has been added 0.2 ml of selenium standard solution (100 ppm
Se) R (10 ppm).
Zinc Not more than 25 ppm of Zn, determined by atomic absorption spectrometry (Method I,
2.2.23).
Test solution. Dilute 2.0 ml of solution S1 to 10.0 ml with water R.
Measure the absorbance at 213.9 nm using a zinc hollow-cathode lamp as source of radiation and an
air-acetylene flame.
Heavy metals (2.4.8). 12 ml of solution S1 complies with limit test A for heavy metals (10 ppm).
ASSAY
Introduce 0.250 g into a 500 ml conical flask containing 50.0 ml of 0.05M iodine. Shake until
completely dissolved. Add 1 ml of starch solution R and titrate the excess of iodine with 0.1M sodium
1 ml of 0.05M iodine is equivalent to 6.30 mg of Na2SO3.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
36-65
Sodium Sulphite Heptahydrate

Sodium Sulphite
Na2SO3,7H2O
252.2
10102-15-5
Sodium Sulphite Heptahydrate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0776]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sodium sulphite heptahydrate contains not less than 48.0 per cent and not more than the equivalent
of 52.5 per cent of Na2SO3.
CHARACTERS
Colourless crystals, freely soluble in water, very slightly soluble in alcohol.
IDENTIFICATION
A. Solution S (see Tests) is slightly alkaline (2.2.4).
B. To 5 ml of solution S, add 0.5 ml of 0.05M iodine. The solution is colourless and gives reaction (a)
of sulphates (2.3.1).
D. It complies with the limits of the Assay.
TESTS
Solution S1 To 20.0 g add 25 ml of water R. Shake until mostly dissolved and carefully and
progressively add 15 ml of hydrochloric acid R. Heat to boiling. Cool and dilute to 100.0 ml with
water R.
Appearance of solution Solution S is clear (2.2.1) and colourless (Method I, 2.2.2).
Thiosulphates To 4.00 g add 100 ml of water R. Shake to dissolve and add 10 ml of formaldehyde
solution R and 10 ml of acetic acid R. Allow to stand for 5 min. Add 0.5 ml of starch solution R and
titrate with 0.05M iodine. Carry out a blank titration. The difference between the volumes used in the
titrations is not more than 0.15 ml (0.05 per cent).
Selenium To 6.0 g add 10 ml of formaldehyde solution R and carefully and progressively add 2 ml of
hydrochloric acid R. Heat on a water-bath for 20 min. Any pink colour in the solution is not more
intense than that of a standard prepared at the same time and in the same manner using 2.0 g of the
substance to be examined to which has been added 0.2 ml of selenium standard solution (100 ppm
Se) R (5 ppm).
Zinc Not more than 12 ppm of Zn, determined by atomic absorption spectrometry (Method I,
2.2.23).
Test solution. Dilute 2.0 ml of solution S1 to 10.0 ml with water R.
Measure the absorbance at 213.9 nm using a zinc hollow-cathode lamp as source of radiation and an
air-acetylene flame.
Heavy metals (2.4.8). 12 ml of solution S1 complies with limit test A for heavy metals (5 ppm).
ASSAY
Introduce 0.500 g into a 500 ml conical flask containing 50.0 ml of 0.05M iodine. Shake until
completely dissolved. Add 1 ml of starch solution R and titrate the excess of iodine with 0.1M sodium
1 ml of 0.05M iodine is equivalent to 6.30 mg of Na2SO3.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
36-66
Sodium Tetradecyl Sulphate Concentrate

Me H H
Me
OSO3ONa
*
H
Me
Et
C14H29NaO4S
316.4
139-88-8
Definition Sodium Tetradecyl Sulphate Concentrate is an aqueous gel containing sodium all-rac-4ethyl-1-isobutyloctyl sulphate. It contains not less than 46.0% w/w and not more than 52.0% w/w of
C14H29NaO4S.
Characteristics A clear, colourless gel.
Identification
A. Carry out the method for gas chromatography, Appendix III B, using the following solutions. For
solution (1) boil 0.2 g under a reflux condenser with 20 ml of 2M hydrochloric acid for 15 minutes,
allow to cool, add 20 ml of ethanol (96%) and extract the mixture with two 10-ml quantities of
n-pentane. Wash the combined pentane extracts with 20 ml of water and dry over anhydrous sodium
sulphate. Solution (2) contains 0.35% w/v of decan-1-ol and 0.7% w/v of dodecan-1-ol in n-pentane.
with acid-washed, silanised diatomaceous support (80 to 100 mesh) coated with 3% w/w of polyethylene
glycol (Carbowax 20M is suitable) and maintained at 120.
The retention time of the principal peak in the chromatogram obtained with solution (1) is less
than the retention time of the peak due to dodecan-1-ol and more than that of the peak due to decan1-ol in the chromatogram obtained with solution (2).
B. Mix 0.1 ml of a 2% w/v solution with 0.1 ml of a 0.1% w/v solution of methylene blue and 2 ml of
1M sulphuric acid, add 2 ml of chloroform and shake. The chloroform layer is intensely blue.
C. Mix 20 mg with 10 ml of ethanol (96%) and heat to boiling on a water bath, shaking frequently.
Filter immediately and evaporate the ethanol. Dissolve the residue in 8 ml of water, add 3 ml of 2M
hydrochloric acid, evaporate the solution to half its volume and cool. Filter to remove the congealed
fatty alcohols and add 1 ml of 0.25M barium chloride to the filtrate. A white, crystalline precipitate is
produced.
D. Yields reaction B characteristic of sodium salts, Appendix VI.
Alkalinity Dissolve 1 g in 100 ml of water and add 0.1 ml of methyl red solution; the solution is
yellow. Not more than 0.5 ml of 0.1M hydrochloric acid VS is required to change the colour of the
solution.
Non-ionic impurities Not more than 3.0% w/w, with respect to the nominal content of Sodium
Tetradecyl Sulphate, when determined by the method for gas chromatography, Appendix III B, using
the following solutions. Dissolve 75 mg of dodecan-1-ol (internal standard) in sufficient n-hexane to
produce 100 ml (solution A). For solution (1) extract 1 g of the concentrate being examined with
10 ml of solution A followed by two 10-ml quantities of n-hexane. Pass 10 ml of the combined
extracts at a rate of about 1.5 ml per minute through a column, 1 cm in diameter, packed with 5 g of
basic aluminium hydroxide (Brockmann grade II is suitable) and pre-washed with 25 ml of n-hexane.
Elute with 20 ml of a mixture of equal volumes of n-hexane and ether, evaporate to dryness using a
rotary evaporator and dissolve the residue in 2 ml of n-hexane. Prepare solution (2) in the same
manner but using 10 ml of n-hexane in place of 10 ml of solution A.
with acid-washed, silanised diatomaceous support (80 to 100 mesh) coated with 3% w/w of polyethylene
glycol (Carbowax 20M is suitable) and maintained at 120.
Allow the chromatography to proceed for the retention time of the peak due to the internal
standard.
In the chromatogram obtained with solution (1) the sum of the areas of any secondary peaks is not
greater than twice the area of the peak due to the internal standard.
Chloride Dissolve 33 mg in sufficient water to produce 100 ml. 15 ml of the resulting solution
complies with the limit test for chlorides, Appendix VII (1%).
Sulphated ash 10.3 to 13.3%, Appendix IX A.
Assay Dissolve 2.4 g in sufficient water to produce 1000 ml. To 20 ml add 15 ml of chloroform and
10 ml of dimidium bromidesulphan blue mixed solution and titrate with 0.004M benzethonium chloride
VS, shaking vigorously and allowing the layers to separate after each addition, until the pink colour of
the chloroform layer is completely discharged and a greyish blue colour is produced. Each ml of
0.004M benzethonium chloride VS is equivalent to 1.266 mg of C14H29NaO4S.
36-67
Storage Sodium Tetradecyl Sulphate Concentrate should be kept in a well-closed container,
protected from light and stored at a temperature not exceeding 25.
Action and use Sclerosant.
Preparation
Sodium Tetradecyl Sulphate Injection
36-68
Sodium Thiosulphate
1/01
Na2S2O3,5H2O
248.2
10102-17-7
Sodium Thiosulphate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Used in treatment of cyanide poisoning.
Preparation
Sodium Thiosulphate Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sodium thiosulphate contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of Na2S2O3,5H2O.
CHARACTERS
Transparent, colourless crystals, efflorescent in dry air, very soluble in water, practically insoluble in
alcohol. It dissolves in its water of crystallisation at about 49C.
IDENTIFICATION
A. It decolourises iodinated potassium iodide solution R.
B. To 0.5 ml of solution S (see Tests) add 0.5 ml of water R and 2 ml of silver nitrate solution R2. A
white precipitate is formed which rapidly becomes yellowish and then black.
C. To 2.5 ml of solution S add 2.5 ml of water R and 1 ml of hydrochloric acid R. A precipitate of
sulphur is formed and gas is evolved which gives a blue colour to starch iodate paper R.
D. 1 ml of solution S gives reaction (a) of sodium (2.3.1).
TESTS
Sulphates and sulphites Dilute 2.5 ml of solution S to 10 ml with distilled water R. To 3 ml of this
solution first add 2 ml of iodinated potassium iodide solution R and continue the addition dropwise until
a very faint persistent yellow colour appears. Dilute to 15 ml with distilled water R. The solution
complies with the limit test for sulphates (2.4.13) (0.2 per cent).
Sulphides To 10 ml of solution S add 0.05 ml of a freshly prepared 50 g/l solution of sodium
nitroprusside R. The solution does not become violet.
Heavy metals To 10 ml of solution S add 0.05 ml of sodium sulphide solution R. After 2 min, any
brown colour in the solution is not more intense than that in a standard prepared at the same time
and in the same manner using 10 ml of lead standard solution (1 ppm Pb) R (10 ppm).
ASSAY
Dissolve 0.500 g in 20 ml of water R and titrate with 0.05M iodine, using 1 ml of starch solution R,
added towards the end of the titration, as indicator.
1 ml of 0.05M iodine is equivalent to 24.82 mg of Na2S2O3,5H2O.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
36-69
Sodium Valproate
H3C
H3C
C8H15NaO2
COONa
H
166.2
1069-66-5
Sodium Valproate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0678].
Preparations
Sodium Valproate Oral Solution
Sodium Valproate Tablets
Enteric-coated Sodium Valproate Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sodium valproate contains not less than 98.5 per cent and not more than the equivalent of 101.0 per
cent of sodium 2-propylpentanoate, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, hygroscopic, very soluble in water, slightly to freely
soluble in alcohol.
IDENTIFICATION
obtained with sodium valproate CRS. If the spectra obtained in the solid state for the substance to be
examined and the reference substance show differences, record further spectra using discs prepared
by placing 50 l of a 100 g/l solution in methanol R on a disc of potassium bromide R and evaporating
the solvent in vacuum. Use the discs immediately.
B. Examine the chromatograms obtained in the test for related substances. The retention time of the
principal peak in the chromatogram obtained with test solution (b) corresponds to that of the
C. 2 ml of solution S (see Tests) gives reaction (a) of sodium (2.3.1).
TESTS
Solution S Dissolve 1.25 g in 20 ml of distilled water R in a separating funnel, add 5 ml of dilute nitric
acid R and shake. Allow the mixture to stand for 12 h. Use the lower layer.
Acidity or alkalinity Dissolve 1.0 g in 10 ml of water R. Add 0.1 ml of phenolphthalein solution R.
Not more than 0.75 ml of 0.1M hydrochloric acid or 0.1M sodium hydroxide is required to change the
Related substances Examine by gas chromatography (2.2.28), using butyric acid R as the internal
standard.
Internal standard solution. Dissolve 10 mg of butyric acid R in heptane R and dilute to 200 ml with the
same solvent.
Test solution (a). Dissolve 0.500 g of the substance to be examined in 10 ml of water R. Add 5 ml of
dilute sulphuric acid R and shake with three quantities, each of 20 ml, of heptane R. Add 10.0 ml of the
internal standard solution to the combined upper layers, shake with anhydrous sodium sulphate R, filter
and evaporate the filtrate, at a temperature not exceeding 30C, using a rotary evaporator. Take up
the residue with the internal standard solution and dilute to 10.0 ml with the same solution. Dilute
1.0 ml of the solution to 10.0 ml with heptane R.
Test solution (b). Dissolve 40 mg of the substance to be examined in 100 ml of water R. To 10 ml of
the solution add 0.5 ml of dilute sulphuric acid R and shake with three quantities, each of 5 ml, of
heptane R. Shake with anhydrous sodium sulphate R, filter and evaporate the filtrate, at a temperature
not exceeding 30C, to a volume of about 10 ml, using a rotatory evaporator.
Reference solution (a). Dissolve 20 mg of 2-(1-methylethyl)pentanoic acid CRS in 5.0 ml of test solution
(b) and dilute to 10 ml with heptane R. Dilute 1 ml of the solution to 10 ml with heptane R.
36-70
Reference solution (b). Prepare as prescribed for test solution (b), using sodium valproate CRS instead
a wide-bore fused-silica column 30 m long and 0.53 mm in internal diameter coated with
macrogol 20,000 2-nitroterephthalate R (film thickness 0.5 m),
Time
(min)
Temperature
(C)
Rate
(C per min)
Column 010
1030
130
130190
Injection
port
220
Detector
220
Comment
isothermal
linear
gradient
Inject 1 l of each solution. Adjust the sensitivity of the system so that the height of the peak due to
the internal standard is at least 20 per cent of the full scale of the recorder. The test is not valid
unless, in the chromatogram obtained with reference solution (a), the resolution between the peaks
corresponding to 2-(1-methylethyl)pentanoic acid and valproic acid is at least 3.0. In the chromatogram obtained with test solution (a): the sum of the areas of the peaks, apart from the principal peak
is not greater than three times the area of the peak due to the internal standard (0.3 per cent); none
of the peaks, apart from the principal peak, has an area greater than that of the peak due to the
internal standard (0.1 per cent). Disregard any peak with an area less than 0.1 times the area of the
peak due to the internal standard.
Chlorides (2.4.4). To 5 ml of solution S add 10 ml of water R. The solution complies with the limit
test for chlorides (200 ppm).
Sulphates (2.4.13). Solution S complies with the limit test for sulphates (200 ppm).
100C to 105C.
ASSAY
Dissolve 0.1500 g in 25 ml of anhydrous acetic acid R. Titrate with 0.1M perchloric acid determining
1 ml of 0.1M perchloric acid, is equivalent to 16.62 mg of C8H15NaO2.
STORAGE
IMPURITIES
H3C
COOH
R R'
A. R = R = H: pentanoic acid (valeric acid),

B. R = H, R = CH2-CH3: (2RS)-2-ethylpentanoic acid,
C. R = H, R = CH(CH3)2: (2RS)-2-(1-methylethyl)pentanoic acid,
D. R = R = CH2-CH2-CH3 = 2,2-dipropylpentanoic acid,
H3C
CONH2
R R'
E. R = R = H: pentanamide (valeramide),
F. R = H, R = CH2-CH2-CH3: 2-propylpentanamide,
G. R = R = CH2-CH2-CH3: 2,2-dipropylpentanamide,
H3C
CN
R R'
H. R = R = H: pentanenitrile (valeronitrile),
I. R = H, R = CH2-CH2-CH3: 2-propylpentanenitrile,
J. R = R = CH2-CH2-CH3: 2,2-dipropylpentanenitrile.
__________________________________________________________________________________________________________ Ph Eur
36-71
Somatostatin
corrected 1/01
Ala-Gly-Cys-Asn-Phe-Phe-Trp-Lys-Thr-PheThr-Ser-Cys
C76H104N18O19S2
1638
38916-34-6
Somatostatin complies with the requirements of the 3rd edition of the European Pharmacopoeia [0949]. These
Action and use Growth hormone release inhibitor.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Somatostatin is a cyclic tetradecapeptide having the structure of the hypothalamic hormone that
inhibits the release of human growth hormone. It is obtained by chemical synthesis. It contains a
variable amount of acetic acid. It is available in the freeze-dried form and contains not less than
95.0 per cent and not more than the equivalent of 103.0 per cent of somatostatin, calculated with
reference to the anhydrous, acetic acid-free substance.
CHARACTERS
A white, amorphous powder, freely soluble in water and acetic acid, practically insoluble in
methylene chloride.
IDENTIFICATION
substance.
Test solution. Dissolve 1.0 mg of the substance to be examined in 1.0 ml of water R.
Reference solution. Dissolve the contents of a vial of somatostatin CRS in water R. Dilute an appropriate
volume of this solution in water R to obtain a final concentration of 1 mg/ml.
Apply separately to the plate 20 l of each solution. Develop over a path of 15 cm using a mixture
of 10 volumes of acetic acid R, 15 volumes of pyridine R, 20 volumes of water R and 45 volumes of
butanol R. Dry the plate in a current of warm air. Spray with a 1 g/l solution of ninhydrin R and heat
the plate in an oven at 110C for about 5 min. The principal spot in the chromatogram obtained with
B. Examine the chromatograms obtained in the Assay. The retention time of the principal peak in the
TESTS
Specific optical rotation (2.2.7). Dissolve 2.0 mg in 1.0 ml of a 1.0 per cent V/V solution of glacial
acetic acid R. The specific optical rotation is 37 to 47, calculated with reference to the anhydrous,
acetic acid-free substance.
Absorbance (2.2.25). Dissolve 5.0 mg in a 9 g/l solution of sodium chloride R and dilute to 100.0 ml
with the same solution. The absorbance at the maximum at 280 nm is not more than 0.20, calculated
with reference to the peptide content as determined in the assay.
Amino acids Examine by means of an amino-acid analyser, using DL-norleucine R as the internal
standard. Standardise the apparatus with a mixture containing equimolar amounts of ammonia,
glycine and the L-form of the following amino acids:
lysine
threonine
alanine
leucine
histidine
serine
valine
tyrosine
arginine
glutamic acid methionine
phenylalanine
aspartic acid
proline
isoleucine
together with half the equimolar amount of L-cystine.
Internal standard solution. Dissolve 30 mg of DL-norleucine R in a mixture of equal volumes of hydrochloric acid R and water R and dilute to 100.0 ml with the same mixture of solvents.
Test solution. Place 1.0 mg of the substance to be examined in a rigorously cleaned suitable hard glass
tube 100 mm long and 6 mm in internal diameter. Add an accurately measured volume of the
internal standard solution containing an amount of DL-norleucine R corresponding to about half the
36-72
expected number of moles of somatostatin. Immerse the tube in a freezing mixture at 5C, reduce
the pressure to below 133 Pa and seal. Heat at 110C to 115C for 16 h. Cool, open the tube,
transfer the contents to a 10 ml flask with the aid of five quantities, each of 0.2 ml, of water R and
evaporate to dryness over potassium hydroxide R under reduced pressure. Take up the residue in
water R and evaporate to dryness over potassium hydroxide R under reduced pressure; repeat these
operations once. Take up the residue in a suitable buffer solution (pH 2.2) and dilute to a suitable
volume with the same buffer solution.
Apply to the amino-acid analyser a suitable, accurately measured volume of the test solution such
that the peak given by the amino acid present in the largest amount occupies most of the chart height
of the recorder.
acids taking one eighth of the sum of the number of moles of aspartic acid, alanine, lysine, glycine
and phenylalanine as equal to one. The values fall within the following limits: aspartic acid 0.95 to
1.05; glycine 0.95 to 1.05; alanine 0.95 to 1.05; phenylalanine 2.85 to 3.15; serine 0.7 to 1.05;
threonine 1.4 to 2.1; half-cystine 1.4 to 2.1; lysine 1.9 to 2.1. Not more than traces of other amino
acids are present.
The test is not valid unless the number of moles of norleucine recovered, corrected for the volume of
test solution applied, is within 5 per cent of the amount taken for hydrolysis.
Related peptides Examine by liquid chromatography (2.2.29).
Test solution. Dissolve 1.5 mg of the substance to be examined in 3.0 ml of water R.
Reference solution (a). Dilute 1.0 ml of the test solution to 50.0 ml with water R.
Reference solution (b). Add 1.0 ml of reference solution (a) to 1.0 ml of water R.
a stainless steel column 50 mm long and 4.6 mm in internal diameter packed with octadecylsilyl
as mobile phase at a flow rate of 1.5 ml per minute, the following elution programme:
Mobile phase A. Dilute 11 ml of phosphoric acid R with water R, adjust to pH 2.3 with
triethylamine R and dilute to 1000 ml with water R,
Interval Mobile phase A
(min)
(per cent V/V)
0 18
18 20
20 21
21 26
Mobile phase B
(per cent V/V)
Comment
79 60
60
21 40
40
60 79
79
40 21
21
linear gradient
isocratic
linear gradient
equilibration

phase A.
Inject reference solution (a) three times; the test is not valid unless the standard deviation of the
area of the principal peak is not greater than 2.5 per cent.
In the chromatogram obtained with the test solution; the area of any peak, apart from the principal
solution (b) (1.0 per cent); the sum of the areas of such peaks is not greater than the area of the
any peak due to the solvent.
Acetic acid Not more than 15.0 per cent m/m, determined by gas chromatography (2.2.28), using
propionic acid R as the internal standard.
Internal standard solution. Dilute 1 ml of propionic acid R to 100 ml with a 1 per cent m/m solution of
anhydrous formic acid R.
Test solution. Dissolve 0.500 mg of the substance to be examined in water R, add 50 l of the internal
Reference solution. Dilute 0.1 ml of acetic acid R to 100 ml with a 1 per cent m/m solution of anhydrous
formic acid R. To 25 l of this solution add 50 l of the internal standard solution and dilute to 1.0 ml
with water R.
a glass column 1 m long and 4 mm in internal diameter packed with graphitised carbon for
chromatography R impregnated with 0.3 per cent of macrogol 20,000 R and 0.1 per cent m/m of
phosphoric acid R,
36-73
maintaining the temperature of the column at 130C, that of the detector at 200C and that of the
injection port at 170C. Inject separately 1.0 l to 2.0 l of the test solution and of the reference
solution.
Water (2.5.12). Not more than 8.0 per cent, determined on 10.0 mg by the semi-micro determination of water.
Pyrogens (2.6.8). It complies with the test for pyrogens. Inject per kilogram of the rabbits mass
2 ml of a solution containing 40 g of the substance to be examined per millilitre in water for injections R.
ASSAY
the same solvent.
Reference solution. Dissolve the contents of a vial of somatostatin CRS in water R. Dilute an appropriate
volume of this solution with water R to obtain a final concentration of 0.05 mg/ml.
a stainless steel column 50 mm long and 4.6 mm in internal diameter packed with octadecylsilyl
as mobile phase at a flow rate of 1.5 ml per minute a mixture of 25 volumes of mobile phase B
and 75 volumes of mobile phase A:
Mobile phase A. Dilute 11 ml of phosphoric acid R with water R, adjust to pH 2.3 with
triethylamine R and dilute to 1000 ml with water R,
phase A.
Inject the reference solution three times; the assay is not valid unless the standard deviation of the
principal peak is not more than 2.5 per cent.
Inject 50 l of the test solution and the reference solution and record each chromatogram for
15 min.
Calculate the content of somatostatin (C76H104N18O19S2) from the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of
C76H104N18O19S2 in somatostatin CRS.
STORAGE
Store in an airtight container protected from light and moisture, at a temperature of 2C to 8C. If
the substance is sterile, store in a sterile, airtight, tamper-proof container.
LABELLING
__________________________________________________________________________________________________________ Ph Eur
36-74
Somatropin
FPTIPLSRLF DNAMLRAHRL HQLAFDTYQE
FEEAYIPKEQ KYSFLQNPQT SLCFSESIPT
PSNREETQQK SNLELLRISL LLIQSWLEPV
QFLRSVFANS LVYGASDSNV YDLLKDLEEG
IQTLMGRLED GSPRTGQIFK QTYSKFDTNS
HNDDALLKNY GLLYCFRKDM DKVETFLRIV
QCRSVEGSCG F
C990H1528N262O300S7
22,125
12629-01-5
Somatropin complies with the requirements of the 3rd edition of the European Pharmacopoeia [0951]. These
Action and use Growth hormone.
Preparation
Somatropin Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Somatropin is a protein having the structure (191 amino-acid residues) of the major component of
growth hormone produced by the human pituitary. It contains not less than 91.0 per cent and not
more than the equivalent of 105.0 per cent of somatropin(1) (C990H1528N262O300S7), calculated with
reference to the anhydrous substance. It complies with the requirements of the monograph
on Recombinant DNA technology, products of (0784).
PRODUCTION
Somatropin is produced by a method based on recombinant DNA (rDNA) technology. During the
course of product development, it must be demonstrated that the manufacturing process produces a
product having a biological activity of not less than 2.5 I.U. per milligram, using a validated bioassay
based on growth promotion and approved by the competent authority.
Somatropin complies with the following additional requirements.
Host-cell-derived proteins The limit is approved by the competent authority.
Host-cell- and vector-derived DNA The limit is approved by the competent authority.
CHARACTERS
A white or almost white powder.
IDENTIFICATION
A. Examine the electrophoretograms obtained in the test for isoform distribution. In the electrophoretogram obtained with test solution (a), the principal band corresponds in position to that in the
electrophoretogram obtained with reference solution (a).
B. Examine the chromatograms obtained in the test for related proteins. The retention time of the
principal peak in the chromatogram obtained with the test solution is similar to that of the principal
peak in the chromatogram obtained with the reference solution.
C. Examine by peptide mapping.
Test solution. Prepare a solution of the substance to be examined in 0.05M tris-hydrochloride buffer
solution pH 7.5 R to obtain a solution containing 2.0 mg/ml of somatropin and transfer about 1.0 ml
to a tube made from suitable material such as polypropylene. Prepare a 1 mg/ml solution of trypsin for
peptide mapping R in 0.05M tris-hydrochloride buffer solution pH 7.5 R and add 30 l to the solution of
the substance to be examined. Cap the tube and place in a water-bath at 37C for 4 h. Remove from
the water-bath and stop the reaction immediately, for example by freezing. If analysed immediately
using an automatic injector, maintain the temperature at 2C to 8C.
36-75
Reference solution. Prepare at the same time and in the same manner as for the test solution but using
somatropin CRS instead of the substance to be examined.
gel for chromatography R (5 m to 10 m),
Mobile phase A. Dilute 1 ml of trifluoroacetic acid R to 1000 ml with water R,
Mobile phase B. To 100 ml of water R add 1 ml of trifluoroacetic acid R and dilute to 1000 ml with
acetonitrile for chromatography R,
following the elution conditions as described in the table below (if necessary, the gradient or the
temperature of the column may be modified to improve separation of the digest):
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
020
2040
4065
10080
020
8075
7550
5020
2025
2550
5080
20100
100
800
0
6570
7071
7185

Equilibrate the column with mobile phase A for at least 15 min. Carry out a blank run using the
above-mentioned gradient.
Inject 100 l of the test solution and 100 l of the reference solution. The test is not valid unless
the chromatogram obtained with each solution is qualitatively similar to the Ph. Eur. reference chromatogram of somatropin digest. The profile of the chromatogram obtained with the test solution corresponds to that of the chromatogram obtained with the reference solution.
D. Examine the chromatograms obtained in the assay. The retention time of the principal peak in the
TESTS
Related proteins Examine by liquid chromatography (2.2.29).
Test solution. Prepare a solution of the substance to be examined in 0.05M tris-hydrochloride buffer
solution pH 7.5 R, containing 2.0 mg/ml of somatropin.
Reference solution. Prepare a solution of somatropin CRS in 0.05M tris-hydrochloride buffer solution pH
7.5 R, containing 2.0 mg/ml of somatropin.
Resolution solution (somatropin/desamido-somatropin resolution mixture). Prepare a solution of somatropin
CRS in 0.05M tris-hydrochloride buffer solution pH 7.5 R to obtain a 2.0 mg/ml solution of somatropin.
Either filter through a sterile filter or add sodium azide R to a concentration of 0.1 mg/ml and allow to
stand at room temperature for 24 h.
Maintain the solutions at 2C to 8C and use within 24 h. If an automatic injector is used, maintain the
temperature at 2C to 8C.
singly end-capped butylsilyl silica gel, with a granulometry of 5 m and a porosity of 30 nm. A
silica saturation column is to be placed between the pump and the injector valve,
as mobile phase at a flow rate of 0.5 ml/min a mixture of 29 volumes of propanol R and 71
volumes of 0.05M tris-hydrochloride buffer solution pH 7.5 R,
Prior to use, rinse the column with 200 ml to 500 ml of a 0.1 per cent V/V solution of trifluoroacetic
acid R in a 50 per cent V/V solution of acetonitrile R. Repeat as necessary, to improve column
performance.
Inject 20 l of the reference solution. If necessary, adjust the concentration of propanol R in the
mobile phase so that the retention time of the principal peak is about 33 min.
Inject 20 l of the resolution solution. Desamido-somatropin appears as a small peak at a retention
time of about 0.85 relative to the principal peak. The test is not valid unless the resolution between
the peaks corresponding to somatropin and desamido-somatropin is at least 1.0 and the symmetry
factor of the somatropin peak is 0.9 to 1.8.
Inject 20 l of the test solution. In the chromatogram obtained, the sum of the areas of all the
36-76
peaks, apart from the principal peak, is not greater than 6.0 per cent of the total area of the peaks.
Disregard any peak due to the solvent.
Dimer and related substances of higher molecular mass Examine by size-exclusion chromatography (2.2.30) as described under Assay.
Inject 20 l of the test solution. In the chromatogram obtained with the test solution, the sum of
the areas of any peak with a retention time less than that of the principal peak is not greater than
4.0 per cent of the total area of the peaks. Disregard any peak due to the solvent.
Isoform distribution Examine by isoelectric focusing.
Test solution (a). Prepare a solution of the substance to be examined in 0.025M phosphate buffer
solution pH 7.0 R, containing 2.0 mg/ml of somatropin.
Test solution (b). Add 0.1 ml of test solution (a) to 1.9 ml of 0.025M phosphate buffer solution pH 7.0 R.
Reference solution (a). Prepare a solution of somatropin CRS in 0.025M phosphate buffer solution pH
Reference solution (b). Use an isoelectric point calibration solution in the pH range of 2.5 to 6.5,
prepared and used according to the manufacturers instructions.
Operate the apparatus in accordance with the manufacturers instructions. The isoelectric focusing
procedure may be carried out using a pre-cast gel 245 mm 110 mm 1 mm, with a pH in the
range 4.0 to 6.5. Apply to the gel 15 l of each solution. Use as the anode solution a 14.7 g/l solution
of glutamic acid R in phosphoric acid (50 g/l H3PO4) and as the cathode solution an 89.1 g/l solution
of alanine R. Adjust the operating conditions to 2000 V and 25 mA. Allow focusing to take place
for 2.5 h at a constant voltage and at a power of not more than 25 W. Immerse the gel for 30 min in
a solution containing 115 g/l of trichloroacetic acid R and 34.5 g/l of sulphosalicylic acid R, and then for
5 min in a mixture of 8 volumes of acetic acid R, 25 volumes of ethanol R and 67 volumes of deionised
water R (de-stain solution). Stain the gel by immersion in a 1.15 g/l solution of acid blue 83 R in destain solution at 60C for 10 min, and then place the gel in de-stain solution until excess stain is
removed.
The test is not valid unless the distribution of bands in the electrophoretogram obtained with
reference solution (b) corresponds to the manufacturers indications. The electrophoretogram
obtained with reference solution (a) contains a major band with an isoelectric point of approximately
five, and a slightly more acidic minor band at approximately 4.8. In the electrophoretogram obtained
with test solution (a), no band apart from the major band is more intense than the major band in the
electrophoretogram obtained with test solution (b) (5 per cent).
Water (2.5.32). Not more than 10.0 per cent, determined by the micro determination of water.
of endotoxin per milligram of somatropin.
ASSAY
Examine by size-exclusion chromatography (2.2.30).
Test solution. Prepare a solution of the substance to be examined in 0.025M phosphate buffer solution
pH 7.0 R, containing 1.0 mg/ml of somatropin.
Reference solution. Dissolve the contents of a vial of somatropin CRS in 0.025M phosphate buffer solution
pH 7.0 R and dilute with the same solvent to obtain a concentration of 1.0 mg/ml.
Resolution solution. Place one vial of somatropin CRS in an oven at 50C for a period (typically
between 12 and 24 h) sufficient to generate 1 per cent to 2 per cent of dimer. Dissolve its contents in
0.025M phosphate buffer solution pH 7.0 R and dilute with the same solvent to obtain a concentration
of 1.0 mg/ml.
a stainless steel column 0.30 m long and 7.8 mm in internal diameter packed with hydrophilic
silica gel for chromatography R of a grade suitable for fractionation of globular proteins in the
molecular mass range 5000 to 150,000,
as mobile phase at a flow rate of 0.6 ml/min a mixture (filtered and degassed) consisting of 3
volumes of 2-propanol R and 97 volumes of 0.063M phosphate buffer solution pH 7.0 R,
Inject 20 l of the resolution solution. In the chromatogram obtained, the main peak elutes at a
retention time of approximately 12 min to 17 min and the peaks corresponding to the somatropin
dimer and to the higher molecular weight proteins at relative retention times of 0.90 and 0.65
respectively, relative to the main peak. The resolution, defined by the ratio of the height above the
baseline of the valley separating the monomer and dimer peaks to the height of the dimer peak, is not
greater than 0.4.
Calculate the content of somatropin (C990H1528N262O300S7) from the peak areas in the chromato-
36-77
grams obtained with the test solution and the reference solution and the declared content of
(C990H1528N262O300S7) in somatropin CRS.
STORAGE
Store in an airtight container at a temperature of 2C to 8C. If the substance is sterile, store in a
LABELLING
The label states:
(1) 1 mg of anhydrous somatropin (C
990H1528N262O300S7) is equivalent to 3.0 I.U. of biological
activity.
__________________________________________________________________________________________________________ Ph Eur
36-78
Somatropin Bulk Solution

Somatropin Bulk Solution complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Growth hormone.
Preparation
Somatropin Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Somatropin bulk solution is a solution containing a protein having the structure (191 amino-acid
residues) of the major component of growth hormone produced by the human pituitary. It may
contain buffer salts and other auxiliary substances. It contains not less than 91.0 per cent and not
more than 105.0 per cent of the amount of somatropin(1) (C990H1528N262O300S7) stated on the label.
Somatropin bulk solution complies with the requirements of the monograph on Recombinant DNA
technology, products of (0784).
PRODUCTION
Somatropin bulk solution is produced by a method based on recombinant DNA (rDNA) technology.
During the course of product development, it must be demonstrated that the manufacturing process
produces a product having a biological activity of at least 2.5 I.U. per milligram, using a validated
bioassay based on growth promotion and approved by the competent authority.
Somatropin bulk solution complies with the following additional requirements.
Host-cell-derived proteins The limit is approved by the competent authority.
Host-cell- and vector-derived DNA The limit is approved by the competent authority.
CHARACTERS
A clear or slightly turbid, colourless solution.
IDENTIFICATION
A. Examine the electrophoretograms obtained in the test for isoform distribution. In the electrophoretogram obtained with test solution (a), the principal band corresponds in position to that in the
electrophoretogram obtained with reference solution (a).
B. Examine the chromatograms obtained in the test for related proteins. The retention time of the
C. Examine by peptide mapping.
Test solution. Dilute the solution to be examined with 0.05M tris-hydrochloride buffer solution pH 7.5 R
so that it contains 2.0 mg/ml of somatropin, and transfer about 1.0 ml to a tube made from suitable
material such as polypropylene. Prepare a 1 mg/ml solution of trypsin for peptide mapping R in 0.05M
tris-hydrochloride buffer solution pH 7.5 R and add 30 l to the solution of the substance to be examined. Cap the tube and place in a water-bath at 37C for 4 h. Remove from the water-bath and stop
the reaction immediately, for example by freezing. If analysed immediately using an automatic
injector, maintain the temperature at 2C to 8C.
Note: If a 2 mg/ml somatropin concentration is not obtainable, a similar digest relationship (g of trypsin
per mg of somatropin) may be used.
7.5 R containing 2.0 mg/ml of somatropin and treat at the same time and in the same manner as for
the test solution.
gel for chromatography R (5 m to 10 m),
Mobile phase A. Dilute 1 ml of trifluoroacetic acid R to 1000 ml with water R,
Mobile phase B. To 100 ml of water R add 1 ml of trifluoroacetic acid R and dilute to 1000 ml with
acetonitrile for chromatography R,
following the elution conditions as described in the table below (if necessary, the gradient or the
temperature of the column may be modified to improve separation of the digest):
36-79
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
020
2040
4065
10080
020
8075
7550
2025
2550
6570
7071
7185
5020
5080
20100
100
800
0

Equilibrate the column with mobile phase A for at least 15 min. Carry out a blank run using the
above-mentioned gradient.
Inject 100 l of the test solution and 100 l of the reference solution. The test is not valid unless
the chromatogram obtained with each solution is qualitatively similar to the Ph. Eur. reference chromatogram of somatropin digest. The profile of the chromatogram obtained with the test solution corresponds to that of the chromatogram obtained with the reference solution.
D. Examine the chromatograms obtained in the assay. The retention time of the principal peak in the
TESTS
Related proteins Examine by liquid chromatography (2.2.29).
Test solution. Dilute the solution to be examined with 0.05M tris-hydrochloride buffer solution pH 7.5 R
so as to contain 2.0 mg/ml of somatropin or less, but then correct the injection volume accordingly.
Resolution solution (somatropin/desamido-somatropin resolution mixture). Prepare a solution of somatropin
CRS in 0.05M tris-hydrochloride buffer solution pH 7.5 R, containing 2.0 mg/ml of somatropin. Either
filter through a sterile filter or add sodium azide R to a concentration of 0.1 mg/ml and allow to stand
at room temperature for 24 h.
Maintain the solutions at 2C to 8C and use within 24 h. If an automatic injector is used, maintain the
temperature at 2C to 8C.
singly end-capped butylsilyl silica gel, with a granulometry of 5 m and a porosity of 30 nm. A
silica saturation column is to be placed between the pump and the injector valve,
as mobile phase at a flow rate of 0.5 ml/min a mixture of 29 volumes of propanol R and 71
volumes of 0.05M tris-hydrochloride buffer solution pH 7.5 R,
Prior to use, rinse the column with 200 ml to 500 ml of a 0.1 per cent V/V solution of trifluoroacetic
acid R in a 50 per cent V/V solution of acetonitrile R. Repeat as necessary, to improve column
performance.
Inject 20 l of the reference solution. If necessary, adjust the concentration of propanol R in the
mobile phase so that the retention time of the principal peak is about 33 min.
Inject 20 l of the resolution solution. Desamido-somatropin appears as a small peak at a retention
time of about 0.85 relative to the principal peak. The test is not valid unless the resolution between
the peaks corresponding to somatropin and desamido-somatropin is at least 1.0 and the symmetry
factor of the somatropin peak is 0.9 to 1.8.
Inject 20 l of the test solution. In the chromatogram obtained, the sum of the areas of all the
peaks, apart from the principal peak, is not greater than 6.0 per cent of the total area of the peaks.
Disregard any peak due to the solvent.
Dimer and related substances of higher molecular mass Examine by size-exclusion chromatography (2.2.30) as described under Assay.
Inject 20 l of the test solution. In the chromatogram obtained with the test solution, the sum of
the areas of any peak with a retention time less than that of the principal peak is not greater than
4.0 per cent of the total area of the peaks. Disregard any peak due to the solvent.
Isoform distribution Examine by isoelectric focusing.
Test solution (a). Dilute the solution to be examined with 0.025M phosphate buffer solution pH 7.0 R so
as to contain 2.0 mg/ml of somatropin.
Test solution (b). Add 0.1 ml of test solution (a) to 1.9 ml of 0.025M phosphate buffer solution pH 7.0 R.
Reference solution (a). Prepare a solution of somatropin CRS in 0.025M phosphate buffer solution pH
36-80
Reference solution (b). Use an isoelectric point calibration solution in the pH range of 2.5 to 6.5,
prepared and used according to the manufacturers instructions.
Operate the apparatus in accordance with the manufacturers instructions. The isoelectric focusing
procedure may be carried out using a pre-cast gel 245 mm 110 mm 1 mm, with a pH in the
range 4.0 to 6.5. Apply to the gel 15 l of each solution. Use as the anode solution a 14.7 g/l solution
of glutamic acid R in phosphoric acid (50 g/l H3PO4) and as the cathode solution an 89.1 g/l solution
of -alanine R. Adjust the operating conditions to 2000 V and 25 mA. Allow focusing to take place
for 2.5 h at a constant voltage and at a power of not more than 25 W. Immerse the gel for 30 min in
a solution containing 115 g/l of trichloroacetic acid R and 34.5 g/l of sulphosalicylic acid R, and then for
5 min in a mixture of 8 volumes of acetic acid R, 25 volumes of ethanol R and 67 volumes of deionised
water R (de-stain solution). Stain the gel by immersion in a 1.15 g/l solution of acid blue 83 R in destain solution at 60C for 10 min, and then place the gel in de-stain solution until excess stain is
removed.
The test is not valid unless the distribution of bands in the electrophoretogram obtained with
reference solution (b) corresponds to the manufacturers indications. The electrophoretogram
obtained with reference solution (a) contains a major band with an isoelectric point of approximately
five, and a slightly more acidic minor band at approximately 4.8. In the electrophoretogram obtained
with test solution (a), no band apart from the major band is more intense than the major band in the
electrophoretogram obtained with test solution (b) (5 per cent).
of endotoxin per milligram of somatropin.
ASSAY
Examine by size-exclusion chromatography (2.2.30).
Test solution. Dilute the solution to be examined with 0.025M phosphate buffer solution pH 7.0 R so as
to contain 1.0 mg/ml of somatropin.
Reference solution. Dissolve the contents of a vial of somatropin CRS in 0.025M phosphate buffer solution
pH 7.0 R and dilute with the same solvent to obtain a concentration of 1.0 mg/ml.
Resolution solution. Place one vial of somatropin CRS in an oven at 50C for a period (typically
between 12 h and 24 h) sufficient to generate 1 per cent to 2 per cent of dimer. Dissolve its contents
in 0.025M phosphate buffer solution pH 7.0 R and dilute with the same solvent to obtain a concentration of 1.0 mg/ml.
a stainless steel column 0.30 m long and 7.8 mm in internal diameter packed with hydrophilic
silica gel for chromatography R of a grade suitable for fractionation of globular proteins in the
molecular mass range 5000 to 150,000,
as mobile phase at a flow rate of 0.6 ml/min a mixture (filtered and degassed) consisting of 3
volumes of 2-propanol R and 97 volumes of 0.063M phosphate buffer solution pH 7.0 R,
Inject 20 l of the resolution solution. In the chromatogram obtained, the main peak elutes at a
retention time of approximately 12 min to 17 min and the peaks corresponding to the somatropin
dimer and to the higher molecular weight proteins at relative retention times of 0.90 and 0.65
respectively, relative to the main peak. The resolution, defined by the ratio of the height above the
baseline of the valley separating the monomer and dimer peaks to the height of the dimer peak, is not
greater than 0.4.
Calculate the content of somatropin (C990H1528N262O300S7) from the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of
(C990H1528N262O300S7) in somatropin CRS.
STORAGE
Store in an airtight container at a temperature of 20C. Avoid repeated freezing and thawing. If the
solution is sterile, store in a sterile, airtight, tamper-proof container.
LABELLING
The label states:
the content of somatropin in milligrams per millilitre,
the name and concentration of any auxiliary substance,
where applicable, that the solution is sterile,
where applicable, that the solution is free from bacterial endotoxins.
(1) 1 mg of anhydrous somatropin (C
990H1528N262O300S7) is equivalent to 3.0 I.U. of biological
activity.
__________________________________________________________________________________________________________ Ph Eur
36-81
Sorbic Acid
H
H3C
COOH
H
C6H8O2
H
112.1
22500-92-1
Sorbic Acid complies with the requirements of the 3rd edition of the European Pharmacopoeia [0592]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sorbic acid contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of (E,E)-hexa-2,4-dienoic acid, calculated with reference to the anhydrous substance.
CHARACTERS
A white or almost white, crystalline powder, slightly soluble in water, freely soluble in alcohol and in
ether.
IDENTIFICATION
B. Dissolve 50.0 mg in water R and dilute to 250.0 ml with the same solvent. Dilute 2.0 ml of this
solution to 200.0 ml with 0.1M hydrochloric acid. Examined between 230 nm and 350 nm (2.2.25),
obtained with sorbic acid CRS.
D. Dissolve 0.2 g in 2 ml of alcohol R and add 0.2 ml of bromine water R. The solution is decolorised.
TESTS
Aldehydes Dissolve 1.0 g in a mixture of 30 ml of water R and 50 ml of 2-propanol R, adjust the
solution to pH 4 with 0.1M hydrochloric acid or 0.1M sodium hydroxide and dilute to 100 ml with
water R. To 10 ml of the solution add 1 ml of decolorised fuchsin solution R and allow to stand for
30 min. Any colour in the solution is not more intense than that in a standard prepared at the same
time by adding 1 ml of decolorised fuchsin solution R to a mixture of 1.5 ml of acetaldehyde standard
solution (100 ppm C2H4O) R, 4 ml of 2-propanol R and 4.5 ml of water R (0.15 per cent, calculated as
C2H4O).
Heavy metals (2.4.8). 12 ml of solution S complies with limit test B for heavy metals (10 ppm).
Prepare the standard using 5 ml of lead standard solution (1 ppm Pb) R and 5 ml of alcohol R.
ASSAY
Dissolve 0.1000 g in 20 ml of alcohol R. Using 0.2 ml of phenolphthalein solution R as indicator, titrate
with 0.1M sodium hydroxide until a pink colour is obtained.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
36-82
Sorbitan Laurate
1338-39-2
Sorbitan Laurate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1040].
When sorbitan monolaurate is demanded, Sorbitan Laurate shall be supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sorbitan laurate is a mixture usually obtained by partial esterification of lauric acid with sorbitol and
its mono- and di-anhydrides.
CHARACTERS
A brownish-yellow, viscous liquid, practically insoluble but dispersible in water, miscible with
alcohol, slightly soluble in cotton-seed oil.
IDENTIFICATION
C. To 4 g add 40 ml of a 50 g/l solution of potassium hydroxide R and boil under a reflux condenser
condenser for about 10 min to break the emulsion. Fatty acids separate at the surface as an oily layer.
Allow to cool. Transfer to a separating funnel and extract the fatty acids with 50 ml of light
petroleum R1, avoiding vigorous shaking. Dry the upper layer with 1 g of anhydrous sodium sulphate R
and filter. Heat on a water-bath until the odour of the solvent is no longer perceptible. The acid value
TESTS
Iodine value (2.5.4). Not more than 10.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
36-83
Sorbitan Oleate
1338-43-8
Sorbitan Oleate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1041].
When sorbitan mono-oleate is demanded, Sorbitan Oleate shall be supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sorbitan oleate is a mixture usually obtained by partial esterification of oleic acid with sorbitol and its
mono- and di-anhydrides.
CHARACTERS
A brownish-yellow, viscous liquid, practically insoluble but dispersible in water, soluble in fatty oils
producing a hazy solution, slightly soluble in ether, miscible with alcohol.
IDENTIFICATION
TESTS
Hydroxyl value (2.5.3). 190 to 210 (Method A), determined on 2.0 g.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
36-84
Sorbitan Palmitate
26266-57-9
Sorbitan Palmitate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1042].
When sorbitan monopalmitate is demanded, Sorbitan Palmitate shall be supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sorbitan palmitate is a mixture usually obtained by partial esterification of palmitic acid with sorbitol
and its mono- and di-anhydrides.
CHARACTERS
A yellowish or yellow powder, waxy flakes or hard masses, practically insoluble in water, soluble in
fatty oils, slightly soluble in alcohol.
IDENTIFICATION
A. Melting point (2.2.15): 44C to 51C. Introduce the melted substance into the glass capillary
tubes and allow to stand at a temperature below 10C for 24 h.
B. It complies with the test for hydroxyl value (see Tests).
TESTS
STORAGE
__________________________________________________________________________________________________________ Ph Eur
36-85
Sorbitan Stearate
1338-41-6
Sorbitan Stearate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1043].
When sorbitan monostearate is demanded, Sorbitan Stearate shall be supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sorbitan stearate is a mixture usually obtained by partial esterification of stearic acid with sorbitol and
its mono- and di-anhydrides.
CHARACTERS
A pale yellow, waxy solid, practically insoluble but dispersible in water, slightly soluble in alcohol.
IDENTIFICATION
A. Melting point (2.2.15): 50C to 55C. Introduce the melted substance into the glass capillary
tubes and allow to stand at a temperature below 10C for 24 h.
B. It complies with the test for hydroxyl value (see Tests).
TESTS
STORAGE
__________________________________________________________________________________________________________ Ph Eur
37-1
Sorbitan Trioleate
26266-58-0
Sorbitan Trioleate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1044].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sorbitan trioleate is a mixture usually obtained by partial esterification of oleic acid with sorbitol and
its mono-anhydride.
CHARACTERS
A pale yellow, light yellowish or brown solid which becomes a viscous, oily, brownish-yellow liquid at
about 25C, practically insoluble but dispersible in water, freely soluble in ether, soluble in fatty oils,
slightly soluble in alcohol.
IDENTIFICATION
TESTS
of water.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
37-2
Sorbitol
1/01
CH 2OH
H
OH
HO
OH
OH
CH 2OH
C6H14O6
182.2
50-70-4
Sorbitol complies with the requirements of the 3rd edition of the European Pharmacopoeia [0435]. These
Action and use Used for parenteral nutrition.
Preparation
Sorbitol Intravenous Infusion
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sorbitol contains not less than 97.0 per cent and not more than the equivalent of 102.0 per cent of
D-glucitol (D-sorbitol), calculated with reference to the anhydrous substance.
CHARACTERS
A white or almost white, crystalline powder, very soluble in water, practically insoluble in alcohol.
IDENTIFICATION
obtained with sorbitol CRS. Examine the substances prepared as discs. If the spectra obtained show
differences, dissolve the substance to be examined and the reference substance separately in water R,
B. Dissolve 0.5 g with heating in a mixture of 0.5 ml of pyridine R and 5 ml of acetic anhydride R.
After 10 min, pour the solution into 25 ml of water R and allow to stand in iced water for 2 h. The
precipitate, recrystallised from a small volume of alcohol R and dried in vacuo, melts (2.2.14) at 98C
to 104C.
same solvent.
Reference solution (a). Dissolve 25 mg of sorbitol CRS in water R and dilute to 10 ml with the same
solvent.
D. Dissolve 5.00 g of the substance to be examined and 6.4 g of disodium tetraborate R in 40 ml of
water R. Allow to stand for 1 h, shaking occasionally, and dilute to 50.0 ml with water R. Filter if
necessary. The specific optical rotation (2.2.7) is +4.0 to +7.0, calculated with reference to the
37-3
TESTS
Appearance of solution Dissolve 5 g in water R and dilute to 50 ml with the same solvent. The
to sorbitol is at least 50 per cent of the full scale of the recorder. Inject 20 l of the test solution and
20 l of reference solution (c) and continue the chromatography for twice the retention time of
sorbitol. In the chromatogram obtained with the test solution: the area of any peak, apart from the
reference solution (b) (2 per cent); the sum of the areas of all the peaks, apart from the principal
reference solution (b) (3 per cent). Disregard any peak with an area less than the area of the principal
peak in the chromatogram obtained with reference solution (c) (0.1 per cent).
of water.
Microbial contamination If intended for use in the manufacture of parenteral dosage forms: the
by plate count; it complies with the tests for Escherichia coli and Salmonella (2.6.13).
sorbitol, and not more than 2.5 I.U. of endotoxin per gram for parenteral dosage forms having a
concentration of 100 g/l or more of sorbitol.
ASSAY
Reference solution (a). Dissolve 0.5 g of sorbitol CRS in 2 ml of water R and dilute to 10.0 ml with the
same solvent.
Reference solution (d). Dissolve 0.5 g of sorbitol CRS and 0.5 g of mannitol CRS in 5 ml of water R and
exchange resin (calcium form) R (9 m) and maintained at a temperature of 85C 1C,
as detector, a refractometer maintained at a constant temperature.
time of sorbitol.
When the chromatograms are recorded in the prescribed conditions, the retention time of sorbitol
is about 27 min and the relative retentions with reference to sorbitol are: maltitol about 0.6, mannitol
about 0.8 and iditol about 1.1. The test is not valid unless the resolution between the peaks due to
sorbitol and to mannitol is at least 2 in the chromatogram obtained with reference solution (d).
for twice the retention time of sorbitol.
37-4
Calculate the percentage content of D-sorbitol from the areas of the peaks and the declared content
of sorbitol CRS.
LABELLING
The label states:
forms.
IMPURITIES
A. mannitol,
OH
O
OH
OH
OH
B. iditol,
C. maltitol.
__________________________________________________________________________________________________________ Ph Eur
37-5
Liquid Sorbitol (Crystallising)

Sorbitol Solution (70 per cent) (Crystallising)
1/01
Liquid Sorbitol (Crystallising) complies with the requirements of the 3rd edition of the European Pharmacopoeia [0436]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Liquid sorbitol (crystallising) is an aqueous solution of a hydrogenated, partly hydrolysed starch. It
contains not less than 68.0 per cent m/m and not more than 72.0 per cent m/m of anhydrous
substance. It contains not less than 92.0 per cent and not more than the equivalent of 101.0 per cent
of D-glucitol (D-sorbitol, C6H14O6), calculated with reference to the anhydrous substance.
CHARACTERS
A clear, colourless, syrupy liquid, miscible with water, miscible with alcohol.
IDENTIFICATION
B. To 7.0 g add 40 ml of water R and 6.4 g of disodium tetraborate R, allow to stand for 1 h, shaking
occasionally, and dilute to 50.0 ml with water R. Filter if necessary. The angle of rotation (2.2.7) is 0
to +1.5.
C. Dry 1 g in vacuo at 80C. Dissolve 0.5 g of the residue with heating in a mixture of 0.5 ml of
pyridine R and 5 ml of acetic anhydride R. After 10 min, pour the mixture into 25 ml of water R and
allow to stand in iced water for 2 h. The precipitate, recrystallised from a small volume of alcohol R
and dried in vacuo, melts (2.2.14) at 98C to 104C.
Test solution. Dilute 70 mg of the substance to be examined to 20 ml with water R.
solvent.
principal spot in the chromatogram obtained with the reference solution (a). The test is not valid
E. It is a clear, syrupy liquid at a temperature of 25C.
TESTS
Appearance of solution Dilute 7.0 g to 50 ml with water R. The solution is clear (2.2.1) and
Conductivity (2.2.38). Not more than 10 Scm1 measured at a temperature of 20C on the
undiluted liquid sorbitol (crystallising) while gently stirring with a magnetic stirrer.
37-6
Water (2.5.12). 28.0 per cent to 32.0 per cent m/m, determined on 0.1 g by the semi-micro determination of water.
ASSAY
Test solution. Mix 1.00 g of the substance to be examined with 20 ml of water R and dilute to 50.0 ml
Reference solution (a). Dissolve 65.0 mg of sorbitol CRS in 2 ml of water R and dilute to 5.0 ml with
the same solvent.
Reference solution (b). Dissolve 65 mg of mannitol CRS and 65 mg of sorbitol CRS in 2 ml of water R
time of sorbitol.
When the chromatogram is recorded in the prescribed conditions, the retention time of sorbitol is
about 27 min and the relative retention of mannitol with reference to sorbitol is about 0.8. The test is
not valid unless the resolution between the peaks due to sorbitol and to mannitol is at least 2 in the
for three times the retention time of sorbitol.
of sorbitol CRS.
__________________________________________________________________________________________________________ Ph Eur
37-7
Liquid Sorbitol (Non-crystallising)

Sorbitol Solution (70 per cent) (Non-crystallising)
1/01
Liquid Sorbitol (Non-crystallising) complies with the requirements of the 3rd edition of the European
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Liquid sorbitol (non crystallising) is an aqueous solution of a hydrogenated, partly hydrolysed starch.
It contains not less than 68.0 per cent m/m and not more than 72.0 per cent m/m of anhydrous
substance. It contains not less than 72.0 per cent and not more than the equivalent of 92.0 per cent
of D-glucitol (D-sorbitol, C6H14O6), calculated with reference to the anhydrous substance.
CHARACTERS
A clear, colourless, syrupy liquid, miscible with water and with alcohol.
IDENTIFICATION
B. To 7.0 g add 40 ml of water R and 6.4 g of disodium tetraborate R. Allow to stand for 1 h, shaking
occasionally, and dilute to 50.0 ml with water R. Filter if necessary. The angle of rotation (2.2.7) is
+1.5 to +3.5.
C. Dry 1 g in vacuo at 80C. Dissolve 0.5 g of the residue with heating in a mixture of 0.5 ml of
pyridine R and 5 ml of acetic anhydride R. After 10 min, pour the mixture into 25 ml of water R and
allow to stand in iced water for 2 h. The precipitate, recrystallised from a small volume of alcohol R
and dried in vacuo, melts (2.2.14) at 98C to 104C.
Test solution. Dilute 70 mg of the substance to be examined to 20 ml with water R.
solvent.
E. It is a clear, syrupy liquid at a temperature of 25C.
TESTS
Appearance of solution Dilute 7.0 g to 50 ml with water R. The solution is clear (2.2.1) and
Conductivity (2.2.38). Not more than 10 Scm-1 measured at a temperature of 20C on the
undiluted liquid sorbitol (non crystallising) while gently stirring with a magnetic stirrer.
Reducing sugars after hydrolysis To 6.0 g add 35 ml of water R, 40 ml of 1M hydrochloric acid and
a few glass beads. Boil under a reflux condenser for 4 h. Cool and neutralise to bromothymol blue by
adding dilute sodium hydroxide solution R. Cool and dilute to 100.0 ml with water R. To 3.0 ml of the
37-8
solution add 5 ml of water R, 20 ml of cupri-citric solution R and a few glass beads. Heat so that boiling
begins after 4 min and maintain boiling for 3 min. Cool rapidly and add 100 ml of a 2.4 per cent V/V
solution of glacial acetic acid R and 20.0 ml of 0.025M iodine. With continuous shaking, add 25 ml of a
mixture of 6 volumes of hydrochloric acid R and 94 volumes of water R. When the precipitate has
dissolved, titrate the excess of iodine with 0.05M sodium thiosulphate using 1 ml of starch solution R,
added towards the end of the titration, as indicator. Not less than 8.0 ml of 0.05M sodium thiosulphate
is required (9.3 per cent calculated as glucose equivalent).
Water (2.5.12). Not less than 28.0 per cent and not more than 32.0 per cent m/m, determined on
0.1 g by the semi-micro determination of water.
ASSAY
Test solution. Mix 1.00 g of the substance to be examined with 20 ml of water R and dilute to 50.0 ml
Reference solution (a). Dissolve 55.0 mg of sorbitol CRS in 2 ml of water R and dilute to 5.0 ml with
the same solvent.
Reference solution (b). Dissolve 55 mg of mannitol CRS and 55 mg of sorbitol CRS in 2 ml of water R
exchange resin (calcium form) R (9 m) and maintained at 85 1C,
time of sorbitol.
When the chromatogram is recorded in the prescribed conditions, the retention time of sorbitol is
about 27 min and the relative retention of mannitol with reference to sorbitol is about 0.8. The test is
not valid unless the resolution between the peaks due to sorbitol and to mannitol is not less than 2 in
for three times the retention time of sorbitol.
of sorbitol CRS.
__________________________________________________________________________________________________________ Ph Eur
37-9
Sotalol Hydrochloride
H
OH
NHPri
,HCl
S
H3C
N
H
and enantiomer
C12H20N2O3S,HCl
308.8
959-24-0
Definition Sotalol Hydrochloride is (RS)-4-(1-hydroxy-2-isopropylaminoethyl)methanesulphonanilide hydrochloride. It contains not less than 99.0% and not more than 101.0% of
C12H20N2O3S,HCl, calculated with reference to the dried substance.
Freely soluble in water; slightly soluble in ethanol (96%); practically insoluble in chloroform.
spectrum of sotalol hydrochloride (RS 319).
phase. For solution (2) dilute 1 volume of solution (1) to 100 volumes with the mobile phase and
further dilute 1 volume of this solution to 3 volumes with the mobile phase. Solution (3) contains
0.0008% w/v of 4-(2-isopropylaminoethyl)methanesulphonanilide hydrochloride BPCRS in the mobile
phase.
(20 cm 4.6 mm) packed with stationary phase C (10 m) (Spherisorb ODS 2 is suitable), (b) as the
mobile phase with a flow rate of 1.5 ml per minute a mixture of 210 volumes of acetonitrile and 790
volumes of water containing 2 g per litre of sodium octanesulphonate the pH of the mixture being
adjusted to 3.0 with orthophosphoric acid and (c) a detection wavelength of 228 nm.
In the chromatogram obtained with solution (1) the area of any peak corresponding to 4-(2isopropylaminoethyl)methanesulphonanilide is not greater than the area of the peak obtained in the
chromatogram obtained with solution (3) (0.4%), the area of any other secondary peak is not greater
than the area of the peak in the chromatogram obtained with solution (2) (0.3%) and the sum of the
areas of any secondary peaks is not more than 1.65 times the area of the peak in the chromatogram
obtained with solution (2) (0.5%).
weight. Use 2 g.
Assay Dissolve 0.25 g in a mixture of 50 ml of glacial acetic acid and 15 ml of mercury(II) acetate
solution and carry out Method I for non-aqueous titration, Appendix VIII A, determining the end point
potentiometrically. Each ml of 0.1M perchloric acid VS is equivalent to 30.88 mg of
C12H20N2O3S,HCl.
Action and use Beta-adrenoreceptor antagonist.
Preparations
Sotalol Injection
Sotalol Tablets
IMPURITIES
A. 4-(2-isopropylaminoethyl)methanesulphonanilide hydrochloride,
B. 4-(isopropylaminoacetyl)methanesulphonanilide hydrochloride,
C. 4-formylmethanesulphonanilide.
37-10
Hydrogenated Soya Oil

Hydrogenated Soyabean Oil
Hydrogenated Soya Oil complies with the requirements of the 3rd edition of the European Pharmacopoeia
[1265] for Hydrogenated Soya-Bean Oil . These requirements are reproduced after the heading Definition
below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Hydrogenated soya-bean oil is the product obtained by refining, bleaching, hydrogenation and
deodorisation of oil obtained from seeds of Glycine soja Sieb. and Zucc. and Glycine max (L.) Merr.
(G. hispida (Moench) Maxim.). The product consists mainly of triglycerides of palmitic and stearic
acids.
CHARACTERS
A white mass or powder which melts to a clear, pale yellow liquid when heated, practically insoluble
in water, freely soluble in methylene chloride, in light petroleum (bp: 65C to 70C) after heating
and in toluene, very slightly soluble in alcohol.
IDENTIFICATION
B. It complies with the test for foreign fatty oils (see Tests).
TESTS
Melting point (2.2.15) 66C to 72C.
Acid value (2.5.1). Not more than 0.5, determined on 10.0 g. Dissolve the substance to be examined in 50 ml of a hot mixture of equal volumes of alcohol R and toluene R, previously neutralised with
0.1M potassium hydroxide using 0.5 ml of phenolphthalein solution R1 as indicator. Titrate the solution
immediately while still hot.
Alkaline impurities in fatty oils (2.4.19). Dissolve 2.0 g with gentle heating in a mixture of 1.5 ml
of alcohol R and 3 ml of toluene R. Add 0.05 ml of a 0.4 g/l solution of bromophenol blue R in alcohol R.
Not more than 0.4 ml of 0.01M hydrochloric acid is required to change the colour to yellow.
Foreign fatty oils Carry out the test for foreign oils in fatty oils by gas chromatography (2.4.22).
a fused-silica column 25 m long and 0.25 mm in internal diameter coated with poly(cyanopropyl)siloxane R (film thickness 0.2 m),
maintaining the temperature of the column at 180C for 20 min and that of the injection port and
The fatty acid fraction of the oil has the following composition:
myristic acid: not more than 0.5 per cent,
oleic acid and isomers (C18:1 equivalent chain length on poly(cyanopropyl)siloxane 18.5 to
18.8): not more than 4.0 per cent,
linoleic acid and isomers (C18:2 equivalent chain length on poly(cyanopropyl)siloxane 19.4 to
linolenic acid and isomers (C18:3 equivalent chain length on poly(cyanopropyl)siloxane 20.3 to
behenic acid: not more than 1.0 per cent.
Nickel Not more than 1 ppm of Ni, determined by atomic absorption spectrometry (Method II,
2.2.23).
Test solution. Introduce 5.0 g into a platinum or silica crucible, previously tared after calcination.
Cautiously heat and introduce into the substance a wick formed from twisted ashless filter paper.
37-11
Light the wick. When the substance is alight stop heating. After combustion, ignite in a muffle
furnace at about 600C. Continue the ignition until white ash is obtained. After cooling, take up the
residue with two quantities, each of 2 ml, of dilute hydrochloric acid R and transfer into a 25 ml
graduated flask. Add 0.3 ml of nitric acid R and dilute to 25.0 ml with water R.
Reference solutions. Prepare three reference solutions by adding 1.0 ml, 2.0 ml and 4.0 ml of nickel
standard solution (0.2 ppm Ni) R to 2.0 ml of the test solution and diluting to 10.0 ml with water R.
Measure the absorbance at 232 nm using a nickel hollow-cathode lamp as a source of radiation, a
graphite furnace as an atomic generator and argon R as the carrier gas.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
37-12
Refined Soya Oil

Refined Soyabean Oil
corrected 1/01
Refined Soya Oil complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Refined Soya-bean Oil [1473]. These requirements are reproduced after the heading Definition below.
When Soya Oil, Soyabean Oil or Soya-bean Oil is demanded, Refined Soya Oil shall be supplied.
When intended for use in the manufacture of a parenteral dosage form, Refined Soya Oil complying
with the requirement below for Water should be used.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Refined soya-bean oil is the fatty oil obtained from seeds of Glycine soja Sieb. and Zucc. and Glycine
max (L.) Merr. (G. hispida (Moench) Maxim.) by extraction and subsequent refining. It may contain
a suitable antioxidant.
CHARACTERS
A clear, pale yellow, liquid, miscible with light petroleum (50C to 70C), practically insoluble in
alcohol.
IDENTIFICATION
obtained is similar to the typical chromatogram for soya-bean oil.
TESTS
Peroxide value (2.5.5, Method A). Not more than 10.0. If intended for use in the manufacture of
parenteral dosage forms, the peroxide value is not more than 5.0.
(2.4.22, Method A). The fatty acid fraction of the oil has the following composition:
0.3 per cent,
cent,
58.0 per cent,
linolenic acid (equivalent chain length on polyethyleneglycol adipate 19.7): 5.0 per cent to
11.0 per cent,
1.0 per cent,
behenic acid: not more than 1.0 per cent.
Brassicasterol (2.4.23). The sterol fraction of the oil contains not more than 0.3 per cent of
brassicasterol.
0.1 per cent, determined on 5.00 g by the coulometric determination of water. Use a mixture of
equal volumes of decanol R and anhydrous methanol R as the solvent.
STORAGE
Store in a well-filled container, protected from light, at a temperature not exceeding 25C.
LABELLING
The label states:
37-13
forms,
__________________________________________________________________________________________________________ Ph Eur
37-14
Spearmint Oil
Definition Spearmint Oil is obtained by distillation from fresh flowering plants of Mentha spicata L.
and Mentha cardiaca (Gray) Bak.
Characteristics A clear, colourless, pale yellow or greenish yellow liquid when recently distilled, but
becoming darker and viscous on keeping; visibly free from water; odour, that of spearmint.
Identification Examine the chromatograms obtained in the test for Chromatographic profile. The
retention times of the principal peaks in the chromatogram obtained with solution (1) are similar to
those of the principal peaks in the chromatogram obtained with solution (2).
Optical rotation American-type oil, 45 to 60; Chinese-type oil, 50 to 62; Appendix V F.
Solubility in ethanol Soluble, at 20, in 1 part of ethanol (80%), Appendix X M. The solution may
become cloudy when diluted.
Weight per ml American-type oil, 0.917 to 0.934 g; Chinese-type oil, 0.935 to 0.952 g, Appendix
V G.
Chromatographic profile Carry out the method for gas chromatography, Appendix III B, using the
following solutions. Solution (1) is the substance being examined. For solution (2) mix carefully 0.1 g
of limonene, 0.2 g of cineole, 0.4 g of menthone, 0.1 g of (+)-isomenthone, 0.4 g of menthyl acetate, 0.2 g
of pulegone, 0.6 g of menthol and 0.1 g of carvone with 1 g of hexane.
The chromatographic procedure may be carried out using (a) a glass capillary column (25 m to
60 m about 0.25 mm) coated with polyethylene glycol 20,000 as bonded phase (Carbowax 20M is
suitable) and (b) helium as the carrier gas at a flow rate of 1.5 ml per minute. Maintain the temperature of the column at 55 for 6 minutes then increase it at the rate of 4 per minute to 180; keep the
injection port temperature at 220 and the detector at 230.
Inject 0.1 l of solution (2). When the chromatograms are recorded in the prescribed conditions,
the components elute in the order indicated in the composition of the reference solution. Record the
retention times of these substances. The test is not valid unless the number of theoretical plates calculated from the limonene peak is at least 30,000 and the resolution factor between the peaks corresponding to limonene and cineole is at least 1.5.
Inject 0.1 l of solution (1). Using the retention times determined from the chromatogram
obtained with solution (2) locate the components of the reference solution on the chromatogram
obtained with solution (1) (disregard the peak due to hexane). Determine the percentage content of
the components by normalisation. The percentages are within the following ranges:
Limonene 2.0 to 25.0%.
Cineole less than 2.5%.
Menthone less than 2.5%.
Isomenthone less than 1.0%.
Menthyl acetate less than 1.0%.
Pulegone less than 0.5%.
Menthol less than 2.0%.
Carvone Not less than 55.0%.
Storage Spearmint Oil should be kept in a well-filled, well-closed container, protected from light and
Labelling The label states whether the oil is American-type oil or Chinese-type oil.
37-15
Spectinomycin Hydrochloride
HO
H
H
MeHN
Me
,2HCl
HO
H
HO
MeHN
C14H24N2O7,2HCl,5H2O
O
495.4
22189-32-8
Spectinomycin Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia [1152]. These requirements are reproduced after the heading Definition below.
Preparation
Spectinomycin Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Spectinomycin hydrochloride is the pentahydrate of the dihydrochloride of
(2R,4aR,5aR,6S,7S,8R,9S,9aR,10aS)-perhydro-4a,7,9-trihydroxy-2-methyl-6,8-bis(methylamino)pyrano[2,3-b][1,4]benzodioxin-4-one, an antimicrobial substance produced by Streptomyces
spectabilis or by any other means. It contains not less than 95.0 per cent and not more than the equivalent of 100.5 per cent of C14H26Cl2N2O7 calculated with reference to the anhydrous substance.
CHARACTERS
A white or almost white powder, slightly hygroscopic, freely soluble in water, very slightly soluble in
IDENTIFICATION
obtained with spectinomycin hydrochloride CRS.
B. Dilute 1.0 ml of solution S (see Tests) to 10 ml with water R. The solution gives reaction (a) of
chlorides (2.3.1).
TESTS
Appearance of solution Dilute 2.0 ml of solution S to 20.0 ml with water R. The solution is clear
Specific optical rotation (2.2.7). +15 to +21, determined on solution S within 20 min of
preparation and calculated with reference to the anhydrous substance.
coating substance.
Test solution. Dilute 2.0 ml of solution S to 10 ml with water R.
5 volumes of glacial acetic acid R, 5 volumes of pyridine R, 40 volumes of water R and 50 volumes of
propanol R. Dry the plate in a current of warm air and spray with a 50 g/l solution of potassium
permanganate R. Allow the plate to stand for 2 min to 3 min. Any spot in the chromatogram obtained
with the test solution, apart from the principal spot, is not more intense than the spot in the chromatogram obtained with the reference solution (1.0 per cent).
37-16
0.09 I.U. of endotoxin per milligram. Prepare the solutions using a 0.42 per cent m/m solution of
sodium hydrogen carbonate R.
ASSAY
Examine by gas chromatography (2.2.28), using phenazone R as the internal standard.
Internal standard solution. Dissolve 0.150 g of phenazone R in dimethylformamide R and dilute to
Allow the test solutions and the reference solution to stand for 1 h. Carry out the assay immediately.
Test solution (a). To 60.0 mg of the substance to be examined in a volumetric flask, add 10 ml of
dimethylformamide R and 2.0 ml of hexamethyldisilazane R. Shake to dissolve and dilute to 20.0 ml
with dimethylformamide R.
Test solution (b). To 60.0 mg of the substance to be examined in a volumetric flask add 10.0 ml of
the internal standard solution and 2.0 ml of hexamethyldisilazane R. Shake to dissolve and dilute to
20.0 ml with dimethylformamide R.
Reference solution. To 60.0 mg of spectinomycin hydrochloride CRS in a volumetric flask add 10.0 ml of
the internal standard solution and 2.0 ml of hexamethyldisilazane R. Shake to dissolve and dilute to
20.0 ml with dimethylformamide R.
earth for gas chromatography R (125 m to 150 m) impregnated with 3 per cent m/m of a mixture
of polyphenylmethylsiloxane R,
an electronic integrator.
Maintain the temperature of the column at 200C and that of the injection port and of the detector
between 200C and 230C. Inject the chosen volumes of the test solutions. The assay is not valid
unless in the chromatogram obtained with test solution (b) the resolution between the main peak and
the peak corresponding to the internal standard is at least 8.0. Inject alternately test solution (b) and
Calculate the percentage content of spectinomycin.
STORAGE
Store in an airtight container, at a temperature not exceeding 30C. If the substance is sterile, store in
a sterile, airtight, tamper-proof container.
LABELLING
The label states:
IMPURITIES
A. actinamine,
B. actinospectinoic acid,
C. dihydrospectinomycins,
D. dihydroxyspectinomycin.
__________________________________________________________________________________________________________ Ph Eur
37-17
Spironolactone
O
Me
O
Me
H
H
SAc
H
C24H32O4S
416.6
52-01-7
Spironolactone complies with the requirements of the 3rd edition of the European Pharmacopoeia [0688].
Preparation
Spironolactone Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Spironolactone contains not less than 97.0 per cent and not more than the equivalent of 102.0 per
cent of 7-acetylthio-17-hydroxy-3-oxo-pregn-4-ene-21-carboxylic acid -lactone, calculated with
CHARACTERS
A white or yellowish-white powder, practically insoluble in water, soluble in alcohol, slightly soluble
in ether.
IDENTIFICATION
obtained with spironolactone CRS. Examine the substances as 50 g/l solutions in chloroform R.
Reference solution. Dissolve 20 mg of spironolactone CRS in methylene chloride R and dilute to 10 ml
volume of water R, 24 volumes of cyclohexane R and 75 volumes of ethyl acetate R. Allow the plate to
obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution.
C. To about 10 mg add 2 ml of a 50 per cent V/V solution of sulphuric acid R and shake. An orange
solution with an intense yellowish-green fluorescence is produced. Heat the solution gently; the
colour becomes deep red and hydrogen sulphide, which blackens lead acetate paper R, is evolved. Add
the solution to 10 ml of water R; a greenish-yellow solution is produced which shows opalescence or a
precipitate.
TESTS
same solvent. The specific optical rotation is 33 to 37, calculated with reference to the dried
substance.
Reference solution (b). Dissolve 25.0 mg of canrenone CRS in 1 ml of tetrahydrofuran R and dilute to
37-18
Reference solution (d). Mix 1 ml of the test solution with 1 ml of reference solution (b) and dilute to
100 ml with the mobile phase.
Reference solution (e). Dilute 0.50 ml of reference solution (a) to 10.0 ml with the mobile phase.
a mixture of 8 volumes of acetonitrile R, 18 volumes of tetrahydrofuran R and 74 volumes of
water R as the mobile phase at a flow rate of 1.8 ml per minute,
a variable wavelength spectrophotometer capable of operating at 254 nm and at 283 nm as
detector.
Inject separately 20 l of the test solution and 20 l of reference solution (a), 20 l of reference
solution (d) and 20 l of reference solution (e) and record the chromatograms with the detector set
for recording at 254 nm. Continue the chromatography for twice the retention time of spironolactone. In the chromatogram obtained with the test solution, the sum of the areas of the peaks,
except those corresponding to spironolactone and canrenone, is not greater than the area of the peak
corresponding to spironolactone in the chromatogram obtained with reference solution (a) (1.0 per
cent). Disregard any peak whose area is less than that of the principal peak in the chromatogram
obtained with reference solution (e). Inject 20 l of the test solution and 20 l of reference solution
(c) and record the chromatogram with the detector set for recording at 283 nm. In the chromatogram obtained with the test solution, the area of any peak corresponding to canrenone is not greater
than the area of the peak corresponding to canrenone in the chromatogram obtained with reference
solution (c) (1.0 per cent). Calculate the percentage content of canrenone found when recording at
283 nm and the percentage content of the other related substances found when recording at 254 nm.
Add together these percentages. The sum is not greater than 1.0 per cent. The test is not valid unless:
the chromatogram obtained with reference solution (d) shows peaks, corresponding to canrenone
and spironolactone, with a resolution greater than 1.4; the principal peak in the chromatogram
obtained with reference solution (e) has a signal-to-noise ratio of at least 6.
Free mercapto compounds To 2.0 g add 20 ml of water R, shake for 1 min and filter. To 10 ml of
the filtrate add 0.05 ml of 0.01M iodine and 0.1 ml of starch solution R and mix. A blue colour
develops.
Chromium To 0.20 g in a platinum crucible add 1 g of potassium carbonate R and 0.3 g of potassium
nitrate R. Heat gently until fused, and ignite at 600C to 650C until carbon is removed. Cool,
dissolve the residue as completely as possible in 10 ml of water R with the aid of gentle heat, filter,
and dilute to 20 ml with water R. To 10 ml of this solution add 0.5 g of urea R, and add a 14 per cent
V/V solution of sulphuric acid R until the solution is just acid. When effervescence ceases, add a
further 1 ml of the sulphuric acid, dilute to 20 ml with water R and add 0.5 ml of diphenylcarbazide
solution R. The solution is not more intensely coloured than a standard prepared by adding 1 ml of a
14 per cent V/V solution of sulphuric acid R to 0.50 ml of a freshly prepared 28.3 mg/l solution of
potassium dichromate R, diluting to 20 ml with water R and adding 0.5 ml of diphenylcarbazide solution R (50 ppm).
100C to 105C for 3 h.
ASSAY
Dissolve 50.0 mg in methanol R and dilute to 250.0 ml with the same solvent. Dilute 5.0 ml of this
solution to 100.0 ml with methanol R. Measure the absorbance (2.2.25) of the solution at the maximum at 238 nm.
Calculate the content of C24H32O4S, taking the specific absorbance to be 470.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
37-19
Squill
Definition Squill consists of the bulb of Drimia maritima (L.) Stearn, collected soon after the plant
has flowered, divested of its dry, outer, membranous coats, cut into transverse slices and dried. It is
known in commerce as white squill.
Characteristics Odourless or almost odourless.
Macroscopical Transverse slices, about 5 to 8 mm thick, occurring as straight or curved triangular
pieces about 5 to 50 mm long and 3 to 8 mm wide at mid-point, tapering towards each end,
yellowish white, texture horny, somewhat translucent, breaking with an almost glassy fracture when
quite dry, but readily absorbing moisture when exposed to the air and becoming tough and flexible;
transversely cut surface showing a single row of prominent, vascular bundles near the concave edge
and numerous smaller bundles scattered throughout the mesophyll.
Microscopical Epidermis: cells polygonal and axially elongated, 1 to 2 times longer than wide, cuticle
thick, stratified; stomata very rare, anomocytic, Appendix XI H, and nearly circular in outline, about
50 to 60 m in diameter; mesophyll of colourless, thin-walled parenchyma containing very occasional
starch granules, many cells containing bundles of acicular crystals of calcium oxalate embedded in
mucilage, crystals up to about 1 mm long and about 1 to 15 m wide; other cells containing sinistrin;
vascular bundles collateral, scattered throughout the mesophyll; xylem vessels with spiral and annular
wall thickening; trichomes absent.
Identification The mucilage contained in the cells of the mesophyll is stained red with alkaline
corallin solution but produces no red colour with ruthenium red solution and no purple colour with
0.01M iodine.
Acid-insoluble ash Not more than 1.5%, Appendix XI K, Method I.
Extractive soluble in ethanol (60%) Not less than 68.0%, Appendix XI B. Use material that has
been dried for 1 hour at 105 and powdered.
Storage Squill should be stored in a dry place at a temperature not exceeding 25.
Preparations
Squill Liquid Extract
Squill Oxymel
When Powdered Squill is prescribed or demanded, material complying with the appropriate requirements above shall be dispensed or supplied.
37-20
Indian Squill
Definition Indian Squill consists of the bulb of Drimia indica (Roxb.) J P Jessop, collected soon after
the plant has flowered, divested of dry, outer membranous coats and usually cut longitudinally into
slices and dried.
Characteristics Odourless or almost odourless.
Macroscopical Curved or irregularly shaped strips, about 10 to 50 mm long, 3 to 10 mm wide and 1
to 3 mm thick, frequently tapering towards the ends, occasionally grouped three or four together and
attached to a portion of the axis; ridged in the direction of their length and varying in colour from
pale yellowish brown to buff; brittle when dry, but tough and flexible when exposed to air.
Microscopical Epidermis: cells tetrahedral to hexahedral, thin-walled, three to five times longer than
wide, having a thick, striated cuticle; stomata rare, anomocytic, Appendix XI H, circular in outline, 40
to 42 m in diameter; mesophyll of thin-walled polygonal cells containing mucilage, some cells also
containing bundles of acicular crystals of calcium oxalate, 20 to 900 m in length; vascular bundles
collateral, scattered throughout the mesophyll; xylem vessels with spiral and annular wall thickening;
trichomes and starch absent.
Identification The mucilage contained in the cells of the mesophyll is stained red with alkaline
corallin solution and reddish purple with 0.01M iodine.
Ash Not more than 6.0%, Appendix XI J.
Storage Indian Squill should be stored in a dry place at a temperature not exceeding 25.
Preparation
Squill Oxymel
When Powdered Indian Squill is prescribed or demanded, material complying with the appropriate
requirements above shall be dispensed or supplied.
37-21
Stannous Chloride Dihydrate

SnCl2,2H2O
225.6
10025-69-1
Stannous Chloride Dihydrate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1266]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Stannous chloride dihydrate contains not less than 98.0 per cent and not more than the equivalent of
101.0 per cent of SnCl2,2H2O.
CHARACTERS
A white, crystalline powder or colourless crystals, efflorescent in air, freely soluble in water (the
solution becomes cloudy after standing or on dilution), freely soluble in alcohol. It dissolves in dilute
hydrochloric acid.
IDENTIFICATION
A. To 1 ml of solution S1 (see Tests) add a mixture of 5 ml of water R and 0.05 ml of mercuric
chloride solution R. A blackish-grey precipitate forms.
B. Dissolve 1.0 g in 3.0 ml of water R. Add 0.5 ml of dilute sodium hydroxide solution R to the cloudy
solution; a yellowish flocculent precipitate is formed. Add 6.5 ml of water R. To 1.0 ml of the
previously shaken suspension add 1.0 ml of strong sodium hydroxide solution R; the precipitate dissolves
and the resulting solution is clear and colourless.
C. Dissolve 10 mg in 2 ml of dilute nitric acid R. The solution gives reaction (a) of chlorides (2.3.1).
TESTS
Solution S1 To 0.40 g add 1 ml of dilute hydrochloric acid R and dilute to 20 ml with distilled water R.
Solution S2 Dissolve 1.0 g in dilute hydrochloric acid R and dilute to 30 ml with the same acid. Heat
to boiling. Add 30 ml of thioacetamide solution R and boil for 15 min (solution A). Use 5 ml, filter and
heat the filtrate to boiling. Add 5 ml of thioacetamide solution R and boil for 15 min. If a precipitate is
formed, add the remainder of solution A (solution A) to the mixture. Add 10 ml of thioacetamide
solution R and boil. Repeat the series of operations from Use 5 ml, until a precipitate is no
longer formed on addition of thioacetamide solution R to the filtrate obtained from the 5 ml of solution
A (solution A, solution A,, respectively). If no precipitate is formed or if no more precipitate is
formed combine the solution obtained with the remainder of solution A (solution A, solution A,,
respectively), filter and wash the precipitate with 10 ml of water R. Heat the filtrate until the resulting
vapour no longer turns a moistened piece of lead acetate paper R blackish-grey. Allow to cool and
dilute to 50 ml with water R.
Appearance of solution Dissolve 10.0 g in dilute hydrochloric acid R and dilute to 20 ml with the
same acid. The solution is clear (2.2.1) and colourless (Method II, 2.2.2).
Substances not precipitated by thioacetamide Evaporate 25 ml of solution S2 to dryness and
ignite at 600C. The residue weighs not more than 1 mg (0.2 per cent).
Sulphates (2.4.13). 15 ml of solution S1 complies with the limit test for sulphates (500 ppm).
Heavy metals Dissolve 1.0 g in 2 ml of a mixture of 1 volume of nitric acid R and 3 volumes of
hydrochloric acid R. Heat the solution on a water-bath until nitrous vapour is no longer evolved.
Dissolve the residue in water R and dilute to 25 ml with the same solvent. To 5 ml of the solution add
3 ml of strong sodium hydroxide solution R and 2 ml of water R. Heat until a clear solution is obtained,
then cool and add 0.5 ml of thioacetamide reagent R. After 2 min, any colour in the solution is not
more intense than that of a mixture of 1.0 ml of lead standard solution (10 ppm Pb) R, 6 ml of water R,
3 ml of strong sodium hydroxide solution R and 0.5 ml of thioacetamide reagent R (50 ppm).
Iron (2.4.9). Dilute 5 ml of solution S2 to 10 ml with water R. The solution complies with the limit
ASSAY
Dissolve 0.100 g in 1.5 ml of hydrochloric acid R1 and dilute to 50 ml with water R. Add 5 g of sodium
potassium tartrate R, 10 g of sodium hydrogen carbonate R and 1 ml of starch solution R. Titrate immediately with 0.05M iodine. Carry out a blank titration.
1 ml of 0.05M iodine is equivalent to 11.28 mg of SnCl2,2H2O.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
37-22
Stanozolol
1/01
Me
Me
OH
Me
N
H
H
N
H
C21H32N2O
328.5
10418-03-8
Stanozolol complies with the requirements of the 3rd edition of the European Pharmacopoeia [1568]. These
Action and use Anabolic steroid; androgen
Preparation
Stanozolol Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Stanozolol contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of
17-methyl-2H-5-androst-2-eno[3,2-c]pyrazol-17-ol, calculated with reference to the dried
substance.
CHARACTERS
A white or almost white crystalline powder, hygroscopic, practically insoluble in water, soluble in
dimethylformamide, slightly soluble in alcohol, very slightly soluble in methylene chloride.
IDENTIFICATION
A. Examine by infrared spectrophotometry (2.2.24), comparing with the spectrum obtained with
stanozolol CRS. If the spectra obtained in the solid state show differences, dissolve the substance to be
examined and the reference substance separately in the minimum volume of methylene chloride,
evaporate to dryness at room temperature under an air-stream and record new spectra using the
residues.
spot in the chromatogram obtained with reference solution (c).
TESTS
substance.
plate R.
solvents.
and 9 volumes of methylene chloride R.
Reference solution (a). Dilute 1.0 ml of test solution (a) to 200 ml with a mixture of 1 volume of
methanol R and 9 volumes of methylene chloride R.
Reference solution (b). Dissolve 5 mg of stanozolol impurity A CRS in reference solution (a) and dilute
Reference solution (c). Dissolve 10 mg of stanozolol CRS in a mixture of 1 volume of methanol R and 9
volumes of methylene chloride R and dilute to 5 ml with the same mixture of solvents.
Apply to the plate 5 l of each solution. Develop over a path corresponding to two thirds of the plate
height using a mixture of 10 volumes of methanol R and 90 volumes of methylene chloride R. Allow the
plate to dry in air, spray with alcoholic solution of sulphuric acid R, heat at 105C for 15 min and
37-23
examine under ultraviolet light at 365 nm. Any secondary spot in the chromatogram obtained with
test solution (a) is not more intense than the spot in the chromatogram obtained with reference
solution (a) (0.5 per cent). The test is not valid unless the chromatogram obtained with reference
solution (b) shows two clearly separated spots.
Loss on drying (2.2.32). Not more than 1.0 per cent, determined on 1.000 g by drying at 100C at
a pressure not exceeding 0.7 kPa.
ASSAY
STORAGE
IMPURITIES
Me Me
OH
Me
H
H
A. 17-hydroxy-17-methyl-5-androstan-3-one (mestanolone),
Me Me
OH
Me
HO
H
H
B. 17-hydroxy-2-(hydroxymethylene)-17-methyl-5-androstan-3-one (oxymetholone).
__________________________________________________________________________________________________________ Ph Eur
37-24
Pregelatinised Starch
Pregelatinised Starch complies with the requirements of the 3rd edition of the European Pharmacopoeia
When Pregelatinised Starch is prepared from Zea mays, the title Pregelatinised Maize Starch may be
used.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Pregelatinised starch is starch, apart from wheat starch, that has been mechanically processed in the
presence of water, with or without heat to rupture all or part of the starch granules and subsequently
dried. It contains no added substances but it may be modified to render it compressible and to
improve its flow characteristics.
CHARACTERS
A white or yellowish-white powder, swelling in cold water.
IDENTIFICATION
A. Examined under a microscope using a mixture of equal volumes of glycerol R and water R it
presents irregular, translucent, white or yellowish-white flakes or pieces with an uneven surface.
Under polarised light, (between crossed nicol prisms), starch granules with a distinct black cross
intersecting at the hilum may be seen.
B. Disperse 0.5 g in 2 ml of water R without heating and add 0.05 ml of iodine solution R1. A reddishviolet to blue colour is produced.
TESTS
Iron (2.4.9). Shake 0.75 g with 15 ml of dilute hydrochloric acid R. Filter. The filtrate complies with
glycerol R and water R, not more than traces of cell walls and of cytoplasmic residues are present.
at 130C for 90 min.
Microbial contamination Not more than 103 bacteria and not more than 102 fungi per gram,
determined by plate-count (2.6.12). It complies with the test for Escherichia coli (2.6.13).
STORAGE
LABELLING
The herbal origin of Starch, pregelatinised is stated.
__________________________________________________________________________________________________________ Ph Eur
37-25
Stearic Acid
Stearic Acid complies with the requirements of the 3rd edition of the European Pharmacopoeia [1474]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Stearic acid is obtained from fat or oils from a vegetable or animal source and is a mixture consisting
mainly of stearic acid (C18H36O2; Mr 284.5) and palmitic acid (C16H32O2; Mr 256.4). It contains
different nominal amounts of C18H36O2; stearic acid 50 contains 40.0 per cent to 60.0 per cent,
stearic acid 70 contains 60.0 per cent to 80.0 per cent and stearic acid 95 contains at least 90.0 per
cent of C18H36O2. The sum of the contents of C18H36O2 and C16H32O2 is not less than 90.0 per cent
for stearic acid 50 and stearic acid 70 and not less than 96.0 per cent for stearic acid 95.
PRODUCTION
CHARACTERS
White, waxy, flaky crystals, white hard masses or a white or yellowish-white powder, insoluble in
water, soluble in alcohol and in light petroleum (50C to 70C).
IDENTIFICATION
A. It complies with the test for freezing point (see Tests).
B. Acid value (2.5.1). 194 to 212, determined on 0.1 g.
C. Examine the chromatograms obtained in the Assay. The retention times of the principal peaks in
the chromatogram obtained with the test solution are approximately the same as those of the principal peaks in the chromatogram obtained with the reference solution.
TESTS
Appearance Heat the substance to be examined to about 75C. The resulting liquid is not more
intensely coloured than reference solution Y7 or BY7 (Method I, 2.2.2).
Acidity Melt 5.0 g, shake for 2 min with 10 ml of hot carbon dioxide-free water R, cool slowly and
filter. To the filtrate add 0.05 ml of methyl orange solution R. No red colour develops.
Iodine value (2.5.4). See Table 1474.-1.
Freezing point (2.2.18). See Table 1474.-1.
Table 14741
Nominal percentage Iodine value
of C18H36O2
Freezing point
50
70
95
53C to 59C
57C to 64C
64C to 69C
Not more than 4.0

Not more than 4.0
Not more than 1.5
Nickel (2.4.27). Not more than 1 ppm of Ni, determined by the method for nickel in hydrogenated
vegetable oils.
ASSAY
Test solution. In a conical flask fitted with a reflux condenser, dissolve 0.100 g of the substance to be
examined in 5 ml of boron trifluoride-methanol solution R. Boil under reflux for 10 min. Add 4.0 ml of
heptane R through the condenser and boil again under reflux for 10 min. Allow to cool. Add 20 ml of
a saturated solution of sodium chloride R. Shake and allow the layers to separate. Remove about 2 ml
of the organic layer and dry it over 0.2 g of anhydrous sodium sulphate R. Dilute 1.0 ml of this solution
to 100.0 ml with heptane R.
Reference solution. Prepare the reference solution in the same manner as the test solution using
50.0 mg of palmitic acid CRS and 50.0 mg of stearic acid CRS instead of the substance to be examined.
a fused-silica column 30 m long and 0.32 mm in internal diameter coated with macrogol
37-26
Time
(min)
Column
Temperature Rate
(C)
(C/min)
02
70
2 36
70 240
36 41 240
Injection port
220
Detector
260
Comment
isothermal
linear gradient
isothermal
Inject 1 l of the reference solution. When the chromatograms are recorded in the prescribed
conditions, the retention time of methyl palmitate relative to that of methyl stearate is about 0.88.
The test is not valid unless, in the chromatogram obtained with the reference solution, the resolution
between the peaks corresponding to methyl stearate and methyl palmitate is at least 5.0.
Inject 1 l of the test solution. Calculate the percentage content of stearic acid and palmitic acid
from the areas of the peaks in the chromatogram obtained with the test solution by the normalisation
procedure, disregarding the peak due to the solvent.
LABELLING
The label states the nominal percentage content of stearic acid (C18H36O2).
__________________________________________________________________________________________________________ Ph Eur
37-27
Stearoyl Macrogolglycerides
Stearoyl Macrogolglycerides comply with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Stearoyl macrogolglycerides are mixtures of monoesters, diesters and triesters of glycerol and monoesters and diesters of macrogols with a mean relative molecular mass between 300 and 4000. They
are obtained by partial alcoholysis of saturated oils containing mainly triglycerides of stearic acid
using macrogol or by esterification of glycerol and macrogol with saturated fatty acids or by mixture
of glycerol esters and condensates of ethylene oxide with the fatty acids of these hydrogenated oils.
The hydroxyl value does not differ by more than 15 units from the nominal value. The saponification
value does not differ by more than 10 units from the nominal value.
PRODUCTION
Where applicable, they comply with the monograph on Products with risk of transmitting agents of
animal spongiform encephalopathies (1483).
CHARACTERS
Pale yellow waxy solids, dispersible in warm water and in warm liquid paraffin, freely soluble in
methylene chloride, soluble in warm ethanol.
IDENTIFICATION
substance.
Apply to the plate 10 l of the test solution. Develop over a path of 15 cm using a mixture of 30
volumes of hexane R and 70 volumes of ether R. Allow the plate to dry in air. Spray with a 0.1 g/l
solution of rhodamine B R in alcohol R and examine in ultraviolet light at 365 nm. The chromatogram
shows a spot corresponding to triglycerides with an Rf value of about 0.9 (Rst 1) and spots corresponding to 1,3-diglycerides (Rst 0.7), to 1,2-diglycerides (Rst 0.6), to monoglycerides (Rst 0.1) and to
esters of macrogol (Rst 0).
B. They comply with the test for hydroxyl value (see Tests).
C. They comply with the test for saponification value (see Tests).
D. They comply with the test for fatty acid composition (see Tests).
TESTS
Hydroxyl value (2.5.3, Method A). Determined on 1.0 g, the hydroxyl value does not differ by more
than 15 units from the nominal value.
Peroxide value (2.5.5). Not more than 6.0, determined on 2.0 g.
Saponification value (2.5.6). Determined on 2.0 g, the saponification value does not differ by more
than 10 units from the nominal value.
Alkaline impurities Into a test-tube introduce 5.0 g and carefully add a mixture, neutralised if
necessary with 0.01M hydrochloric acid or with 0.01M sodium hydroxide, of 0.05 ml of a 0.4 g/l solution
of bromophenol blue R in alcohol R, 0.3 ml of water R and 10 ml of alcohol R. Shake and allow to stand.
Not more than 1.0 ml of 0.01M hydrochloric acid is required to change the colour of the upper layer to
yellow.
Free glycerol Not more than 3.0 per cent. Dissolve 1.20 g in 25.0 ml of methylene chloride R. Heat if
necessary. After cooling, add 100 ml of water R. Shake and add 25.0 ml of a 6 g/l solution of periodic
acid R. Shake and allow to stand for 30 min. Add 40 ml of a 75 g/l solution of potassium iodide R.
Allow to stand for 1 min. Add 1 ml of starch solution R. Titrate the iodine with 0.1M sodium
1 ml of 0.1M sodium thiosulphate is equivalent to 2.3 mg of glycerol.
Fatty acid composition Examine by gas chromatography (2.4.22). The constitutive fatty acid
composition is the following:
37-28
different nominal amounts of stearic acid and of palmitic acid. The sum of C18H36O2 and
C16H32O2 is not less than 90.0 per cent.
correction factor of 1/5 to the calculation.
of water using a mixture of 30 volumes of anhydrous methanol R and 70 volumes of methylene
chloride R as solvent.
STORAGE
LABELLING
The label states the nominal hydroxyl value, the nominal saponification value and the type of the
macrogol used (mean relative molecular mass) or the number of ethylene oxide units per molecule
(nominal value).
__________________________________________________________________________________________________________ Ph Eur
37-29
Stearyl Alcohol
112-92-5
Stearyl Alcohol complies with the requirements of the 3rd edition of the European Pharmacopoeia [0753].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Stearyl alcohol is a mixture of solid alcohols; it contains not less than 95.0 per cent of octadecanol
(C18H38O; Mr 270.5).
CHARACTERS
White, unctuous flakes, granules or mass, practically insoluble in water, soluble in alcohol, freely
soluble in ether. When melted, it is miscible with fatty oils, with liquid paraffin and with melted wool
fat.
IDENTIFICATION
Examine the chromatograms obtained in the Assay. The retention time and size of the principal peak
in the chromatogram obtained with the test solution are approximately the same as those of the
TESTS
Appearance of solution Dissolve 0.50 g in 20 ml of alcohol R by heating to boiling. Allow to cool.
The solution is clear (2.2.1) and not more intensely coloured than reference solution B6 (Method II,
2.2.2).
Melting point (2.2.14). 57C to 60C.
Iodine value (2.5.4). Not more than 2.0, determined on 2.00 g dissolved in 25 ml of chloroform R,
warming if necessary.
ASSAY
Test solution. Dissolve 0.100 g of the substance to be examined in ethanol R and dilute to 10.0 ml with
the same solvent.
Reference solution (a). Dissolve 0.100 g of stearyl alcohol CRS in ethanol R and dilute to 10.0 ml with
the same solvent.
Reference solution (b). Dissolve 5 mg of cetyl alcohol CRS in 4.5 ml of reference solution (a) and dilute
to 5.0 ml with ethanol R.
a stainless steel column 2 m long and 2 mm in internal diameter packed with diatomaceous earth
for chromatography R impregnated with 10 per cent m/m of polydimethylsiloxane R,
the detector at 250C. Inject 2 l of reference solution (b). The assay is not valid unless the resolution between the peaks corresponding to cetyl alcohol and stearyl alcohol is at least 4.0. Inject 2 l of
the test solution and 2 l of reference solution (a). Calculate the percentage content of octadecanol
using the normalisation procedure.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
37-30
Sterculia
Sterculia Gum; Karaya Gum
Definition Sterculia is the gum obtained from Sterculia urens Roxb. and other species of Sterculia.
Characteristics It has the macroscopical and microscopical characters described under Identification tests A and B. Odour, that of acetic acid.
Sparingly soluble in water, but swells into a homogeneous, adhesive, gelatinous mass. Practically
insoluble in ethanol (96%).
Identification A. Irregular or vermiform pieces, about 5 to 20 mm thick; greyish white with a brown
or pink tinge; surface striated.
B. When powdered and mounted in ethanol (96%) it appears as small, transparent, angular particles
of various sizes and shapes; the particles lose their sharp edges when water is added and each
gradually swells until a large, indefinite, almost structureless mass results; when mounted in
ruthenium red solution the particles are stained red; no blue coloured particles (starch) are visible when
mounted in iodine solution R1.
C. Add 1 g to 80 ml of water and allow to stand for 24 hours, shaking occasionally. A tacky and
viscous granular mucilage is produced. Retain the mucilage for use in test D.
D. Boil 4 ml of the mucilage obtained in test C with 0.5 ml of hydrochloric acid, add 1 ml of 5M
sodium hydroxide, filter, add 3 ml of cupri-tartaric solution R1 to the filtrate and heat. A red precipitate
is produced.
E. Warm 0.5 g with 2 ml of 5M sodium hydroxide. A brown colour is produced.
Acid-insoluble ash Not more than 1.0%, Appendix XI K.
Foreign matter Complies with the test for foreign matter, Appendix XI D, Test B.
Volatile acid Not less than 14.0%, calculated as acetic acid, C2H4O2, when determined by the
following method. To 1 g contained in a 700-ml Kjeldahl flask add 100 ml of water and 5 ml of
orthophosphoric acid, allow to stand for several hours, or until the gum is completely swollen, and boil
gently under a reflux condenser for 2 hours. Steam distil until 800 ml of distillate is obtained and the
acid residue measures about 20 ml and titrate the distillate with 0.1M sodium hydroxide VS using
phenolphthalein solution R1 as indicator. Repeat the operation without the substance being examined.
The difference between the titrations represents the amount of alkali required to neutralise the
volatile acid. Each ml of 0.1M sodium hydroxide VS is equivalent to 6.005 mg of volatile acid, calculated as C2H4O2.
Microbial contamination 1.0 g is free from Escherichia coli, Appendix XVI B1.
Storage Sterculia should be stored in a dry place at a temperature not exceeding 25.
Action and use Bulk-forming laxative; pharmaceutical aid.
Preparation
Sterculia Granules
When Powdered Sterculia is prescribed or demanded, material complying with the appropriate
requirements above and containing not less than 10.0% of volatile acid shall be dispensed or
supplied.
37-31
Stramonium Leaf
Stramonium Leaf complies with the requirements of the 3rd edition of the European Pharmacopoeia [0246].
Preparation
Prepared Stramonium
When Stramonium Leaf or Powdered Stramonium Leaf is prescribed, Prepared Stramonium shall be
dispensed.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Stramonium leaf consists of the dried leaf or of the dried leaf, flowering tops and occasionally, fruitbearing tops of Datura stramonium L. and its varieties. It contains not less than 0.25 per cent of total
alkaloids, calculated as hyoscyamine (C17H23NO3; Mr 289.4) with reference to the drug dried at
100C to 105C. The alkaloids consist mainly of hyoscyamine with a small quantity of hyoscine
(scopolamine).
CHARACTERS
Stramonium leaf has an unpleasant odour.
IDENTIFICATION
A. The leaves are dark brownish-green to dark greyish-green, often much twisted and shrunken
during drying, thin and brittle, ovate or triangular-ovate, dentately lobed with an acuminate apex and
often unequal at the base. Young leaves are pubescent on the veins, older leaves are nearly glabrous.
Stems are green or purplish-green, slender, curved and twisted, wrinkled longitudinally and
sometimes wrinkled transversely, branched dichasially, with a single flower or an immature fruit in
the fork. Flowers, on short pedicels, have a gamosepalous calyx with five lobes and trumpet-shaped
brownish-white or purplish corolla. The fruit is a capsule, usually covered with numerous short, stiff
emergences; seeds are brown to black with a minutely pitted testa.
B. Reduce to a powder (355). The powder is greyish-green. Examine under a microscope using
chloral hydrate solution R. The powder shows the following diagnostic characters: fragments of leaf
lamina showing epidermal cells with slightly wavy anticlinal walls and smooth cuticle; stomata are
more frequent on the lower epidermis (anisocytic and anomocytic) (2.8.3); covering trichomes are
conical, uniseriate with three to five cells and warty walls; glandular trichomes are short and clavate
with heads formed by 2 to 7 cells; dorsiventral mesophyll, with a single layer of palisade cells and a
spongy parenchyma containing cluster crystals of calcium oxalate; annularly and spirally thickened
vessels. The powdered drug may also show the following: fibres and reticulately thickened vessels
from the stem; subspherical pollen grains usually about 60 m to 80 m in diameter with three
germinal pores and nearly smooth exine; fragments of the corolla with papillose epidermis; seed
fragments containing yellowish-brown, sinuous, thick-walled sclereids of testa; occasional prisms and
microsphenoidal crystals of calcium oxalate.
C. Examine the chromatograms obtained in the chromatography test. The principal zones in the
chromatogram obtained with the test solution are similar in position, colour and size to the principal
zones in the chromatogram obtained with the same volume of the reference solution.
D. Shake 1 g of the powdered drug (180) with 10 ml of 0.05M sulphuric acid for 2 min. Filter and add
to the filtrate 1 ml of concentrated ammonia R and 5 ml of water R. Shake cautiously with 15 ml of
peroxide-free ether R to avoid the formation of an emulsion. Separate the ether layer and dry over
anhydrous sodium sulphate R. Filter and evaporate the ether in a porcelain dish. Add 0.5 ml of nitric
acid R and evaporate to dryness on a water-bath. Add 10 ml of acetone R and, dropwise, a 30 g/l
solution of potassium hydroxide R in alcohol R. A deep violet colour develops.
TESTS
Chromatography Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance.
Test solution. To 1.0 g of the powdered drug (180) add 10 ml of 0.05M sulphuric acid, shake for
15 min and filter. Wash the filter with 0.05M sulphuric acid until 25 ml of filtrate is obtained. To the
filtrate add 1 ml of concentrated ammonia R and shake with two quantities, each of 10 ml, of peroxidefree ether R. If necessary, separate by centrifugation. Dry the combined ether layers over anhydrous
sodium sulphate R, filter and evaporate to dryness on a water-bath. Dissolve the residue in 0.5 ml of
methanol R.
37-32
Reference solution. Dissolve 50 mg of hyoscyamine sulphate R in 9 ml of methanol R. Dissolve 15 mg of
hyoscine hydrobromide R in 10 ml of methanol R. Mix 3.8 ml of the hyoscyamine sulphate solution and
4.2 ml of the hyoscine hydrobromide solution and dilute to 10 ml with methanol R.
Apply separately to the plate as bands 20 mm by 3 mm 10 l and 20 l of each solution, leaving 1 cm
between the bands. Develop over a path of 10 cm using a mixture of 3 volumes of concentrated
ammonia R, 7 volumes of water R and 90 volumes of acetone R. Dry the plate at 100C to 105C for
15 min, allow to cool and spray with potassium iodobismuthate solution R2, using about 10 ml for a
plate 200 mm square, until the orange or brown zones become visible against a yellow background.
The zones in the chromatograms obtained with the test solution are similar to those in the chromatograms obtained with the reference solution with respect to their position (hyoscyamine in the lower
third, hyoscine in the upper third of the chromatogram) and their colour. The zones in the chromatograms obtained with the test solution are at least equal in size to the corresponding zones in the
chromatogram obtained with the same volume of the reference solution. Faint secondary zones may
appear, particularly in the middle of the chromatogram obtained with 20 l of the test solution or
near the starting point in the chromatogram obtained with 10 l of the test solution. Spray the plate
with sodium nitrite solution R until the coating is transparent. Examine after 15 min. The zones corresponding to hyoscyamine in the chromatograms obtained with the test solution and the reference
solution change from brown to reddish-brown but not to greyish-blue (atropine) and any secondary
zones disappear.
Foreign matter (2.8.2). Not more than 3 per cent of stem having a diameter exceeding 5 mm.
ASSAY
Take about 50 g of the drug and completely reduce it to a powder (180). Using the powder, determine the loss on drying and the total alkaloids.
a) Determine the loss on drying (2.2.32) on 2.000 g by drying in an oven at 100C to 105C.
b) Moisten 10.0 g of the powder with a mixture of 5 ml of ammonia R, 10 ml of alcohol R and 30 ml
of peroxide-free ether R and mix thoroughly. Transfer the mixture to a suitable percolator, if necessary
with the aid of the extracting mixture. Allow to macerate for 4 h and percolate with a mixture of 1
volume of chloroform R and 3 volumes of peroxide-free ether R until the alkaloids are completely
extracted. Evaporate to dryness a few millilitres of the liquid flowing from the percolator, dissolve the
residue in 0.25M sulphuric acid and verify the absence of alkaloids using potassium tetraiodomercurate
solution R. Concentrate the percolate to about 50 ml by distilling on a water-bath and transfer it to a
separating funnel, rinsing with peroxide-free ether R. Add a quantity of peroxide-free ether R equal to at
least 2.1 times the volume of the percolate to produce a liquid of a density well below that of water.
Shake the solution with no fewer than three quantities, each of 20 ml, of 0.25M sulphuric acid,
separate the two layers by centrifugation if necessary and transfer the acid layers to a second separating funnel. Make the acid layer alkaline with ammonia R and shake with three quantities, each of
30 ml, of chloroform R. Combine the chloroform layers, add 4 g of anhydrous sodium sulphate R and
allow to stand for 30 min with occasional shaking. Decant the chloroform and wash the sodium
sulphate with three quantities, each of 10 ml, of chloroform R. Add the washings to the chloroform
extract, evaporate to dryness on a water-bath and heat in an oven at 100C to 105C for 15 min.
Dissolve the residue in a few millilitres of chloroform R, add 20.0 ml of 0.01M sulphuric acid and
remove the chloroform by evaporation on a water-bath. Titrate the excess of acid with 0.02M sodium
hydroxide using methyl red mixed solution R as indicator.
Calculate the percentage content of total alkaloids, expressed as hyoscyamine, from the expression:
57.88 (20 n )
(100 d ) m
d = loss on drying as a percentage,
n = volume of 0.02M sodium hydroxide used in millilitres,
m = mass of drug used in grams.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
37-33
Prepared Stramonium
Prepared Stramonium complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Prepared stramonium is stramonium leaf powder (180) adjusted, if necessary, by the addition of
powdered lactose or stramonium leaf of lower alkaloidal content to contain 0.23 per cent to 0.27 per
cent of total alkaloids, calculated as hyoscyamine (C17H23NO3; Mr 289.4) with reference to the dried
drug.
CHARACTERS
It presents the characters of the powder described in the monograph on Stramonium leaf (246).
IDENTIFICATION
It complies with identification tests B, C and D in the monograph of Stramonium leaf (246). Examined in glycerol (85 per cent) R, it may be seen to contain lactose crystals.
TESTS
It complies with the tests for chromatography, for total ash and for ash insoluble in hydrochloric acid
prescribed in the monograph on Stramonium leaf (246) and with the following additional test.
100C to 105C.
ASSAY
Carry out the assay prescribed in the monograph on Stramonium leaf (246).
Calculate the percentage content of total alkaloids, expressed as hyoscyamine, from the expression:
57.88 (20 n )
(100 d ) m
d = loss on drying as a percentage,
n = volume of 0.02M sodium hydroxide used in millilitres,
m = mass of drug used in grams.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
37-34
Streptokinase
Streptokinase complies with the requirements of the 3rd edition of the European Pharmacopoeia [0356]. These
Action and use Fibrinolytic enzyme.
Preparation
Streptokinase Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Streptokinase is a preparation of a protein obtained from culture filtrates of certain strains of
haemolytic Streptococcus group C; it has the property of combining with human plasminogen to form
plasminogen activator.
It is purified so that, before any addition of stabiliser or carrier, the potency is not less than
600 I.U. of streptokinase activity per microgram of nitrogen. It usually contains a buffer and may be
stabilised by the addition of suitable substances such as human albumin.
CHARACTERS
A white powder or a white, friable solid, hygroscopic, freely soluble in water.
IDENTIFICATION
A. Place 0.5 ml of citrated human, canine or rabbit plasma in a haemolysis tube maintained in a
water-bath at 37C. Add 0.1 ml of a solution of the preparation to be examined containing
10,000 I.U. of streptokinase activity per millilitre in phosphate buffer solution pH 7.2 R and 0.1 ml of a
solution of human thrombin R containing 20 I.U. per millilitre in phosphate buffer solution pH 7.2 R.
Shake immediately. A clot forms and lyses within 30 min. Repeat the procedure using citrated bovine
plasma. The clot does not lyse within 60 min.
B. Dissolve 0.6 g of agar in 50.0 ml of barbital buffer solution pH 8.6 R1, heating until a clear solution
is obtained. Use glass plates 50 mm square (transparency mounts) free from traces of grease. Using a
pipette, apply to each plate 4 ml of the agar solution described below. Maintain the plates horizontal.
Allow to cool. Pierce a cavity 6 mm in diameter in the centre of the agar and an appropriate number
of cavities (not exceeding six) at distances of 11 mm from the central cavity. Remove the residual
agar from the cavities using a cannula connected to a vacuum pump. Using pipettes graduated in
microlitres, place in the central cavity about 80 l of goat or rabbit antistreptokinase serum containing 10,000 units of antistreptokinase activity per millilitre; place in each of the surrounding cavities
about 80 l of a solution of the preparation to be examined containing 125,000 I.U. of streptokinase
activity per millilitre. Allow the plates to stand in a humidified tank for 24 h. Only one precipitation
arc appears and it is well defined and localised between the application point of the serum and each
cavity containing the solution of the preparation to be examined.
TESTS
pH (2.2.3). Dissolve the preparation to be examined in carbon dioxide-free water R to obtain a solution
containing 5000 I.U. of streptokinase activity per millilitre. The pH of the solution is 6.8 to 7.5.
Streptodornase
Test solution. Dissolve the preparation to be examined in imidazole buffer solution pH 6.5 R to obtain a
solution containing 150,000 I.U. of streptokinase activity per millilitre.
Reference solution. Dissolve in imidazole buffer solution pH 6.5 R a reference preparation of streptodornase, calibrated in International Units against the International Standard of Streptodornase, to
obtain a solution containing 20 I.U. of streptodornase activity per millilitre. The equivalence in
International Units of the International Standard is stated by the World Health Organisation.
To each of eight numbered centrifuge tubes add 0.5 ml of a 1 g/l solution of sodium deoxyribonucleate R in imidazole buffer solution pH 6.5 R. To tube number 1 and tube number 2 add 0.25 ml of
imidazole buffer solution pH 6.5 R, 0.25 ml of the test solution and, immediately, 3.0 ml of perchloric
acid (25 g/l HClO4). Mix, centrifuge at about 3000 g for 5 min and measure the absorbances (2.2.25)
of the supernatant liquids at 260 nm, using as the compensation liquid a mixture of 1.0 ml of
imidazole buffer solution pH 6.5 R and 3.0 ml of perchloric acid (25 g/l HClO4) (absorbances A1 and
A2). To the other six tubes (numbers 3 to 8) add 0.25 ml, 0.25 ml, 0.125 ml, 0.125 ml, 0 ml and
0 ml respectively of imidazole buffer solution pH 6.5 R; add to each tube 0.25 ml of the test solution
and 0 ml, 0 ml, 0.125 ml, 0.125 ml, 0.25 ml and 0.25 ml respectively of the reference solution. Mix
the contents of each tube and heat at 37C for 15 min. To each tube add 3.0 ml of perchloric acid
(25 g/l HClO4), mix and centrifuge. Measure the absorbances (2.2.25) of the supernatant liquids at
260 nm using the compensation liquid described above (absorbances A3 to A8).
37-35
( A3 + A4 ) ( A1 + A2 ) <
( A5 + A6 + A7 + A8 )
( A3 + A4 )
2
(equivalent to a maximum of 10 I.U. of streptodornase activity per 100,000 I.U. of streptokinase

activity).
Streptolysin In a haemolysis tube, dissolve a quantity of the preparation to be examined equivalent
to 500,000 I.U. of streptokinase activity in 0.5 ml of a mixture of 1 volume of phosphate buffer solution
pH 7.2 R and 9 volumes of a 9 g/l solution of sodium chloride R. Add 0.4 ml of a 23 g/l solution of
sodium thioglycollate R. Heat in a water-bath at 37C for 10 min. Add 0.1 ml of a solution of a reference preparation of human antistreptolysin O containing 5 I.U. per millilitre. Heat at 37C for 5 min.
Add 1 ml of rabbit erythrocyte suspension R. Heat at 37C for 30 min. Centrifuge at about 1000 g. In
the same manner, prepare a haemolysis tube in which the solution of the preparation to be examined
has been replaced by 0.5 ml of a mixture of 1 volume of phosphate buffer solution pH 7.2 R and 9
volumes of a 9 g/l solution of sodium chloride R. Measure the absorbances (2.2.25) of the supernatant
liquids at 550 nm. The absorbance of the test solution is not more than 50 per cent greater than that
of the reference solution.
Loss on drying (2.2.32). Not more than 4.0 per cent, determined by drying over diphosphorus
pentoxide R at a pressure not exceeding 2.7 Pa for 24 h.
further appropriate procedure for removal of pyrogens, it complies with the test for pyrogens. Inject
per kilogram of the rabbits mass a quantity of the preparation to be examined equivalent to 20,000
I.U. of streptokinase activity dissolved in not more than 1 ml of water for injections R.
Abnormal toxicity (2.6.9). If intended for use in the manufacture of parenteral dosage forms, it
complies with the test for abnormal toxicity. Inject into each mouse a quantity of the preparation to
be examined equivalent to 50,000 I.U. of streptokinase activity dissolved in 0.5 ml of water for injections R, the injection lasting 15 s to 20 s.
ASSAY
The potency of streptokinase is determined by comparing its capacity to activate plasminogen to form
plasmin with the same capacity of a reference preparation of streptokinase calibrated in International
Units; the formation of plasmin is measured by the determination of the lysis time of a fibrin clot in
given conditions.
The International Unit is the streptokinase activity contained in a stated amount of the International Standard, which consists of freeze-dried streptokinase with lactose. The equivalence in International Units of the International Standard is stated by the World Health Organisation.
Unless otherwise prescribed, use phosphate buffer solution pH 7.2 R containing 30 g/l bovine albumin R for
the preparation of the solutions and dilutions used in the assay.
Test solution. Prepare a solution of the preparation to be examined expected to contain 1000 I.U. of
streptokinase activity per millilitre.
Reference solution. Prepare a solution of a reference preparation containing 1000 I.U. of streptokinase
activity per millilitre.
Keep the test solution and the reference solution in iced water and use within 6 h.
Prepare three 1.5-fold dilutions of the reference preparation such that the longest clot-lysis time is
less than 20 min. Prepare three similar dilutions of the test solution.
Keep the solutions in iced water and use within 1 h.
Use twenty-four tubes 8 mm in diameter. Label the tubes T1, T2 and T3 for the dilutions of the
test solution and S1, S2 and S3 for the dilutions of the reference solution, allocating four tubes to each
dilution. Place the tubes in iced water. Into each tube, introduce 0.2 ml of the appropriate dilution,
0.2 ml of phosphate buffer solution pH 7.2 R containing 30 g/l of bovine albumin R and 0.1 ml of a
solution of human thrombin R containing 20 I.U. per millilitre. Place the tubes in a water-bath at
37C and allow to stand for 2 min to attain temperature equilibrium. Using an automatic pipette,
introduce into the bottom of the first tube 0.5 ml of a 10 g/l solution of human euglobulins R, ensuring
mixing. At intervals of 5 s, introduce successively into the remaining tubes 0.5 ml of a 10 g/l solution
of human euglobulins R. Using a stop-watch, measure for each tube the time in seconds that elapses
between the addition of the euglobulins solution and the lysis of the clot.
Using the logarithms of the lysis times, calculate the activity of the preparation to be examined with
reference to the activity of the reference preparation using the usual statistical methods.
The estimated potency is not less than 90 per cent and not more than 111 per cent of the stated
potency. The fiducial limits of error (P = 0.95) of the estimated potency are not less than 80 per cent
and not more than 125 per cent of the stated potency.
37-36
STORAGE
Store in a sealed container, protected from light. If the substance is sterile, store in a sterile,
LABELLING
The label states:
the number of International Units of streptokinase activity per milligram, calculated with reference to the dried preparation,
the name and quantity of any added substance,
where applicable, that the substance is free from pyrogens,
forms.
__________________________________________________________________________________________________________ Ph Eur
37-37
Streptomycin Sulphate
NH
NH
NHCNH2
H2NCNH
OH HO
OH
O
,3H 2SO4
CHO
Me
HO
HO
O
O
CH2OH
MeHN
OH
(C21H39N7O12)2,3H2SO4
1457
3810-74-0
Streptomycin Sulphate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Streptomycin Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Streptomycin sulphate is di[O-2-deoxy-2-methylamino--L-glucopyranosyl-(12)-O-5-deoxy-3-Cformyl--L-lyxofuranosyl-(14)-N,N-diamidino-D-streptamine] trisulphate, a substance produced
by the growth of certain strains of Streptomyces griseus or obtained by any other means. Stabilisers may
be added. The potency is not less than 720 I.U. per milligram calculated with reference to the dried
substance.
PRODUCTION
It is produced by methods of manufacture designed to eliminate or minimise substances lowering
blood pressure. The method of manufacture is validated to demonstrate that the product if tested
would comply with the following test:
Abnormal toxicity (2.6.9). Inject into each mouse 1 mg of the substance to be examined dissolved
in 0.5 ml of water for injections R.
CHARACTERS
A white or almost white powder, hygroscopic, very soluble in water, practically insoluble in ethanol
and in ether.
IDENTIFICATION
A. Examine by thin-layer chromatography (2.2.27), using a plate coated with a 0.75 mm layer of the
following mixture: mix 0.3 g of carbomer R with 240 ml of water R and allow to stand, with moderate
shaking, for 1 h; adjust to pH 7 by the gradual addition, with continuous shaking, of dilute sodium
hydroxide solution R and add 30 g of silica gel H R.
Heat the plate at 110C for 1 h, allow to cool and use immediately.
same solvent.
Reference solution (a). Dissolve 10 mg of streptomycin sulphate CRS in water R and dilute to 10 ml with
the same solvent.
Reference solution (b). Dissolve 10 mg of kanamycin monosulphate CRS, 10 mg of neomycin sulphate
CRS and 10 mg of streptomycin sulphate CRS in water R and dilute to 10 ml with the same solvent.
37-38
Apply separately to the plate 10 l of each solution. Develop over a path of 12 cm using a 70 g/l
solution of potassium dihydrogen phosphate R. Dry the plate in a current of warm air, and spray with a
mixture of equal volumes of a 2 g/l solution of 1,3-dihydroxynaphthalene R in alcohol R and a 460 g/l
solution of sulphuric acid R. Heat at 150C for 5 min to 10 min. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained
with reference solution (b) shows three clearly separated spots.
B. Dissolve 5 mg to 10 mg in 4 ml of water R and add 1 ml of 1M sodium hydroxide. Heat in a waterbath for 4 min. Add a slight excess of dilute hydrochloric acid R and 0.1 ml of ferric chloride solution R1.
A violet colour develops.
C. Dissolve 0.1 g in 2 ml of water R, add 1 ml of -naphthol solution R and 2 ml of a mixture of equal
volumes of strong sodium hypochlorite solution R and water R. A red colour develops.
D. Dissolve about 10 mg in 5 ml of water R and add 1 ml of 1M hydrochloric acid. Heat in a waterbath for 2 min. Add 2 ml of a 5 g/l solution of -naphthol R in 1M sodium hydroxide and heat in a
water-bath for 1 min. A faint yellow colour develops.
E. It gives the reactions of sulphates (2.3.1).
TESTS
Appearance of solution Solution S is not more intensely coloured than intensity 3 of the range of
reference solutions of the most appropriate colour (Method II, 2.2.2). Allow to stand protected from
light, at a temperature of about 20C for 24 h. Solution S is not more opalescent than reference
suspension II (2.2.1).
Methanol Examine by gas chromatography (2.2.28).
the same solvent.
Reference solution. Dilute 12.0 mg of methanol R to 100 ml with water R.
a column 1.5 m to 2.0 m long and 2 mm to 4 mm in internal diameter, packed with
ethylvinylbenzene-divinylbenzene copolymer R (150 m to 180 m),
nitrogen for chromatography R as the carrier gas at a constant flow rate of 30 ml to 40 ml per
minute,
Maintain the column at a constant temperature between 120C and 140C and the injection port
and the detector at a temperature at least 50C higher than that of the column. Inject the test solution and the reference solution. The area of the peak corresponding to methanol in the chromatogram obtained with the test solution is not greater than the area of the peak in the chromatogram
Streptomycin B Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating
substance.
Test solution. Dissolve 0.2 g of the substance to be examined in a freshly prepared mixture of 3
volumes of sulphuric acid R and 97 volumes of methanol R and dilute to 5 ml with the same mixture of
solvents. Heat under a reflux condenser for 1 h, cool, rinse the condenser with methanol R and dilute
to 20 ml with the same solvent (10 g/l solution).
Reference solution. Dissolve 36 mg of mannose R in a freshly prepared mixture of 3 volumes of sulphuric
acid R and 97 volumes of methanol R and dilute to 5 ml with the same mixture of solvents. Heat
under a reflux condenser for 1 h, cool, rinse the condenser with methanol R and dilute to 50 ml with
the same solvent. Dilute 5 ml of the solution to 50 ml with methanol R (0.3 g/l solution expressed as
streptomycin B; 1 mg of mannose R is equivalent to 4.13 mg of streptomycin B).
Apply separately to the plate 10 l of each solution. Develop over a path of 13 cm to 15 cm using a
mixture of 25 volumes of glacial acetic acid R, 25 volumes of methanol R and 50 volumes of toluene R.
Allow the plate to dry in air and spray with a freshly prepared mixture of equal volumes of a 2 g/l
solution of dihydroxynaphthalene R in alcohol R and a 20 per cent V/V solution of sulphuric acid R and
heat at 110C for 5 min. Any spot corresponding to streptomycin B in the chromatogram obtained
with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution (3.0 per cent).
37-39
0.1M sodium edetate adding 50 ml of alcohol R when the colour of the solution begins to change and
continue the titration until the violet-blue colour disappears.
Colorimetric test Dry the substance to be examined and streptomycin sulphate CRS at 60C over
diphosphorus pentoxide R at a pressure not exceeding 0.1 kPa for 24 h. Dissolve 0.100 g of the dried
substance to be examined in water R and dilute to 100.0 ml with the same solvent. Prepare a reference solution in the same manner using 0.100 g of the dried streptomycin sulphate CRS. Place 5.0 ml
of each solution separately in two volumetric flasks and in a third flask place 5 ml of water R. To each
flask add 5.0 ml of 0.2M sodium hydroxide and heat for exactly 10 min in a water-bath. Cool in ice for
exactly 5 min, add 3 ml of a 15 g/l solution of ferric ammonium sulphate R in 0.5M sulphuric acid, dilute
to 25.0 ml with water R and mix. Exactly 20 min after the addition of the ferric ammonium sulphate
solution measure the absorbance (2.2.25) of the test solution and the reference solution in a 2 cm cell
at the maximum at 525 nm, using as compensation liquid the solution prepared from 5 ml of
water R. The absorbance of the test solution is not less than 90.0 per cent of that of the reference
solution.
ASSAY
STORAGE
Store in an airtight container, protected from moisture. If the substance is sterile, store in a sterile,
LABELLING
The label states:
where applicable, the name and quantity of any added stabiliser,
__________________________________________________________________________________________________________ Ph Eur
37-40
Succinylsulfathiazole
O
O
S
N
H
HN
O
COOH
C13H13N3O5S2,H2O
373.4
116-43-8
Succinylsulfathiazole complies with the requirements of the 3rd edition of the European Pharmacopoeia
When succinylsulphathiazole is prescribed or demanded, Succinylsulfathiazole shall be dispensed or
supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Succinylsulfathiazole contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of 4-(thiazol-2-ylsulphamoyl)succinanilic acid, calculated with reference to the dried
substance.
CHARACTERS
A white or yellowish-white, crystalline powder, very slightly soluble in water, slightly soluble in
acetone and in alcohol, practically insoluble in ether. It dissolves in solutions of alkali hydroxides and
carbonates.
IDENTIFICATION
obtained with succinylsulfathiazole CRS. If the spectra obtained in the solid state with the substance to
be examined and the reference substance show differences, dissolve in hot water R, allow to
crystallise, dry the crystals carefully between two sheets of filter paper and prepare new discs.
B. To 2 g add 10 ml of water R and 10 ml of strong sodium hydroxide solution R and boil for 10 min.
Cool and adjust to pH 3.0 with hydrochloric acid R1. Cool, adjust to pH 7.0 with sodium hydrogen
carbonate solution R and filter. The precipitate, washed with water R and dried at 100C to 105C,
melts (2.2.14) at 196C to 204C. Introduce the capillary containing the precipitate into the bath at a
temperature of 190C.
C. Heat 0.1 g in a test-tube over a small flame. Fumes are evolved which blacken lead acetate paper R.
D. Place 0.1 g in a borosilicate-glass tube of about 30 ml capacity and add 0.5 g of hydroquinone R
and 1 ml of sulphuric acid R. Heat in a glycerin bath at 135C for 10 min, mixing at the beginning of
heating to obtain a homogeneous liquid phase. Allow to cool and place in iced water. Add carefully
and with shaking 15 ml of water R. Add 5 ml of toluene R, shake for 5 s to 10 s and allow to stand for
2 min; promote separation of the two layers using a stirrer. The upper (toluene) layer shows an
intense pink colour.
E. Dissolve about 10 mg of the precipitate obtained in identification test B in 200 ml of 0.1M hydrochloric acid. 2 ml of the solution gives the reaction of primary aromatic amines (2.3.1) with formation
of an orange precipitate.
TESTS
15 ml of water R. The solution is clear (2.2.1) and not more intensely coloured than reference solution Y4 or BY4 (Method II, 2.2.2).
Acidity To 2.0 g add 20 ml of water R, shake continuously for 30 min and filter. To 10 ml of the
filtrate add 0.1 ml of phenolphthalein solution R. Not more than 2 ml of 0.1M sodium hydroxide is
Sulfathiazole and other primary aromatic amines Dissolve 20 mg in a mixture of 3.5 ml of
water R, 6 ml of dilute hydrochloric acid R and 25 ml of alcohol R, previously cooled to 15C. Place
37-41
immediately in iced water and add 1 ml of a 2.5 g/l solution of sodium nitrite R. Allow to stand for
3 min, add 2.5 ml of a 40 g/l solution of sulphamic acid R and allow to stand for 5 min. Add 1 ml of a
4 g/l solution of naphthyl-ethylenediamine dihydrochloride R and dilute to 50 ml with water R. Measured
at 550 nm, the absorbance (2.2.25) is not greater than that of a standard prepared at the same time
and in the same manner using a mixture of 1.5 ml of a solution containing 10 mg of sulfathiazole R
and 0.5 ml of hydrochloric acid R per 100 ml; 2 ml of water R; 6 ml of dilute hydrochloric acid R; and
25 ml of alcohol R.
100C to 105C.
ASSAY
Dissolve 0.300 g in 100 ml of a mixture of 1 volume of hydrochloric acid R and 2 volumes of water R.
Heat under a reflux condenser for 1 h. Carry out the determination of primary aromatic aminonitrogen (2.5.8), determining the end-point electrometrically.
1 ml of 0.1M sodium nitrite is equivalent to 35.54 mg of C13H13N3O5S2.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
37-42
Sucrose
Refined Sugar
HOCH2
CH2OH
O
OH
HO
HO
C12H22O11
CH2OH
O
OH
OH
342.3
57-30-1
Sucrose complies with the requirements of the 3rd edition of the European Pharmacopoeia [0204]. These
Preparation
Syrup
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sucrose is D-fructofuranosyl -D-glucopyranoside. It contains no additives.
CHARACTERS
A white, crystalline powder or lustrous, dry, colourless or white crystals, very soluble in water, slightly
soluble in alcohol, practically insoluble in ethanol.
IDENTIFICATION
obtained with sucrose CRS.
Test solution. Dissolve 10 mg of the substance to be examined in a mixture of 2 volumes of water R
Reference solution (a). Dissolve 10 mg of sucrose CRS in a mixture of 2 volumes of water R and 3
Reference solution (b). Dissolve 10 mg each of fructose CRS, glucose CRS, lactose CRS and sucrose CRS
in a mixture of 2 volumes of water R and 3 volumes of methanol R and dilute to 20 ml with the same
volumes of anhydrous acetic acid R and 50 volumes of ethylene chloride R, measured accurately as a
slight excess of water causes cloudiness of the solution. Dry the plate in a current of warm air. Repeat
the development immediately, after renewing the mobile phase. Dry the plate in a current of warm air
and spray evenly with a solution of 0.5 g of thymol R in a mixture of 5 ml of sulphuric acid R and
95 ml of alcohol R. Heat at 130C for 10 min. The principal spot in the chromatogram obtained with
reference solution (b) shows four clearly separated spots.
C. Dilute 1 ml of solution S (see Tests) to 100 ml with water R. To 5 ml of the solution add 0.15 ml
of freshly prepared copper sulphate solution R and 2 ml of freshly prepared dilute sodium hydroxide
solution R. The solution is blue and clear and remains so after boiling. To the hot solution add 4 ml
of dilute hydrochloric acid R and boil for 1 min. Add 4 ml of dilute sodium hydroxide solution R. An
orange precipitate is formed immediately.
TESTS
Solution S Dissolve 50.0 g in carbon dioxide free water R prepared from distilled water R and dilute to
37-43
colourless. Not more than 0.3 ml of 0.01M sodium hydroxide is required to change the colour of the
indicator to pink.
Conductivity (2.2.38). Not more than 35 Scm-1. Dissolve 31.3 g in carbon dioxide-free water R
prepared from distilled water R and dilute to 100.0 ml with the same solvent. Measure the
conductivity of the solution (C1), while gently stirring with a magnetic stirrer, and that of the water
used for preparing the solution (C2). The readings must be stable within 1 per cent over a period of
30 s. Calculate the conductivity of the solution of the substance to be examined from the expression:
C1 0.35C2
solvent. The specific optical rotation is +66.3 to +67.0.
Dextrins If intended for use in the preparation of large volume infusions, it complies with the test for
dextrins. To 2 ml of solution S add 8 ml of water R, 0.05 ml of dilute hydrochloric acid R and 0.05 ml
of 0.05M iodine. The solution remains yellow.
Glucose and invert sugar To 5 ml of solution S in a test-tube about 150 mm long and 16 mm in
diameter add 5 ml of water R, 1.0 ml of 1M sodium hydroxide and 1.0 ml of a 1 g/l solution of
methylene blue R. Mix and place in a water-bath. After exactly 2 min, take the tube out of the bath
and examine the solution immediately. The blue colour does not disappear completely (0.04 per
cent). Ignore any blue colour at the air/solution interface.
Sulphites Dissolve 5.0 g in 40 ml of water R, add 2.0 ml of 0.1M sodium hydroxide and dilute to
50.0 ml with water R. To 10.0 ml of the solution, add 1 ml of 3M hydrochloric acid, 2.0 ml of
decolorised fuchsin solution R1 and 2.0 ml of a 0.5 per cent V/V solution of formaldehyde R. Allow to
stand for 30 min and measure the absorbance (2.2.25) at the maximum at 583 nm. Prepare a
standard as follows. Dissolve 76 mg of sodium metabisulphite R in water R and dilute to 50.0 ml with
the same solvent. Dilute 5.0 ml of this solution to 100.0 ml with water R. To 3.0 ml of this solution
add 4.0 ml of 0.1M sodium hydroxide and dilute to 100.0 ml with water R. To 10.0 ml of this solution
add immediately 1 ml of 3M hydrochloric acid, 2.0 ml of decolorised fuchsin solution R1 and 2.0 ml of a
0.5 per cent V/V solution of formaldehyde R. Allow to stand for 30 min and measure the absorbance
at the maximum at 583 nm. Use as the compensation liquid for both measurements a solution
prepared in the same manner using 10.0 ml of water R. The absorbance of the test solution is not
greater than that of the standard (15 ppm of SO2). The test is not valid unless the standard shows a
clearly visible violet-red colour.
Lead Not more than 0.5 ppm of Pb, determined by atomic absorption spectrometry (Method II,
2.2.23), using an apparatus equipped with a graphite furnace.
Test solution. Dissolve 50 mg in 0.5 ml of lead-free nitric acid R in a polyfluorocarbon-lined digestion
bomb and heat at 150C for 5 h. Allow to cool and dilute to 5.0 ml with water R.
Measure the absorbance at 283.3 nm, maintaining the drying temperature of the furnace at 110C,
the ashing temperature at 600C and the atomising temperature at 2100C.
Loss on drying (2.2.32). Not more than 0.1 per cent, determined on 2.000 g, by heating in an oven
at 105C for 3 h.
Bacterial endotoxins (2.6.14). If intended for use in the preparation of large-volume infusions, not
more than 0.25 I.U. of bacterial endotoxins per milligram.
STORAGE
LABELLING
The label states, where applicable, that the substance is suitable for use in the manufacture of largevolume parenteral dosage forms.
__________________________________________________________________________________________________________ Ph Eur
37-44
Sufentanil
1/01
S
O
N
N
CH3
MeO
C22H30N2O2S
386.6
56030-54-7
Sufentanil complies with the requirements of the 3rd edition of the European Pharmacopoeia [1569]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sufentanil contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of
N-[4-(methoxymethyl)-1-[2-(thiophen-2-yl)ethyl]piperidin-4-yl]-N-phenylpropanamide, calculated
CHARACTERS
A white or almost white powder, practically insoluble in water, freely soluble in alcohol and in
methanol.
IDENTIFICATION
spectrum of sufentanil.
TESTS
Reference solution (a). In order to prepare in situ the degradation compound (sufentanil impurity E),
dissolve 10 mg of the substance to be examined in 10.0 ml of dilute hydrochloric acid R. Heat on a
water-bath under a reflux condenser for 4 h. Add 10.0 ml of dilute sodium hydroxide R. Evaporate to
dryness on a water-bath. Cool and take up the residue in 10 ml of methanol R. Filter.
Mobile phase A. A 5 g/l solution of ammonium carbonate R in a mixture of 10 volumes of tetrahydrofuran R and 90 volumes of water R,
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
0 15
90 40
10 60
Linear gradient
15 20
40
60
20 21
40 90
60 10
Isocratic
Switch to initial
eluent composition
21 25
90
10
Re-equilibration
37-45
Equilibrate the column for at least 30 min with acetonitrile R and then equilibrate with the initial
conditions, the retention times are: impurity E about 12 min and sufentanil about 13 min. The test
is not valid unless the resolution between the peaks corresponding to sufentanil and impurity E is at
least 4.0. If necessary, adjust the concentration of acetonitrile in the mobile phase or adjust the time
programme for the linear gradient elution.
Inject 10 l of methanol R as a blank, 10 l of the test solution and 10 l of reference solution (b).
solution (b) (0.25 per cent); the sum of the areas of all the peaks, apart from the principal peak, is
solution (b) (0.5 per cent). Disregard any peak due to the blank and any peak with an area less than
0.2 times the area of the principal peak in the chromatogram obtained with reference solution (b)
(0.05 per cent).
60C for 2 h.
ASSAY
Dissolve 0.300 g in 50 ml of a mixture of 1 volume of anhydrous acetic acid R and 7 volumes of
methyl ethyl ketone R and titrate with 0.1M perchloric acid, using 0.2 ml of naphtholbenzein solution R as
indicator.
STORAGE
IMPURITIES
Qualified impurities: D, F, H.
Other detectable impurities: A, B, C, E, G, I.
Ar =
R=
NH
O
Me
N
OMe
Ar
A. N-[4-(methoxymethyl)piperidin-4-yl]-N-phenylpropanamide,
O
N
O
Me
N
OMe
Ar
B. cis-N-[4-(methoxymethyl)-1-[2-(thiophen-2-yl)ethyl]piperidin-4-yl 1-oxide]-Nphenylpropanamide,
N
Ar
N
H
OH
C. [4-(phenylamino)-1-[2-(thiophen-2-yl)ethyl]piperidin-4-yl]methanol,
N
O
Me
N
Ar
OMe
D. N-[4-(methoxymethyl)-1-[2-(thiophen-2-yl)ethyl]piperidin-4-yl]-N-phenylacetamide,
37-46
HN
Ar
OMe
E. 4-(methoxymethyl)-N-phenyl-1-[2-(thiophen-2-yl)ethyl]piperidin-4-amine,
S
N
O
Me
N
Ar
OMe
F. N-[4-(methoxymethyl)-1-[2-(thiophen-3-yl)ethyl]piperidin-4-yl]-N-phenylpropanamide,
R
O
Me
Et
O
Ar
G. [4-(phenylpropanoylamino)-1-[2-(thiophen-2-yl)ethyl]piperidin-4-yl]methyl propanoate,
N
Me
Ar
OMe
H. N-[4-(methoxymethyl)-1-[2-(thiophen-2-yl)ethyl]piperidin-4-yl]-N-phenylbutanamide,
O
N
O
Me
N
Ar
OMe
I. trans-N-[4-(methoxymethyl)-1-[2-(thiophen-2-yl)ethyl]piperidin-4-yl 1-oxide]-Nphenylpropanamide.
__________________________________________________________________________________________________________ Ph Eur
37-47
Sufentanil Citrate
corrected 1/01
N
O
CH3
CH2COOH
,HO C
MeO
COOH
CH2COOH
C28H38N2O9S
578.7
60561-54-7
Sufentanil Citrate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1269].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sufentanil citrate contains not less than 99.0 per cent and not more than the equivalent of 101.0 per
cent of N-[4-(methoxymethyl)-1-[2-(thiophen-2-yl)ethyl]piperidin-4-yl]-N-phenylpropanamide
citrate, calculated with reference to the dried substance.
CHARACTERS
A white or almost white powder, soluble in water and in alcohol, freely soluble in methanol.
IDENTIFICATION
spectrum of sufentanil citrate.
TESTS
Appearance of solution Dissolve 0.2 g of the substance to be examined in water R and dilute to
20 ml with the same solvent. The solution is clear (2.2.1) and colourless (Method II, 2.2.2).
Reference solution (a). In order to prepare in situ the degradation compound (sufentanil impurity E),
dissolve 10 mg of the substance to be examined in 10.0 ml of dilute hydrochloric acid R. Heat on a
water-bath under a reflux condenser for 4 h. Add 10.0 ml of dilute sodium hydroxide solution R.
Evaporate to dryness on a water-bath. Cool and take up the residue in 10 ml of methanol R. Filter.
as mobile phase at a flow rate of 1.5 ml/min the following mixtures:
Mobile phase A. A 5 g/l solution of ammonium carbonate R in a mixture of 10 volumes of tetrahydrofuran R and 90 volumes of water R,
Time
(min)
Mobile phase A
(per cent V/V)

(per cent V/V)
0 15
15 20
20 25
90 40
40
90
10 60
60
10
25 = 0
90
10
linear gradient
isocratic elution
switch to initial
eluent composition
restart gradient
37-48
Equilibrate the column for at least 30 min with acetonitrile R and then equilibrate at the initial
conditions, the retention times are: sufentanil impurity E about 12 min and sufentanil about 13 min.
The test is not valid unless the resolution between the peaks corresponding to sufentanil and
sufentanil impurity E is at least 4.0. If necessary, adjust the concentration of acetonitrile in the mobile
phase or adjust the time programme for the linear gradient elution.
with reference solution (b) (0.5 per cent); the sum of the areas of all of the peaks, apart from the
principal peak, is not greater than twice the area of the principal peak in the chromatogram obtained
with reference solution (b) (1 per cent). Disregard any peak obtained with the blank run, any peak
with a retention time relative to the main compound of 0.05 or less, and any peak with an area less
(b).
60C.
ASSAY
ethyl ketone R and titrate with 0.1M perchloric acid, using 0.2 ml of naphtholbenzein solution R as
indicator.
STORAGE
IMPURITIES
Ar =
R=
NH
O
Me
N
OMe
Ar
A. N-[4-(methoxymethyl)piperidin-4-yl]-N-phenylpropanamide,
O
N
O
Me
N
OMe
Ar
B. cis-N-[4-(methoxymethyl)-1-[2-(thiophen-2-yl)ethyl] piperidin-4-yl 1-oxide]-Nphenylpropanamide,
N
Ar
N
H
OH
C. [4-(phenylamino)-1-[2-(thiophen-2-yl)ethyl]piperidin-4-yl]methanol,
N
O
Me
N
Ar
OMe
D. N-[4-(methoxymethyl)-1-[2-(thiophen-2-yl)ethyl]piperidin-4-yl]-N-phenylacetamide,
37-49
HN
Ar
OMe
E. 4-(methoxymethyl)-N-phenyl-1-[2-(thiophen-2-yl)ethyl] piperidin-4-amine,
S
N
O
Me
N
Ar
OMe
F. N-[4-(methoxymethyl)-1-[2-(thiophen-3-yl)ethyl]piperidin-4-yl]-N-phenylpropanamide,
N
O
Me
R
Et
Ar
G. [4-(phenylpropanoylamino)-1-[2-(thiophen-2-yl)ethyl] piperidin-4-yl]methyl propanoate,

N
Me
Ar
OMe
H. N-[4-(methoxymethyl)-1-[2-(thiophen-2-yl)ethyl]piperidin-4-yl]-N-phenylbutanamide,
O
N
O
Me
N
Ar
OMe
I. trans-N-[4-(methoxymethyl)-1-[2-(thiophen-2-yl)ethyl] piperidin-4-yl 1-oxide]-Nphenylpropanamide.

__________________________________________________________________________________________________________ Ph Eur
38-1
Sugar Spheres
1/01
Sugar Spheres comply with the requirements of the 3rd edition of the European Pharmacopoeia [1570]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sugar spheres contain not more than 92 per cent of sucrose, calculated on the dried basis. The
remainder consists of maize starch and may also contain starch hydrolysates and colour additives.
The diameter of sugar spheres varies usually from 200 m to 2000 m and the upper and lower limits
of the size of the sugar spheres are stated on the label.
IDENTIFICATION
Test solution. Mix 2 ml of solution S (see Tests) with 3 ml of methanol R. Dilute to 20 ml with a
mixture of 2 volumes of water R and 3 volumes of methanol R.
Reference solution (a). Dissolve 10 mg of sucrose CRS in a mixture of 2 volumes of water R and 3
Reference solution (b). Dissolve 10 mg of fructose CRS, 10 mg of glucose CRS, 10 mg of lactose CRS and
10 mg of sucrose CRS in a mixture of 2 volumes of water R and 3 volumes of methanol R and dilute to
20 ml with the same mixture of solvents.
Apply to the plate 2 l of each solution and thoroughly dry the starting points. Develop over a path of
15 cm using a mixture of 10 volumes of water R, 15 volumes of methanol R, 25 volumes of anhydrous
acetic acid R and 50 volumes of ethylene chloride R, measured accurately as a slight excess of water
causes cloudiness of the solution. Dry the plate in a current of warm air. Repeat the development
immediately after renewing the mobile phase. Dry the plate in a current of warm air and spray evenly
with a 5 g/l solution of thymol R in a mixture of 5 volumes of sulphuric acid R and 95 volumes of
alcohol R. Heat at 130C for 10 min. The principal spot in the chromatogram obtained with the test
solution is similar in position, colour and size to the principal spot in the chromatogram obtained
with reference solution (a). The test is not valid unless the chromatogram obtained with reference
solution (b) shows 4 clearly separated spots.
B. To water slurry of the insoluble portion obtained (see Assay), add 0.05 ml of iodine solution R1. A
dark-blue is produced which disappears on heating.
C. To 5 ml of solution S add 0.15 ml of freshly prepared copper sulphate solution R and 2 ml of freshly
prepared dilute sodium hydroxide solution R. The solution is blue and clear and remains so after boiling.
To the hot solution add 4 ml of dilute hydrochloric acid R and boil for 1 min. Add 4 ml of dilute sodium
hydroxide solution R. An orange precipitate is formed immediately.
TESTS
Solution S To 0.5 g in a 100 ml volumetric flask add 80 ml of water R and shake until the sucrose is
dissolved. Dilute to 100.0 ml with water R. Filter under vacuum to obtain a clear solution.
Fineness (2.9.12). Minimum of 90 per cent (m/m) of the sugar spheres are between the lower and
the upper limits of the size of the sugar spheres stated on the label.
100C to 105C for 4 h.
Sulphated ash (2.4.14). Not more than 0.2 per cent, determined on 2 g.
Microbial contamination Total viable aerobic count (2.6.12) not more than 103 bacteria and 102
fungi per gram, determined by plate count. It complies with the tests for Escherichia coli and
Salmonella (2.6.13).
ASSAY
Sucrose content
Weigh 10.000 g of ground sugar spheres in a 100 ml graduated flask and complete to 100.0 ml with
water R. Stir and decant. Filter under vacuum to obtain a clear solution (the insoluble portion is used
for the identification test B). Measure the angle of optical rotation (2.2.7) and calculate the sucrose
percentage content as follows:
38-2
106
66.5 l m (100 H )
= angle of rotation,
l = length of the polarimeter tube, in decimetres,
m = exact weight of the granules sample, in grams,
H = loss on drying.
LABELLING
The label states:
the upper and the lower limits of the size of the sugar spheres,
any added colour additives,
any added starch hydrolysates.
__________________________________________________________________________________________________________ Ph Eur
38-3
Compressible Sugar
Definition Compressible Sugar contains Sucrose and maltodextrin or dried glucose syrup that are
co-crystallised. It contains 95.0 to 98.0% of Sucrose and 2 to 5% of dried glucose syrup or
maltodextrin. It may contain a suitable lubricant, invert sugar or suitable colouring matter.
Characteristics Dry free-flowing powder or microcrystalline agglomerates.
Dissolve 20 g in 80 ml of water, dilute to 100 ml with water and filter (solution S).
Identification
compressible sugar (RS 401).
B. The specific optical rotation of the uninverted solution obtained in the Assay is not less than 62.6
and the acid-inverted solution is laevorotatory.
Conductivity Not more than 35 Scm-1, Appendix V O. Dissolve 31.3 g in carbon dioxide-free water
prepared from distilled water and dilute to 100 ml with the same solvent. Measure the conductivity of
the solution (C1), while gently stirring with a magnetic stirrer, and that of the water used for preparing the solution (C2). The readings must be stable within 1% over a period of 30 seconds. Calculate
the conductivity of the solution of the substance being examined from the expression C10.35C2.
Calcium Add 1 ml of ammonium oxalate to 5 ml of solution S. The solution remains clear for not less
than 1 minute.
Lead Not more than 0.5 ppm of Pb when determined by atomic absorption spectrometry, Appendix
II D, Method II, using an apparatus equipped with a graphite furnace and the following solution.
Dissolve 50 mg in 0.5 ml of lead-free nitric acid in a polyfluorocarbon-lined digestion bomb and heat
at 150 for 5 hours. Allow to cool and dilute to 5 ml with water. Measure the absorbance at
283.3 nm, maintaining the drying temperature of the furnace at 110, the ashing temperature at 600
and the atomising temperature at 2100.
Chloride 2 ml of solution S diluted to 15 ml with water complies with the limit test for chlorides,
Sulphate 7.5 ml of solution S diluted to 15 ml with water complies with the limit test for sulphates,
Loss on drying When dried at 105 for 4 hours, loses 0.25 to 1.0% of its weight. Use 1 g.
Assay To 26 g of the substance being examined, previously dried, add 0.3 ml of a saturated aqueous
solution of lead(II) acetate and 90 ml of water, shake, dilute to 100 ml with water and mix. Distribute
evenly on the surface of a sheet of medium-fast filter paper 8 g of chromatographic siliceous earth
and filter the solution with the aid of vacuum, discarding the first 20 ml of the filtrate. Transfer 25 ml
of the filtrate into each of two 50-ml flasks. Slowly add 6 ml of 2M hydrochloric acid to one flask while
rotating it, add 10 ml of water, mix, place the flask in a water bath at 60, continuously shake for 3
minutes and allow the flask to stand in the water bath for a further 7 minutes. Immediately cool to
20 and dilute the solution with water to 50 ml and mix. Cool the contents of the second flask to 20,
dilute with water to volume and mix. Maintain both flasks at 20 for 30 minutes. Determine the
specific optical rotation, Appendix V F, of each solution at 20. Calculate the percentage of C12H22O11
using the expression 100(a0 - ai)/88.3 where a0 and ai are the specific optical rotations of the
uninverted and acid-inverted solutions respectively.
Storage Compressible Sugar should be kept in a well-closed container.
38-4
Sulfacetamide Sodium
Soluble Sulfacetamide
O
O
S
N
CH3
Na
H2N
C8H9N2NaO3S,H2O
254.2
6209-17-2
Sulfacetamide Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia
When sulphacetamide sodium is prescribed or demanded, Sulfacetamide Sodium shall be dispensed
or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sulfacetamide sodium contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of the sodium derivative of N-[(4-aminophenyl)sulphonyl]acetamide, calculated with
CHARACTERS
A white or yellowish-white, crystalline powder, freely soluble in water, slightly soluble in ethanol,
IDENTIFICATION
First identification: B, F.
A. Dissolve 0.1 g in phosphate buffer solution pH 7.0 R and dilute to 100.0 ml with the same buffer
solution. Dilute 1.0 ml of the solution to 100.0 ml with phosphate buffer solution pH 7.0 R. Examined
between 230 nm and 350 nm (2.2.25), the solution shows an absorption maximum at 255 nm. The
specific absorbance at the maximum is 660 to 720, calculated with reference to the anhydrous
substance.
obtained with sulfacetamide sodium CRS.
C. Dissolve 1 g in 10 ml of water R, add 6 ml of dilute acetic acid R and filter. The precipitate, washed
with a small quantity of water R and dried at 100C to 105C for 4 h, melts (2.2.14) at 181C to
185C.
D. Dissolve 0.1 g of the precipitate obtained in identification test C in 5 ml of alcohol R. Add 0.2 ml
of sulphuric acid R and heat. The odour of ethyl acetate is perceptible.
E. Dissolve about 1 mg of the precipitate obtained in identification test C, with heating, in 1 ml of
water R. The solution gives the reaction of primary aromatic amines (2.3.1) with formation of an
orange-red precipitate.
F. Solution S (see Tests) gives the reactions of sodium (2.3.1).
TESTS
coating substance.
same solvent.
Reference solution (a). Dissolve 5 mg of sulphanilamide R in water R and dilute to 10 ml with the same
solvent.
38-5
Reference solution (c). Dissolve 5 mg of sulphanilamide R in 10 ml of the test solution.
10 volumes of concentrated ammonia R, 25 volumes of ethanol R, 25 volumes of water R and 50
volumes of butanol R. Allow the plate to dry in air and spray with dimethylaminobenzaldehyde solution R2. Any spot in the chromatogram obtained with the test solution, apart from the principal spot,
is not more intense than the spot in the chromatogram obtained with reference solution (a) (0.5 per
cent), and not more than one such spot is more intense than the spot in the chromatogram obtained
with reference solution (b) (0.25 per cent). The test is not valid unless the chromatogram obtained
with reference solution (c) shows two clearly separated spots.
Sulphates (2.4.13). Dissolve 2.5 g in distilled water R and dilute to 25 ml with the same solvent. Add
25 ml of dilute acetic acid R, shake for 30 min and filter. 15 ml of the filtrate complies with the limit
Heavy metals (2.4.8). 12 ml of the filtrate obtained in the test for sulphates complies with limit test
A for heavy metals (20 ppm). Prepare the standard using lead standard solution (1 ppm Pb) R.
ASSAY
Dissolve 0.500 g in a mixture of 50 ml of water R and 20 ml of dilute hydrochloric acid R. Cool the
solution in iced water and carry out the determination of primary aromatic amino-nitrogen (2.5.8),
determining the end-point electrometrically.
1 ml of 0.1M sodium nitrite is equivalent to 23.62 mg of C8H9N2NaO3S.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
38-6
Sulfadiazine
O
S
N
H
H2N
C10H10N4O2S
250.3
68-35-9
Sulfadiazine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0294]. These
Preparation
Sulfadiazine Injection
When sulphadiazine is prescribed or demanded, Sulfadiazine shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sulfadiazine contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of 4-amino-N-pyrimidin-2-ylbenzenesulphonamide, calculated with reference to the dried substance.
CHARACTERS
White, yellowish-white or pinkish-white, crystalline powder or crystals, practically insoluble in water,
slightly soluble in acetone, very slightly soluble in alcohol. It dissolves in solutions of alkali hydroxides
and in dilute mineral acids.
IDENTIFICATION
obtained with sulfadiazine CRS. Examine the substances prepared as discs.
chromatogram obtained with test solution (a) corresponds in position and size to the principal spot in
C. Place 3 g in a dry tube. Immerse the lower part of the tube, inclined at 45, in a silicone oil bath
and heat to about 270C. The substance to be examined decomposes and a white or yellowish-white
sublimate is formed which, after recrystallisation from toluene R and drying at 100C, melts (2.2.14)
at 123C to 127C.
D. Dissolve about 5 mg in 10 ml of 1M hydrochloric acid. Dilute 1 ml of the solution to 10 ml with
water R. The solution, without further acidification, gives the reaction of primary aromatic amines
(2.3.1).
TESTS
5 ml of water R. The solution is not more intensely coloured than reference solution Y5, BY5 or GY5
(Method II, 2.2.2).
Acidity To 1.25 g, finely powdered, add 25 ml of carbon dioxide-free water R. Heat at about 70C for
5 min. Cool in iced water for about 15 min and filter. To 20 ml of the filtrate add 0.1 ml of bromothymol blue solution R1. Not more than 0.2 ml of 0.1M sodium hydroxide is required to change the
coating substance.
Test solution (a). Dissolve 20 mg of the substance to be examined in 3 ml of a mixture of 2 volumes of
concentrated ammonia R and 48 volumes of methanol R and dilute to 5.0 ml with the same mixture of
solvents.
Test solution (b). Dissolve 0.10 g of the substance to be examined in 0.5 ml of concentrated ammonia R
and dilute to 5.0 ml with methanol R. If the solution is not clear, heat gently until dissolution is
complete.
38-7
Reference solution (a). Dissolve 20 mg of sulfadiazine CRS in 3 ml of a mixture of 2 volumes of concentrated ammonia R and 48 volumes of methanol R and dilute to 5.0 ml with the same mixture of
solvents.
Reference solution (b). Dilute 1.25 ml of test solution (a) to 50 ml with a mixture of 2 volumes of
concentrated ammonia R and 48 volumes of methanol R.
volumes of dilute ammonia R1, 5 volumes of water R, 40 volumes of nitromethane R and 50 volumes of
dioxan R. Dry the plate at 100C to 105C and examine in ultraviolet light at 254 nm. Any spot in
the chromatogram obtained with test solution (b), apart from the principal spot, is not more intense
100C to 105C.
ASSAY
Dissolve 0.200 g in a mixture of 20 ml of dilute hydrochloric acid R and 50 ml of water R. Cool the
solution in iced water. Carry out the determination of primary aromatic amino-nitrogen (2.5.8),
1 ml of 0.1M sodium nitrite is equivalent to 25.03 mg of C10H10N4O2S.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
38-8
Sulfadimidine
Me
O
O
S
N
H
Me
H2N
C12H14N4O2S
278.3
57-68-1
Sulfadimidine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0295].
When sulphadimidine is prescribed or demanded, Sulfadimidine shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sulfadimidine contains not less than 99.0 per cent and not more than the equivalent of 101.0 per
cent of 4-amino-N-(4,6-dimethylpyrimidin-2-yl)benzenesulphonamide, calculated with reference to
CHARACTERS
White or almost white powder or crystals, very slightly soluble in water and in ether, soluble in
acetone, slightly soluble in alcohol. It dissolves in solutions of alkali hydroxides and in dilute mineral
acids.
IDENTIFICATION
obtained with sulfadimidine CRS. Examine the substances prepared as discs.
chromatogram obtained with test solution (a) corresponds in position and size to the principal spot in
C. Place 3 g in a dry tube. Immerse the lower part of the tube, inclined at 45, in a silicone oil bath
and heat to about 270C. The substance to be examined decomposes and a white or yellowish-white
sublimate is formed which, after recrystallisation from toluene R and drying at 100C, melts (2.2.14)
at 150C to 154C.
(2.3.1).
TESTS
(Method II, 2.2.2).
Acidity To 1.25 g, finely powdered, add 25 ml of carbon dioxide-free water R. Heat at about 70C for
5 min. Cool in iced water for about 15 min and filter. To 20 ml of the filtrate add 0.1 ml of
bromothymol blue solution R1. Not more than 0.2 ml of 0.1M sodium hydroxide is required to change the
coating substance.
Test solution (a). Dissolve 20 mg of the substance to be examined in 3 ml of a mixture of 2 volumes of
solvents.
Test solution (b). Dissolve 0.10 g of the substance to be examined in 0.5 ml of concentrated ammonia R
and dilute to 5.0 ml with methanol R. If the solution is not clear, heat gently until dissolution is
complete.
38-9
Reference solution (a). Dissolve 20 mg of sulfadimidine CRS in 3 ml of a mixture of 2 volumes of
solvents.
Reference solution (b). Dilute 1.25 ml of test solution (a) to 50 ml with a mixture of 2 volumes of
the chromatogram obtained with test solution (b), apart from the principal spot, is not more intense
100C to 105C.
ASSAY
Dissolve 0.250 g in a mixture of 20 ml of dilute hydrochloric acid R and 50 ml of water R. Cool the
solution in iced water. Carry out the determination of primary aromatic amino-nitrogen (2.5.8),
STORAGE
__________________________________________________________________________________________________________ Ph Eur
38-10
Sulfadimidine Sodium
Me
O
O
S
N
Me
Na
H2N
C12H13N4NaO2S
300.3
1981-58-4
Definition Sulfadimidine Sodium is the sodium salt of N 1-(4,6-dimethylpyrimidin-2-yl)sulphanilamide. It contains not less than 98.0% and not more than 101.0% of C12H13N4NaO2S, calculated
Characteristics White or creamy white crystals or powder; odourless or almost odourless; hygroscopic.
Freely soluble in water; sparingly soluble in ethanol (96%).
Identification
A. Dissolve 0.1 g in 10 ml of water, acidify with 1M hydrochloric acid, filter, wash the precipitate with
water and dry the residue at 105. The infrared absorption spectrum of the residue, Appendix II A, is
concordant with the reference spectrum of sulfadimidine (RS 325).
B. Acidify a solution with 6M acetic acid. A precipitate is produced which, after washing with cold
water and drying at 105, yields the reaction characteristic of primary aromatic amines, Appendix VI,
producing a bright orange-red precipitate.
C. Incinerate 0.5 g. The residue, when moistened with hydrochloric acid and introduced on a
platinum wire into the flame of a Bunsen burner, imparts a yellow colour to the flame.
Clarity and colour of solution A 33.3% w/v solution is clear, Appendix IV A, and not more
intensely coloured than reference solution Y4, Appendix IV B, Method I.
gel H as the coating substance and a mixture of 18 volumes of 10M ammonia and 90 volumes of
butan-1-ol as the mobile phase. Apply separately to the plate 10 l of each of the following solutions.
Prepare solution (1) by dissolving the substance being examined in 1 volume of 13.5M ammonia and
then diluting with 9 volumes of ethanol (96%) to produce a 1.0% w/v solution. Solution (2) contains
0.0050% w/v of sulphanilamide in a mixture of 1 volume of 13.5M ammonia and 9 volumes of ethanol
(96%). After removal of the plate, heat it at 105 for 10 minutes and spray with a 0.1% w/v solution
of 4-dimethylaminobenzaldehyde in ethanol (96%) containing 1% v/v of hydrochloric acid. Any secondary
spot in the chromatogram obtained with solution (1) is not more intense than the spot in the chromatogram obtained with solution (2) (0.5%).
1 g.
Assay Dissolve 0.5 g in a mixture of 75 ml of water and 10 ml of hydrochloric acid, add 3 g of
potassium bromide, cool in ice and titrate slowly with 0.1M sodium nitrite VS, stirring constantly and
determining the end point electrometrically. Each ml of 0.1M sodium nitrite VS is equivalent to
30.03 mg of C12H13N4NaO2S.
Storage Sulfadimidine Sodium should be protected from light.
When sulphadimidine sodium is prescribed or demanded, Sulfadimidine Sodium shall be dispensed
or supplied.
38-11
Sulfadoxine
S
N
H
OMe
OMe
H2N
C12H14N4O4S
310.3
2447-57-6
Sulfadoxine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0740]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sulfadoxine contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of 4-amino-N-(5,6-dimethoxypyrimidin-4-yl)benzenesulphonamide, calculated with reference to the
dried substance.
CHARACTERS
White or yellowish-white crystalline powder or crystals, very slightly soluble in water, slightly soluble
in alcohol and in methanol, practically insoluble in ether. It dissolves in solutions of alkali hydroxides
and in dilute mineral acids.
IDENTIFICATION
obtained with sulfadoxine CRS. Examine the substances prepared as discs.
C. Dissolve 0.5 g in 1 ml of a 40 per cent V/V solution of sulphuric acid R, heating gently. Continue
heating until a crystalline precipitate appears (about 2 min). Allow to cool and add 10 ml of dilute
sodium hydroxide solution R. Cool again. Add 25 ml of ether R and shake for 5 min. Separate the ether
layer, dry over anhydrous sodium sulphate R and filter. Evaporate the solvent by heating in a waterbath. The residue melts (2.2.14) at 80C to 82C or at 90C to 92C.
(2.3.1).
TESTS
(Method II, 2.2.2).
Acidity To 1.25 g, finely powdered, add 25 ml of carbon dioxide-free water R. Heat at 70C for 5 min.
Cool in a bath of iced water for about 15 min and filter. To 20 ml of the filtrate add 0.1 ml of
bromothymol blue solution R1. Not more than 0.2 ml of 0.1M sodium hydroxide is required to change the
coating substance.
Test solution (a). Dissolve 0.10 g of the substance to be examined in 3 ml of a mixture of 2 volumes of
concentrated ammonia R and 48 volumes of methanol R and dilute to 5 ml with the same mixture of
solvents.
Test solution (b). Dilute 1 ml of test solution (a) to 5 ml with a mixture of 2 volumes of concentrated
Reference solution (a). Dissolve 20 mg of sulfadoxine CRS in 3 ml of a mixture of 2 volumes of concen-
38-12
trated ammonia R and 48 volumes of methanol R and dilute to 5 ml with the same mixture of solvents.
Reference solution (b). Dilute 2.5 ml of test solution (b) to 100 ml with a mixture of 2 volumes of
100C to 105C.
ASSAY
Carry out the determination of primary aromatic amino-nitrogen (2.5.8), using 0.250 g and determining the end-point electrometrically.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
38-13
Sulfafurazole
Me
O
Me
O
S
N
O
N
H
H2N
C11H13N3O3S
267.3
127-69-5
Sulfafurazole complies with the requirements of the 3rd edition of the European Pharmacopoeia [0741]. These
When sulphafurazole or sulphisoxazole is prescribed or demanded, Sulfafurazole shall be dispensed
or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sulfafurazole contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of 4-amino-N-(3,4-dimethylisoxazol-5-yl)benzenesulphonamide, calculated with reference to the
dried substance.
CHARACTERS
White or yellowish-white, crystalline powder or crystals, practically insoluble in water, sparingly
soluble in alcohol, slightly soluble in ether and in methylene chloride. It dissolves in solutions of alkali
hydroxides and in dilute mineral acids.
IDENTIFICATION
obtained with sulfafurazole CRS. Examine the substances prepared as discs.
C. To 0.5 g add 1 ml of a 40 per cent V/V solution of sulphuric acid R and heat over a low flame to
dissolve. Continue heating until a crystalline precipitate appears (about 2 min). Allow to cool and
add 10 ml of dilute sodium hydroxide solution R. Cool. Shake the solution for 5 min with 25 ml of
ether R. Separate the ether layer, dry over anhydrous sodium sulphate R and filter. Evaporate the
solvent by heating on a water-bath. The residue melts (2.2.14) at 119C to 123C.
(2.3.1).
TESTS
5 ml of water R, with gently warming if necessary. The solution is not more intensely coloured than
reference solution Y6, BY6 or GY6 (Method II, 2.2.2).
Cool in iced water for about 15 min and filter. To 20 ml of the filtrate add 0.1 ml of bromothymol blue
solution R1. Not more than 0.2 ml of 0.1M sodium hydroxide is required to change the colour of the
indicator.
coating substance.
solvents.
38-14
Reference solution (a). Dissolve 20 mg of sulfafurazole CRS in 3 ml of a mixture of 2 volumes of
solvents.
volume of concentrated ammonia R, 25 volumes of methanol R and 75 volumes of methylene chloride R.
Dry the plate at 100C to 105C and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with test solution (a), apart from the principal spot, is not more intense than the
100C to 105C.
ASSAY
Dissolve 0.200 g in 50 ml of acetone R. Titrate with 0.1M tetrabutylammonium hydroxide using a 4 g/l
solution of thymol blue R in methanol R as indicator.
1 ml of 0.1M tetrabutylammonium hydroxide is equivalent to 26.73 mg of C11H13N3O3S.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
38-15
Sulfaguanidine
O
NH
O
S
N
H
NH2
H2N
C7H10N4O2S
214.3
56-67-0
Sulfaguanidine complies with the requirements of the 3rd edition of the European Pharmacopoeia [1476].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sulfaguanidine contains not less than 99.0 per cent and not more than the equivalent of 101.0 per
cent of (4-aminophenylsulphonyl)guanidine, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, fine crystalline powder, very slightly soluble in water, slightly soluble in
acetone, very slightly soluble in alcohol, practically insoluble in methylene chloride. It dissolves in
dilute solutions of mineral acids.
IDENTIFICATION
A. Melting point (2.2.14): 189C to 193C, determined on the dried substance.
obtained with sulfaguanidine CRS.
(2.3.1).
E. Suspend 0.1 g in 2 ml of water R, add 1 ml of -naphthol solution R and 2 ml of a mixture of equal
volumes of water R and strong sodium hypochlorite solution R. A red colour develops.
TESTS
Solution S To 2.5 g, add 40 ml of carbon dioxide-free water R. Heat at about 70C for 5 min. Cool
while stirring in iced water for about 15 min, filter and dilute to 50 ml with carbon dioxide-free
water R.
Acidity To 20 ml of solution S, add 0.1 ml of bromothymol blue solution R1. Not more than 0.2 ml of
plate R.
Test solution (a). Dissolve 50 mg of the substance to be examined in acetone R and dilute to 5 ml with
the same solvent.
Reference solution (a). Dissolve 10 mg of sulfaguanidine CRS in acetone R and dilute to 5 ml with the
same solvent.
Reference solution (d). Dissolve 10 mg of sulphanilamide R in test solution (b) and dilute to 5 ml with
the same solution.
10 volumes of anhydrous formic acid R, 20 volumes of methanol R and 70 volumes of methylene
chloride R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Any spot in the
38-16
the spot in the chromatogram obtained with reference solution (b) (0.5 per cent) and at most one
such spot is more intense that the spot in the chromatogram obtained with reference solution (c)
(0.25 per cent). The test is not valid unless the chromatogram obtained with reference solution (d)
Heavy metals (2.4.8). 12 ml of solution S complies with limit test F for heavy metals (20 ppm).
100C to 105C.
ASSAY
Dissolve 0.175 g in 50 ml of dilute hydrochloric acid R. Cool the solution in iced water. Carry out the
determination of primary aromatic amino-nitrogen (2.5.8), determining the end-point electrometrically.
STORAGE
IMPURITIES
O O
S
NH2
H2N
A. sulfanilamide,
O O O
S
N
NH2
H
H2N
B. sulfacarbamide.
__________________________________________________________________________________________________________ Ph Eur
38-17
Sulfamethizole
Me
O
O
S
N
H
S
N
N
H2N
C9H10N4O2S2
270.3
144-82-1
Sulfamethizole complies with the requirements of the 3rd edition of the European Pharmacopoeia [0637].
When sulphamethizole is prescribed or demanded, Sulfamethizole shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sulfamethizole contains not less than 99.0 per cent and not more than the equivalent of 101.0 per
cent of 4-amino-N-(5-methyl-1,3,4-thiadiazol-2-yl)benzenesulphonamide, calculated with reference
CHARACTERS
White or yellowish-white crystalline powder or crystals, very slightly soluble in water, soluble in
acetone, sparingly soluble in alcohol, very slightly soluble in ether. It dissolves in dilute solutions of
alkali hydroxides and in dilute mineral acids.
IDENTIFICATION
obtained with sulfamethizole CRS. Examine the substances prepared as discs.
C. Dissolve 50 mg in 4 ml of methanol R and add 0.2 ml of a 40 g/l solution of copper acetate R. A
flocculent, yellowish-green precipitate is formed, changing to dark green.
D. Dissolve about 5 mg in 1M hydrochloric acid and dilute to 10 ml with the same solvent. Dilute 1 ml
of this solution to 10 ml with water R. The solution, without further acidification, gives the reaction
of primary aromatic amines (2.3.1).
TESTS
(Method II, 2.2.2).
Acidity To 1.25 g add 25 ml of carbon dioxide-free water R and heat at 70C for 5 min. Cool for
about 15 min in iced water and filter. To 20 ml of the filtrate add 0.1 ml of bromothymol blue solution R1. Not more than 0.5 ml of 0.1M sodium hydroxide is required to change the colour of the
indicator.
coating substance.
Reference solution (a). Dissolve 30 mg of sulfamethizole CRS in acetone R and dilute to 10 ml with the
same solvent.
38-18
15 volumes of methanol R and 80 volumes of chloroform R. Dry the plate at 100C to 105C and
examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with test solution (a),
100C to 105C.
ASSAY
STORAGE
__________________________________________________________________________________________________________ Ph Eur
38-19
Sulfamethoxazole
O
Me
S
N
H
H 2N
C10H11N3O3S
253.3
723-46-6
Sulfamethoxazole complies with the requirements of the 3rd edition of the European Pharmacopoeia [0108].
Preparations
Co-trimoxazole Intravenous Infusion
Co-trimoxazole Oral Suspension
Paediatric Co-trimoxazole Oral Suspension
Co-trimoxazole Tablets
Dispersible Co-trimoxazole Tablets
Paediatric Co-trimoxazole Tablets
When sulphamethoxazole is prescribed or demanded, Sulfamethoxazole shall be dispensed or
supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sulfamethoxazole contains not less than 99.0 per cent and not more than the equivalent of 101.0 per
cent of 4-amino-N-(5-methyl-3-isoxazolyl)benzenesulphonamide, calculated with reference to the
dried substance.
CHARACTERS
sparingly soluble in alcohol, slightly soluble in ether. It dissolves in dilute solutions of sodium
hydroxide.
IDENTIFICATION
obtained with sulfamethoxazole CRS.
(2.3.1).
TESTS
(Method II, 2.2.2).
Acidity To 1.25 g, finely powdered, add 25 ml of water R. Heat at 70C for 5 min. Cool in iced
water for about 15 min and filter. To 20 ml of the filtrate add 0.1 ml of bromothymol blue solution R1.
Not more than 0.3 ml of 0.1M sodium hydroxide is required to change the colour of the indicator.
coating substance.
solvents.
38-20
Reference solution (a). Dissolve 20 mg of sulfamethoxazole CRS in 3 ml of a mixture of 2 volumes of
solvents.
100C to 105C.
ASSAY
Carry out the assay of primary aromatic amino-nitrogen (2.5.8), using 0.2000 g determining the endpoint electrometrically.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
38-21
Sulfasalazine
COOH
OH
N
N
N
H
N
O
C18H14N4O5S
S
O
398.4
599-79-1
Sulfasalazine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0863].
Action and use Used in treatment of ulcerative colitis.
Preparation
Sulfasalazine Tablets
When sulphasalazine is prescribed or demanded, Sulfasalazine shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sulfasalazine contains not less than 97.0 per cent and not more than the equivalent of 101.5 per cent
of 2-hydroxy-5-[[4-[[(pyridin-2-yl)amino]sulphonyl]phenyl]azo]benzoic acid, calculated with
CHARACTERS
A bright yellow or brownish-yellow, fine powder, practically insoluble in water, very slightly soluble in
alcohol, practically insoluble in methylene chloride. It dissolves in dilute solutions of alkali
hydroxides.
IDENTIFICATION
with sulfasalazine CRS. Examine the substances prepared as discs.
TESTS
Test solution. Dissolve 25.0 mg of the substance to be examined in dilute ammonia R3 and dilute to
Reference solution (a). Dilute 1.0 ml of the test solution to 100.0 ml with dilute ammonia R3.
Reference solution (b). Dissolve 1.0 mg of sulfasalazine derivative for resolution CRS in 10.0 ml of
reference solution (a). Dilute 1.0 ml of this solution to 10.0 ml with reference solution (a).
Mobile phase A. In a 1000 ml volumetric flask dissolve 1.13 g of sodium dihydrogen phosphate R
and 2.5 g of sodium acetate R in 900 ml of water R. Adjust to pH 4.8 with glacial acetic acid R and
adjust the volume to 1000 ml with water R,
Mobile phase B. Mix 1 volume of mobile phase A with 4 volumes of methanol R,
38-22
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
0 15
15 25
25 60
60 65
65 67
60 45
45
45 0
0
0 60
40 55
55
55 100
100
100 40
67 77
60
40
linear gradient
isocratic
linear gradient
isocratic
switch to initial
composition
re-equilibration

Inject 20 l of reference solution (a). Adjust the sensitivity of the detector so that the height of the
Inject 20 l of reference solution (b). The test is not valid unless the resolution between the peaks
corresponding to sulfasalazine and sulfasalazine derivative for resolution is at least 3.0.
Inject 20 l of the test solution and 20 l of reference solution (a). When the chromatograms are
recorded in the prescribed conditions, the approximate retention times relative to sulfasalazine are the
following:
impurity A 2.00
impurity B 1.85
impurity C 0.80
impurity D 1.90
impurity E 1.63
impurity F 0.85
impurity G 1.39
impurity H 0.16
impurity I 0.28
solution (a) (1 per cent); the sum of the areas of all peaks, apart from the principal peak is not greater
than four times the area of the principal peak in the chromatogram obtained with reference solution
(a) (4 per cent). Disregard any peak with a retention time shorter than 6 min (corresponding to
salicylic acid and sulfapyridine) and any peak with an area less than 0.05 times the area of the
Salicylic acid and sulfapyridine Examine by liquid chromatography (2.2.29).
Test solution. Dissolve 25.0 mg of the substance to be examined in dilute ammonia R3 and dilute to
Reference solution (a). Dissolve 5.0 mg of salicylic acid R and 5.0 mg of sulfapyridine CRS in dilute
ammonia R3 and dilute to 10.0 ml with the same solvent.
Reference solution (b). Dilute 2.0 ml of reference solution (a) to 100.0 ml with dilute ammonia R3.
as mobile phase at a flow rate of 1 ml/min a mixture of 70 volumes of mobile phase A (described
in the test for related substances) and 30 volumes of mobile phase B (described in the test for
related substances),
principal peaks in the chromatogram obtained is at least 50 per cent of the full scale of the recorder.
When the chromatogram is recorded in the prescribed conditions, the retention times are: salicylic
acid about 6 min and sulfapyridine about 7 min. The test is not valid unless the resolution between
the peaks corresponding to salicylic acid and sulfapyridine is at least 2.
for 10 min. In the chromatogram obtained with the test solution the area of the peak corresponding
to salicylic acid is not greater than 0.5 times the area of the first peak in the chromatogram obtained
with reference solution (b) (0.5 per cent) and the peak corresponding to sulfapyridine is not greater
than 0.5 times the area of the second peak in the chromatogram obtained with reference solution (b)
Chlorides (2.4.4.). To 1.25 g add 50 ml of distilled water R. Heat at about 70C for 5 min. Cool and
filter. To 20 ml of the filtrate add 1 ml of nitric acid R, allow to stand for 5 min and filter to obtain a
clear solution. 15 ml of the filtrate complies with the limit test for chlorides (140 ppm).
38-23
Sulphates (2.4.13). To 20 ml of the filtrate prepared for the test for chlorides add 1 ml of dilute
hydrochloric acid R, allow to stand for 5 min and filter. 15 ml of the filtrate complies with the limit test
for sulphates (400 ppm).
100C to 105C for 2 h.
ASSAY
Dissolve 0.150 g in 0.1M sodium hydroxide and dilute to 100.0 ml with the same solvent. Transfer
5.0 ml of this solution to a 1000 ml volumetric flask containing about 750 ml of water R. Add
20.0 ml of 0.1M acetic acid and dilute to 1000.0 ml with water R. Prepare a standard solution at the
same time and in the same manner using 0.150 g of sulfasalazine CRS. Measure the absorbance
(2.2.25) of the two solutions at the maximum at 359 nm.
Calculate the content of C18H14N4O5S from the absorbances measured and the concentration of
the solutions.
STORAGE
IMPURITIES
H
N S
R=
O
OH
A. 2,4-bis[[4-[[(pyridin-2-yl)amino]sulphonyl]phenyl]azo]phenol,
COOH
OH
N
B. 2-hydroxy-3,5-bis[[4-[[(pyridin-2-yl)amino]sulphonyl]phenyl]azo]benzoic acid,
COOH
OH
N
NH
C. 2-hydroxy-5-[[4-(2-iminopyridin-1-yl)phenyl]azo]benzoic acid,
OH
N
D. 2-[[4-[[(pyridin-2-yl)amino]sulphonyl]phenyl]azo]phenol,
COOH
OH
N
E. 2-hydroxy-3-[-4-[[(pyridin-2-yl)amino]sulphonyl]phenyl]-5-[[4-[[(pyridin-2yl)amino]sulphonyl]phenyl]azo]benzoic acid,
COOH
OH
N
F. 2-hydroxy-3-[[4-[[(pyridin-2-yl)amino]sulphonyl]phenyl]azo]benzoic acid,
38-24
COOH
OH
N
N
H
N S
O
R
O
G. 2-hydroxy-3-[4-[[(pyridin-3-yl)amino]sulphonyl]phenyl]-5-[[4-[[(pyridin-2yl)amino]sulphonyl]phenyl]azo]benzoic acid,
H. 2-hydroxybenzoic acid (salicylic acid),
COOH
OH
N
HO3S
I. 2-hydroxy-5-[(4-sulphophenyl)azo]benzoic acid.
__________________________________________________________________________________________________________ Ph Eur
38-25
Sulfathiazole
O
O
S
N
H
H2N
C9H9N3O2S2
255.3
72-14-0
Sulfathiazole complies with the requirements of the 3rd edition of the European Pharmacopoeia [0742]. These
When sulphathiazole is prescribed or demanded, Sulfathiazole shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sulfathiazole contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of 4-amino-N-(thiazol-2-yl)benzenesulphonamide, calculated with reference to the dried substance.
CHARACTERS
A white or slightly yellowish, crystalline powder, practically insoluble in water, slightly soluble in
alcohol, practically insoluble in ether and in methylene chloride. It dissolves in dilute solutions of
alkali hydroxides and in dilute mineral acids.
IDENTIFICATION
A. Melting point (2.2.14): 200C to 203C. Melting may occur at about 175C, followed by
solidification and a second melting between 200C and 203C.
obtained with sulfathiazole CRS. Examine the substances prepared as discs. If the spectra obtained
alcohol R, evaporate to dryness in vacuo and record the spectra again using the residues.
D. Dissolve about 10 mg in a mixture of 10 ml of water R and 2 ml of 0.1M sodium hydroxide and add
0.5 ml of copper sulphate solution R. A greyish-blue or purple precipitate is formed.
E. Dissolve about 5 mg in 10 ml of 1M hydrochloric acid. Dilute 1 ml of the solution to 10 ml with
water R. The solution, without further addition of acid, gives the reaction of primary aromatic amines
(2.3.1).
TESTS
Appearance of solution Dissolve 1.0 g in 10 ml of 1M sodium hydroxide. The solution is clear
(2.2.1) and not more intensely coloured than reference solution GY4 (Method II, 2.2.2).
Acidity To 1.0 g add 50 ml of carbon dioxide-free water R. Heat to 70C for 5 min. Cool rapidly to
20C and filter. To 25 ml of the filtrate add 0.1 ml of bromothymol blue solution R1. Not more than
0.1 ml of 0.1M sodium hydroxide is required to change the colour of the indicator.
coating substance.
Test solution (a). Dissolve 0.10 g of the substance to be examined in a mixture of 1 volume of concentrated ammonia R and 9 volumes of alcohol R and dilute to 10 ml with the same mixture of solvents.
Test solution (b). Dilute 1 ml of test solution (a) to 5 ml with a mixture of 1 volume of concentrated
ammonia R and 9 volumes of alcohol R.
Reference solution (a). Dissolve 20 mg of sulfathiazole CRS in a mixture of 1 volume of concentrated
ammonia R and 9 volumes of alcohol R and dilute to 10 ml with the same mixture of solvents.
Reference solution (b). Dissolve 50 mg of sulphanilamide R in a mixture of 1 volume of concentrated
ammonia R and 9 volumes of alcohol R and dilute to 100 ml with the same mixture of solvents. Dilute
1 ml of this solution to 10 ml with the same mixture of solvents.
38-26
18 volumes of ammonia R and 90 volumes of butanol R. Dry the plate at 100C to 105C for 10 min
and spray with a 1 g/l solution of dimethylaminobenzaldehyde R in alcohol R containing 1 per cent V/V
of hydrochloric acid R. Any spot in the chromatogram obtained with test solution (a), apart from the
principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (b) (0.5 per cent).
100C to 105C.
ASSAY
Carry out the determination of primary aromatic amino-nitrogen (2.5.8), using 0.200 g, determining
the end-point electrometrically.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
38-27
Sulfinpyrazone
O
S
N
H
N
O
C23H20N2O3S
404.5
57-96-5
Sulfinpyrazone complies with the requirements of the 3rd edition of the European Pharmacopoeia [0790].
Action and use Used in treatment of gout.
Preparation
Sulfinpyrazone Tablets
When sulphinpyrazone is prescribed or demanded, Sulfinpyrazone shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sulfinpyrazone contains not less than 99.0 per cent and not more than the equivalent of 101.0 per
cent of 1,2-diphenyl-4-(2-phenylsulphinylethyl)pyrazolidine-3,5-dione, calculated with reference to
CHARACTERS
A white or almost white powder, very slightly soluble in water, sparingly soluble in alcohol, slightly
soluble in ether. It dissolves in dilute solutions of alkali hydroxides.
IDENTIFICATION
B. Dissolve 30.0 mg in 0.01M sodium hydroxide and dilute to 100.0 ml with the same alkaline solution. Dilute 1.0 ml of this solution to 20.0 ml with 0.01M sodium hydroxide. Examined between
230 nm and 350 nm (2.2.25), the solution shows an absorption maximum at 260 nm. The specific
absorbance at the maximum is 530 to 580.
obtained with sulfinpyrazone CRS. Examine the substances prepared as discs.
D. Dissolve about 10 mg in 3 ml of acetone R and add a mixture of 0.2 ml of ferric chloride solution R2
and 3 ml of water R. A red to violet colour develops.
TESTS
Appearance of solution in acetone Dissolve 1.25 g in acetone R and dilute to 25 ml with the same
solvent. The solution is clear (2.2.1). The absorbance (2.2.25) of the solution, measured at 420 nm
with a path-length of 4 cm, is not greater than 0.10.
Appearance of solution in 1M sodium hydroxide Dissolve 1.25 g, heating gently if necessary, in
25 ml of 1M sodium hydroxide. The solution is clear (2.2.1). The absorbance (2.2.25) of the solution,
measured at 420 nm with a path-length of 4 cm, is not greater than 0.15.
Related substances Examine by thin-layer chromatography (2.2.27), using a suitable silica gel with
a fluorescent indicator having an optimal intensity at 254 nm. Expose the plate, in a tank, to the
vapour from glacial acetic acid R. After 10 min, remove the plate and heat at 60C for 40 min. Cool
and pass a current of nitrogen over the plate for 10 min.
same solvent.
Reference solution (a). Dilute 2 ml of the test solution to 100 ml with acetone R. Dilute 1 ml of this
solution to 10 ml with acetone R.
Reference solution (b). Dissolve 10 mg of sulfinpyrazone impurity A CRS in acetone R and dilute to
38-28
Reference solution (d). Dissolve 10 mg of sulfinpyrazone impurity B CRS in acetone R and dilute to
Reference solution (e). Dilute 5 ml of reference solution (d) to 10 ml with acetone R.
Apply separately to the plate, under an atmosphere of nitrogen, 2 l of each solution. Develop over a
path of 15 cm using a mixture of 20 volumes of glacial acetic acid R and 80 volumes of chloroform R.
Allow the plate to dry in air and examine in ultraviolet light at 254 nm. In the chromatogram
obtained with the test solution: any spots corresponding to sulfinpyrazone impurity A and
sulfinpyrazone impurity B are not more intense than the spots in the chromatograms obtained with
reference solutions (b) (2.0 per cent) and (d) (2.0 per cent), respectively, and at most one of the
spots is more intense than the corresponding spot in the chromatogram obtained with reference
solution (c) (1.0 per cent) or (e) (1.0 per cent); any spot apart from the principal spot and the two
spots previously cited is not more intense than the spot in the chromatogram obtained with reference
solution (a) (0.2 per cent).
100C to 105C.
ASSAY
Dissolve 0.300 g in 25 ml of acetone R. Add 0.5 ml of bromothymol blue solution R1. Titrate with 0.1M
sodium hydroxide until the colour changes from yellow to blue.
STORAGE
IMPURITIES
A. 1,2-diphenyl-4-(2-phenylsulphonylethyl)pyrazolidine-3,5-dione,
B. 1,2-diphenyl-4-(2-phenylthioethyl)pyrazolidine-3,5-dione.
__________________________________________________________________________________________________________ Ph Eur
38-29
Sulfisomidine
Me
O
S
N
H
Me
H2N
C12H14N4O2S
278.3
515-64-0
Sulfisomidine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0639]. These
When sulphasomidine is prescribed or demanded, Sulfisomidine shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sulfisomidine contains not less than 99.0 per cent and not more than the equivalent of 101.0 per
cent of 4-amino-N-(2,6-dimethylpyrimidin-4-yl)benzenesulphonamide, calculated with reference to
CHARACTERS
White or yellowish-white powder or crystals, very slightly soluble in water, slightly soluble in acetone
and in alcohol. It dissolves in dilute solutions of the alkali hydroxides and in dilute mineral acids.
IDENTIFICATION
obtained with sulfisomidine CRS. Examine the substances prepared as discs.
C. Dissolve 50 mg with heating in 4 ml of methanol R. Cool and filter. Add to the filtrate 0.2 ml of a
40 g/l solution of copper acetate R. The solution becomes yellowish-green.
(2.3.1).
TESTS
(Method II, 2.2.2).
Cool in a bath of iced water for about 15 min and filter. To 20 ml of the filtrate add 0.1 ml of bromothymol blue solution R1. Not more than 0.2 ml of 0.1M sodium hydroxide is required to change the
coating substance.
solvents.
Reference solution (a). Dissolve 20 mg of sulfisomidine CRS in 3 ml of a mixture of 2 volumes of
solvents.
38-30
100C to 105C.
ASSAY
STORAGE
__________________________________________________________________________________________________________ Ph Eur
38-31
Sulindac
Me
COOH
Me
S
O
F
C20H17FO3S
356.4
38194-50-2
Sulindac complies with the requirements of the 3rd edition of the European Pharmacopoeia [0864]. These
Preparation
Sulindac Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sulindac contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of
(Z)-[5-fluoro-2-methyl-1-[4-(methylsulphinyl)benzylidene]-1H-inden-3-yl]acetic acid, calculated
CHARACTERS
A yellow, crystalline powder, very slightly soluble in water, soluble in methylene chloride, sparingly
soluble in alcohol, very slightly soluble in ether. It dissolves in dilute solutions of alkali hydroxides.
IDENTIFICATION
B. Dissolve 50 mg in a 0.3 per cent V/V solution of hydrochloric acid R in methanol R and dilute to
100 ml with the same acid solution. Dilute 2 ml of the solution to 50 ml with a 0.3 per cent V/V
solution of hydrochloric acid R in methanol R. Examined between 230 nm and 350 nm (2.2.25), the
solution shows two absorption maxima, at 284 nm and 327 nm, and a shoulder at about 258 nm.
The ratio of the absorbance measured at the maximum at 284 nm to that measured at the maximum
at 327 nm is 1.10 to 1.20.
obtained with sulindac CRS. Examine the substances prepared as discs. If the spectra obtained show
minimum volume of hot methanol R, evaporate to dryness and record new spectra using the residues.
Reference solution (a). Dissolve 10 mg of sulindac CRS in methylene chloride R and dilute to 10 ml with
the same solvent.
Reference solution (b). Dissolve 10 mg of diflunisal CRS in methylene chloride R and dilute to 10 ml with
the same solvent. Dilute 1 ml of the solution to 2 ml with reference solution (a).
volume of glacial acetic acid R, 49 volumes of methylene chloride R and 50 volumes of acetone R. Dry
the plate in a current of warm air and examine in ultraviolet light at 254 nm. The principal spot in
the chromatogram obtained with the test solution is similar in position and size to the principal spot
in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows two clearly separated principal spots.
E. Mix about 5 mg with 45 mg of heavy magnesium oxide R and ignite in a crucible until an almost
phenolphthalein solution R1 and about 1 ml of dilute hydrochloric acid R to render the solution colour-
38-32
less. Filter. Add 1.0 ml of the filtrate to a freshly prepared mixture of 0.1 ml of alizarin S solution R
and 0.1 ml of zirconyl nitrate solution R. Mix, allow to stand for 5 min and compare the colour of the
solution with that of a blank prepared in the same manner. The test solution is yellow and the blank
is red.
TESTS
Related substances Examine by liquid chromatography (2.2.29)
Reference solution (b). Dissolve 20.0 mg of sulindac CRS (which contains 0.5 per cent m/m of the
E-isomer) in the mobile phase and dilute to 10.0 ml with the same solvent.
as mobile phase at a flow rate of 2 ml per minute a mixture of 1 volume of glacial acetic acid R, 4
volumes of alcohol R, 100 volumes of ethyl acetate R and 400 volumes of ethanol-free chloroform R,
Inject 20 l of reference solution (a) and adjust the sensitivity of the system so that the height of the
Inject 20 l of reference solution (b) and continue the chromatography for twice the retention time of
the principal peak. The chromatogram obtained shows a principal peak corresponding to sulindac
and a peak corresponding to the E-isomer, with a retention time relative to sulindac of about 1.75.
solution (b). In the chromatogram obtained with the test solution: the area of any peak corresponding
to the E-isomer is not greater than the area of the corresponding peak in the chromatogram obtained
with reference solution (b) (0.5 per cent); the area of any peak, apart from the principal peak and any
peak corresponding to the E-isomer, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent); the sum of the areas of all the peaks,
apart from the principal peak, is not greater than twice the area of the principal peak in the chromatogram obtained with reference solution (a) (1 per cent).
100C to 105C at a pressure not exceeding 700 Pa.
ASSAY
Dissolve 0.300 g in 50 ml of methanol R. Titrate with 0.1M sodium hydroxide, determining the endpoint potentiometrically (2.2.20).
1 ml of 0.1M sodium hydroxide is equivalent to 35.64 mg of C20H17FO3S.
STORAGE
IMPURITIES
O
S
Me
Me
COOH
A. (E)-[5-fluoro-2-methyl-1-[4-(methylsulphinyl)benzylidene]-1H-inden-3 -yl]acetic
acid,
38-33
Me
COOH
Me
O
F
B. (Z)-[5-fluoro-2-methyl-1-[4-(methylsulphonyl)benzylidene]-1H-inden-3-yl]acetic
acid,
Me
COOH
Me
C. (Z)-[5-fluoro-2-methyl-1-[4-methylsulphanyl)benzylidene]-1H-inden-3-yl]acetic
acid.
__________________________________________________________________________________________________________ Ph Eur
38-34
Sulphur for External Use

S
32.07
7704-34-9
Sulphur for External Use complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sulphur contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of S.
CHARACTERS
A yellow powder, practically insoluble in water, soluble in carbon disulphide, slightly soluble in
vegetable oils. The size of most of the particles is not greater than 20 m and that of almost all the
particles is not greater than 40 m.
IDENTIFICATION
A. Heated in the presence of air, it burns with a blue flame, emitting sulphur dioxide which changes
the colour of moistened blue litmus paper R to red.
B. Heat 0.1 g with 0.5 ml of bromine water R until decolorised. Add 5 ml of water R and filter. The
solution gives reaction (a) of sulphates (2.3.1).
TESTS
Solution S To 5 g add 50 ml of carbon dioxide-free water R prepared from distilled water R. Allow to
stand for 30 min with frequent shaking and filter.
Odour (2.3.4). The substance to be examined has no perceptible odour of hydrogen sulphide.
Acidity or alkalinity To 5 ml of solution S add 0.1 ml of phenolphthalein solution R1. The solution is
colourless. Add 0.2 ml of 0.01M sodium hydroxide. The solution is red. Add 0.3 ml of 0.01M hydrochloric acid. The solution is colourless. Add 0.15 ml of methyl red solution R. The solution is orangered.
Sulphides To 10 ml of solution S add 2 ml of buffer solution pH 3.5 R and 1 ml of a freshly prepared
1.6 g/l solution of lead nitrate R in carbon dioxide-free water R. Shake. After 1 min any colour in the
solution is not more intense than that in a reference solution prepared at the same time using 1 ml of
lead standard solution (10 ppm Pb) R, 9 ml of carbon dioxide-free water R, 2 ml of buffer solution pH
3.5 R and 1.2 ml of thioacetamide reagent R.
ASSAY
Carry out the oxygen-flask method (2.5.10), using 60.0 mg of the substance to be examined in a
1000 ml combustion flask. Absorb the combustion products in a mixture of 5 ml of dilute hydrogen
peroxide solution R and 10 ml of water R. Heat to boiling, boil gently for 2 min and cool. Using 0.2 ml
of phenolphthalein solution R as indicator, titrate with 0.1M sodium hydroxide until the colour changes
from colourless to red. Carry out a blank titration under the same conditions.
1 ml of 0.1M sodium hydroxide is equivalent to 1.603 mg of S.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
38-35
Sulphuric Acid
1/01
H2SO4
98.1
7664-93-9
Sulfuric Acid complies with the requirements of the 3rd edition of the European Pharmacopoeia [1572]. These
Preparation
Dilute Sulphuric Acid
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sulphuric acid contains not less than 95.0 per cent m/m and not more than 100.5 per cent m/m of
H2SO4.
CHARACTERS
A colourless, oily liquid, very hygroscopic, miscible with water and with alcohol producing intense
heat.
The relative density is about 1.84.
IDENTIFICATION
A. Carefully add 1 ml to 100 ml of water R. The solution is strongly acid (2.2.4).
B. The solution obtained in identification test A gives reaction (a) of sulphates (2.3.1).
TESTS
Appearance of solution Carefully pour, while cooling, 5 ml into 30 ml of water R and dilute to
50 ml with the same solvent. The solution is clear (2.2.1) and colourless (Method II, 2.2.2).
Chlorides (2.4.4). Mix carefully, while cooling, 3.3 g with 30 ml of water R. Neutralise with
ammonia R and dilute to 50 ml with water R. 15 ml of the solution complies with the limit test for
chlorides (50 ppm).
Nitrates Add 5 ml to 5 ml of water R. Cool to room temperature and add 0.5 ml of indigo carmine
solution R. The blue colour persists for at least 1 min.
Arsenic (2.4.2). Mix, while cooling, 1 g with 20 ml of water R and dilute to 25 ml with the same
solvent. The solution complies with limit test A for arsenic (1 ppm).
Iron (2.4.9). Cautiously evaporate 10.0 g and ignite to dull redness. Dissolve the ignition residue in
1 ml of dilute hydrochloric acid R with gentle heating and dilute to 25 ml with water R. Dilute 1 ml of
the solution to 10 ml with water R. The solution complies with the limit test for iron (25 ppm).
ASSAY
Weigh accurately a ground-glass-stoppered flask containing 30 ml of water R. Introduce 0.2 ml of the
acid, cool and weigh again. Titrate with 1M sodium hydroxide, determining the end-point potentiometrically (2.2.20).
1 ml of 1M sodium hydroxide is equivalent to 49.04 mg of H2SO4.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
38-36
Dilute Sulphuric Acid

Definition Dilute Sulphuric Acid is prepared by adding 104 g of Sulphuric Acid to 896 g of Purified
Water with constant stirring and cooling. It contains not less than 9.5% and not more than 10.5%
w/w of H2SO4.
Weight per ml 1.062 g to 1.072 g, Appendix V G.
Assay To 10 g add 40 ml of water and titrate with 1M sodium hydroxide VS using methyl orange solution as indicator. Each ml of 1M sodium hydroxide VS is equivalent to 49.04 mg of H2SO4.
38-37
Sulpiride
H
N
N
OM e
Et
H2N
S
O
C15H23N3O4S
341.4
15676-16-1
Sulpiride complies with the requirements of the 3rd edition of the European Pharmacopoeia [1045]. These
Preparation
Sulpiride Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sulpiride contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of
(RS)-5-sulphamoyl-N-[(1-ethylpyrrolidin-2-yl)methyl]-2-methoxybenzamide, calculated with reference to the dried substance.
CHARACTERS
methanol, slightly soluble in alcohol and in methylene chloride. It dissolves in dilute solutions of
mineral acids and alkali hydroxides.
IDENTIFICATION
obtained with sulpiride CRS. Examine the substances prepared as discs.
C. Examine the chromatograms obtained in test A for related substances (see Tests) in ultraviolet
light at 254 nm. The principal spot in the chromatogram obtained with test solution (b) is similar in
position and size to the principal spot in the chromatogram obtained with reference solution (a).
D. To about 1 mg in a porcelain dish, add 0.5 ml of sulphuric acid R and 0.05 ml of formaldehyde
solution R. Examined in ultraviolet light at 365 nm, the solution shows blue fluorescence.
TESTS
Appearance of solution Dissolve 1.0 g in dilute acetic acid R and dilute to 10 ml with the same acid.
The solution is clear (2.2.1) and not more intensely coloured than reference solution Y6 (Method I,
2.2.2).
Related substances
A. Examine by thin-layer chromatography (2.2.27), using silica gel HF254 R as the coating substance.
Test solution (a). Dissolve 0.20 g in methanol R and dilute to 10 ml with the same solvent.
Reference solution (a). Dissolve 20 mg of sulpiride CRS in methanol R and dilute to 10 ml with the
same solvent.
Reference solution (b). Dissolve 5 mg of sulpiride impurity A CRS in methanol R and dilute to 25 ml
Reference solution (c). Dilute 1.0 ml of reference solution (b) to 10 ml with methanol R.
2 volumes of concentrated ammonia R, 10 volumes of dioxan R, 14 volumes of methanol R and 90
volumes of methylene chloride R. Allow the plate to dry in air. Examine in ultraviolet light at 254 nm
38-38
for identification test C and then spray with ninhydrin solution R. Heat at 100C to 105C for 15 min.
Examine in daylight. Any spot in the chromatogram obtained with test solution (a) corresponding to
the principal spot in the chromatogram obtained with reference solution (b) is not more intense than
the spot in the chromatogram obtained with reference solution (c) (0.1 per cent).
Test solution. Dissolve 0.100 g in the mobile phase and dilute to 100.0 ml with the mobile phase.
Reference solution (b). Dissolve 10 mg of sulpiride CRS and 10 mg of sulpiride impurity B CRS in the
mobile phase and dilute to 100.0 ml with the same solvent.
a column 0.25 m long and 4.6 mm in internal diameter, packed with octylsilyl silica gel for chromatography R (5 m) in spherical micro-particles,
as mobile phase at a flow rate of 1.5 ml per minute a mixture of 10 volumes of acetonitrile R, 10
volumes of methanol R and 80 volumes of a solution containing 6.8 g/l of potassium dihydrogen
phosphate R and 1 g/l of sodium octanesulphonate R, adjusted to pH 3.3 using phosphoric acid R,
a loop injector.
Adjust the sensitivity of the detector so that the height of the principal peak in the chromatogram
obtained with reference solution (a) is not less than 5 per cent of the full scale of the recorder. Inject
20 l of reference solution (b). The test is not valid unless in the chromatogram obtained the resolution between the two principal peaks is at least 2.5. Inject separately 20 l of the test solution and
20 l of reference solution (a). Continue the chromatography for 2.5 times the retention time of
sulpiride. In the chromatogram obtained with the test solution, the sum of the areas of any peaks,
apart from the principal peak, is not greater than the area of the peak in the chromatogram obtained
with reference solution (a) (0.3 per cent).
Chlorides (2.4.4).Shake 1.0 g with 20 ml of water R. Filter through a sintered-glass filter (40). To
10 ml of the solution add 5 ml of water R. The solution complies with the limit test for chlorides
(100 ppm).
Iron (2.4.9). Ignite 1.0 g in a silica crucible. To the residue add 1 ml of 1M hydrochloric acid, 3 ml of
water R and 0.1 ml of nitric acid R. Heat on a water-bath for a few minutes. Place the solution in a
test-tube. Rinse the crucible with 4 ml of water R. Collect the rinsings in the test-tube and dilute to
10 ml with water R. The solution complies with the limit test for iron (10 ppm).
100C to 105C.
ASSAY
IMPURITIES
H
2N
and enantiomer
N
Et
A. (2RS)-2-aminomethyl-1-ethylpyrrolidine,
COOMe
OMe
H2N
B. methyl 2-methoxy-5-sulphamoylbenzoate,
COOEt
OMe
H2N
O
S
O
C. ethyl 2-methoxy-5-sulphamoylbenzoate,
38-39
COOH
OMe
H2N
D. 2-methoxy5-sulphamoylbenzoic acid,
CONH2
OMe
H2N
E. 5-sulphamoyl-2-methoxybenzamide,
O
H
N
O
Et
OMe
H2N
S
O O
F. N-[(1-ethylpyrrolidin-2-yl)methyl]-2-methoxy-5-sulphamoylbenzamide 1N-oxide,
O
H H
N
OMe
N
Et
and enantiomer
H2N
O O
G. (RS)-N-[(1-ethylpyrrolidin-2-yl)methyl]-2-hydroxy-5-sulphamoylbenzamide.
__________________________________________________________________________________________________________ Ph Eur
38-40
Sumatriptan Succinate
1/01
O
H
N
COOH
,
S
NMe2
MeHN
C18H27N3O6S
413.5
COOH
103628-48-4
Sumatritan Succinate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use 5HT1D-Serotonin receptor agonist.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sumatriptan succinate contains not less than 97.5 per cent and not more than the equivalent of
102.0 per cent of [3-[2-(dimethylamino)ethyl]-1H-indol-5-yl]-N-methylmethanesulphonamide
hydrogen butanedioate, calculated with reference to the anhydrous substance.
CHARACTERS
A white or almost white powder, freely soluble in water, sparingly soluble in methanol, practically
IDENTIFICATION
with sumatriptan succinate CRS. Examine the substances prepared as discs.
TESTS
Absorbance (2.2.25). The absorbance of solution S measured at 440 nm is not more than 0.10.
pH (2.2.3). Dilute 2.5 ml of solution S to 10 ml with carbon dioxide-free water R. The pH of the
Related substances
A. Examine by liquid chromatography (2.2.29).
Reference solution (a). Dissolve 2 mg of sumatriptan impurity A CRS in the mobile phase, add 1 ml of
the test solution and dilute to 100 ml with the mobile phase. Dilute 1 ml to 10 ml with the mobile
phase.
Reference solution (c). Dissolve 3 mg of sumatriptan for system suitability CRS in the mobile phase and
as mobile phase at a flow rate of 2.0 ml/min a mixture of 10 volumes of a 771 g/l solution of
ammonium acetate R and 90 volumes of methanol R,
principal peak is at least 20 per cent of the full scale of the recorder.
Inject 20 l of reference solution (c). The resulting chromatogram is similar to the chromatogram
provided with sumatriptan for system suitability CRS.
Inject 20 l of reference solution (a). The test is not valid unless, in the chromatogram obtained,
the resolution between the peaks corresponding to sumatriptan (retention time = about 3 min) and
impurity A (relative retention = about 2.5) is at least 1.5.
Inject 20 l of the test solution. Continue the chromatography for 5 times the retention time of the
principal peak. In the chromatogram obtained with the test solution: the area of any peak corresponding to impurity A is not greater than 6 times the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.6 per cent); the area of any peak corresponding to impurity H
38-41
(relative retention = about 3.5) is not greater than 3 times the area of the principal peak in the
chromatogram obtained with reference solution (b) (0.3 per cent); the area of any other peak, apart
from the principal peak and the peaks due to impurity A and impurity H, is not greater than the area
of the principal peak in the chromatogram obtained with reference solution (b) (0.1 per cent). The
sum of the areas of all the peaks, apart from the principal peak, is not greater than 9 times the area of
the principal peak in the chromatogram obtained with reference solution (b) (0.9 per cent).
B. Examine by liquid chromatography (2.2.29) as described under Assay.
Reference solution (a). Dissolve 2 mg of sumatriptan impurity C CRS in the mobile phase, add 1 ml of
the test solution and dilute to 100 ml with the mobile phase. Dilute 1 ml to 10 ml with the mobile
phase.
Reference solution (c). Dissolve 30 mg of sumatriptan impurity mixture CRS in the mobile phase and
principal peak is at least 50 per cent of the full scale of the recorder. Determine the retention time of
sumatriptan.
Inject 10 l of reference solution (a). The test is not valid unless, in the chromatogram obtained,
the resolution between the peaks corresponding respectively to sumatriptan and impurity C is at least
1.5.
Inject 10 l of reference solution (c). In the chromatogram obtained 5 principal peaks are observed.
The area of the peak due to impurity E is about twice the area of the peak due to impurity B, impurity C or impurity D (the retention time of impurity E may vary depending on the column used). The
relative retention with reference to sumatriptan is about 0.6 for impurity B, 0.9 for impurity C and
0.3 for impurity D. Determine the retention times of impurity E, impurity B, impurity C and impurity D.
Inject 10 l of the test solution. Continue the chromatography for 4 times the retention time of the
principal peak. In the chromatogram obtained with the test solution: the area of any peak due to
impurity B, impurity C or impurity D is not greater than 5 times the area of the principal peak in the
chromatogram obtained with reference solution (b) (0.5 per cent); the area of the peak due to
impurity E and the area of any other peak, apart from the principal peak and the peaks due to
impurity B, impurity C or impurity D, is not greater than the area of the principal peak in the
chromatogram obtained with reference solution (b) (0.1 per cent); the sum of the areas of all the
peaks, apart from the principal peak, is not greater than 6 times the area of the principal peak in the
chromatogram obtained with reference solution (b) (0.6 per cent). Disregard any peak with an area
less than 0.5 times that of the principal peak in the chromatogram obtained with reference solution
(b) (0.05 per cent).
ASSAY
Solution A. Dissolve 2.925 g of sodium dihydrogen phosphate R in 600 ml of water R, adjust to pH 6.5
with strong sodium hydroxide solution R, dilute to 750 ml with water R, add 250 ml of acetonitrile R and
mix.
Test solution. Dissolve 15.0 mg of the substance to be examined in solution A and dilute to 100.0 ml
with solution A.
Reference solution (a). Dissolve 15.0 mg of sumatriptan succinate CRS in solution A and dilute to
100.0 ml with solution A.
Reference solution (b). Dissolve 3 mg of sumatriptan impurity C CRS in solution A, add 40 ml of
reference solution (a) and dilute to 100 ml with solution A.
volumes of a solution prepared as follows: dissolve 0.970 g of dibutylamine R, 0.735 g of
phosphoric acid R and 2.93 g of sodium dihydrogen phosphate R in 750 ml of water R, adjust to pH
6.5 with strong sodium hydroxide solution R and dilute to 1000 ml with water R,
38-42
principal peak is at least 50 per cent of the full scale of the recorder. Inject reference solution (b). The
test is not valid unless, in the chromatogram obtained, the resolution between the peaks corresponding
to sumatriptan and impurity C is at least 1.5. Inject 10 l of the test solution and 10 l of reference
solution (a) and calculate the percentage content of sumatriptan succinate.
STORAGE
IMPURITIES
O O
S
MeHN
H
N
NMe2
N
H
NMe2
A. [3-[2-(dimethylamino)ethyl]-2-[[3-[2-(dimethylamino)ethyl]-1H-indol-5-yl]methyl]-1Hindol-5-yl]-N-methylmethanesulphonamide,
R1
O O
S
eHN
N
N
R2
Me
B. R1 = R2 = H: [3-[2-(methylamino)ethyl]-1H-indol-5-yl]-N-methylmethanesulphonamide,
C. R1 = CH2-OH, R2 = CH3: [3-[2-(dimethylamino)ethyl]-1-(hydroxymethyl)-1H-indol-5-yl]-Nmethylmethanesulphonamide,
O O
S
eHN
H
N
NMe2
O
D. [3-[2-(dimethylamino N-oxide)ethyl]-1H-indol-5-yl]-N-methylmethanesulphonamide,
O O
S
eHN
H
N
NH2
E. [3-(2-aminoethyl)-1H-indol-5-yl]-N-methylmethanesulphonamide,
O
H
N
NR
S
eHN
F. R = H: N-methyl(2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-6-yl)methanesulphonamide,
G. R = CH3: N-methyl(2-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-6yl)methanesulphonamide,
H
N
NMe2
O O
S
MeHN
NMe2
H. [3-[2-(dimethylamino)ethyl]-1-[[3-[2-(dimethylamino)ethyl]-1H-indol-5-yl]methyl]-1Hindol-5-yl]-N-methylmethanesulphonamide.
__________________________________________________________________________________________________________ Ph Eur
38-43
Refined Sunflower Oil

Refined Sunflower Oil complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Sunflower oil is the fatty oil obtained from the seeds of Helianthus annuus C. by mechanical
expression or by extraction. It is then refined. A suitable antioxidant may be added.
CHARACTERS
(bp: 40C to 60C).
IDENTIFICATION
obtained is similar to the typical chromatogram for sunflower oil.
TESTS
Composition of fatty acids Carry out the test for foreign fatty oils in fatty oils by gas chromatography (2.4.22). The fatty-acid fraction of the oil has the following composition:
linoleic acid: 48.0 per cent to 74.0 per cent.
STORAGE
LABELLING
The label states:
__________________________________________________________________________________________________________ Ph Eur
38-44
Suxamethonium Chloride
1/01
O
Me3N
NMe3 2Cl
C14H30Cl2N2O4,2H2O
397.3
6101-15-1
Suxamethonium Chloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Skeletal muscle relaxant.
Preparation
Suxamethonium Chloride Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Suxamethonium chloride contains not less than 98.0 per cent and not more than the equivalent of
102.0 per cent of 2,2-succinyldioxybis(N,N,N-ethyltrimethylammonium) dichloride, calculated with
CHARACTERS
A white or almost white, crystalline powder, hygroscopic, freely soluble in water, slightly soluble in
alcohol.
It melts at about 160C, determined without previous drying.
IDENTIFICATION
obtained with suxamethonium chloride CRS. Examine the substances prepared as discs.
B. To 1 ml of solution S (see Tests) add 9 ml of water R, 10 ml of dilute sulphuric acid R and 30 ml of
ammonium reineckate solution R. A pink precipitate is formed. Allow to stand for 30 min, filter, wash
with water R, with alcohol R and then with ether R and dry at 80C. The melting point (2.2.14) of the
precipitate is 180C to 185C.
C. Dissolve about 25 mg in 1 ml of water R and add 0.1 ml of a 10 g/l solution of cobalt chloride R and
0.1 ml of potassium ferrocyanide solution R. A green colour is produced.
D. About 20 mg gives reaction (a) of chlorides (2.3.1).
TESTS
Appearance of solution Solution S is clear (2.2.1). Dilute 4 ml of solution S to 10 ml with water R.
The solution is colourless (Method II, 2.2.2).
Choline chloride Examine by thin-layer chromatography (2.2.27), using cellulose for chromatography R1 as the coating substance.
the same solvent.
Reference solution. Dissolve 0.4 g of suxamethonium chloride CRS and 2 mg of choline chloride R in
Apply to the plate 5 l of each solution. Prepare the mobile phase as follows: shake together for
10 min, 10 volumes of anhydrous formic acid R, 40 volumes of water R and 50 volumes of butanol R;
allow to stand and use the upper layer. Develop over a path of 15 cm. Dry the plate in a current of air
and spray with potassium iodobismuthate solution R. Any spot in the chromatogram obtained with the
test solution, apart from the principal spot, is not more intense than the spot corresponding to
choline chloride in the chromatogram obtained with the reference solution (0.5 per cent). The test is
not valid unless the chromatogram obtained with the reference solution shows two clearly separated
spots.
38-45
ASSAY
STORAGE
__________________________________________________________________________________________________________ Ph Eur
39-1
Suxibuzone
1/01
N
N
HOOC
O
Bu n
C24H26N2O6
438.5
O
27470-51-5
Suxibuzone complies with the requirements of the 3rd edition of the European Pharmacopoeia [1574]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
4-[(4-Butyl-3,5-dioxo-1,2-diphenylpyrazolidin-4-yl)methoxy]-4-oxobutanoic acid.
CHARACTERS
Appearance: white crystalline powder.
Solubility: practically insoluble in water, freely soluble in acetone, soluble in alcohol, practically
insoluble in cyclohexane.
IDENTIFICATION
Comparison: suxibuzone CRS.
TESTS
Appearance of solution The solution is clear (2.2.1) and colourless (2.2.2, Method II).
Dissolve 1 g in ethanol R and dilute to 20 ml with the same solvent.
Test solution. Dissolve 0.10 g of the substance to be examined in acetonitrile R and dilute to 25.0 ml
Reference solution (a). Dissolve 2.8 mg of suxibuzone impurity B CRS and 2.8 mg of suxibuzone impurity
C CRS in acetonitrile R and dilute to 10.0 ml with the same solvent. Dilute 1.0 ml of this solution to
Reference solution (b). Dissolve 4 mg of phenylbutazone CRS (impurity A of suxibuzone) in acetonitrile R and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml of this solution to 10.0 ml with
acetonitrile R.
Reference solution (c). Dissolve 10 mg of phenylbutazone CRS in acetonitrile R and dilute to 25.0 ml
with the same solvent. Dilute the mixture of 10.0 ml of this solution and 1.0 ml of the test solution to
Column:
size: l = 0.125 m, = 4.0 mm,
stationary phase: octadecylsilyl silica gel for chromatography R (5 m).
Mobile phase: mix 44 volumes of acetonitrile R and 56 volumes of a solution prepared as follows:
dissolve 6.7 g of citric acid R and 2.4 g of tris(hydroxymethyl)aminomethane R in 950 ml of water R,
adjust to pH 3.0 with citric acid R and dilute to 1000 ml with water R (44:56 V/V).
Flow rate: 1 ml/min.
Injection: 10 l.
Relative retention with reference to suxibuzone (retention time = about 7 min): impurity C = 0.7;
impurity A = 1.4; impurity B = 3.3.
39-2
System suitability:
resolution: minimum of 2.0 between the peaks due to suxibuzone and to impurity A in the
chromatogram obtained with the reference solution (c).
Limits:
impurity A: not more than the area of the principal peak in the chromatogram obtained with
impurity C: not more than the area of the principal peak in the chromatogram obtained with
any other impurity: not more than the area of the principal peak in the chromatogram obtained
with reference solution (b) (0.1 per cent),
Heavy metals (2.4.8): maximum of 10 ppm.
2.0 g complies with the limit test C. Prepare the standard using 2 ml of lead standard solution (10 ppm
Pb) R.
Loss on drying (2.2.32): maximum of 0.5 per cent, determined on 1.000 g by drying in an oven in
vacuo at 60C.
Sulphated ash (2.4.14): maximum of 0.1 per cent, determined on 1.0 g.
ASSAY
Dissolve 0.400 g in previously neutralised ethanol R, and dilute to 10 ml with the same solvent. Carry
out a potentiometric titration (2.2.20) using 0.1M sodium hydroxide.
STORAGE
IMPURITIES
Qualified impurities: B, C.
Other detectable impurities: A.
A. phenylbutazone,
RO
Bun
N
O
B. R = CO-CH2-CH2-CO-O-CH2-CH3: (4-butyl-3,5-dioxo-1,2-diphenylpyrazolidin-4-yl)methyl
ethyl butanedioate,
C. R = H: 4-butyl-4-(hydroxymethyl)-1,2-diphenyl-1,2-dihydro-4H-pyrazole-3,5-dione.
__________________________________________________________________________________________________________ Ph Eur
39-3
Purified Talc
14807-96-6
Purified Talc complies with the requirements of the 3rd edition of the European Pharmacopoeia for Talc
Preparation
Talc Dusting Powder
PhEur ___________________________________________________________________________________________________________
DEFINITION
Talc is a powdered, selected, natural, hydrated magnesium silicate. Pure talc has the formula
[Mg3Si4O10(OH)2; Mr 379.3]. It may contain variable amounts of associated minerals among which
chlorites (hydrated aluminium and magnesium silicates), magnesite (magnesium carbonate), calcite
(calcium carbonate) and dolomite (calcium and magnesium carbonate) are predominant.
PRODUCTION
Talc derived from deposits that are known to contain associated asbestos is not suitable for
pharmaceutical use. The manufacturer is responsible for demonstrating by the test for amphiboles
and serpentines that the product is free from asbestos. The presence of amphiboles and of serpentines
is revealed by X-ray diffraction or by infrared spectrophotometry (see A and B). If detected, the
specific morphological criteria of asbestos are investigated by a suitable method of optical microscopy
to determine whether tremolite asbestos or chrysotile is present, as described below.
A. Examine by infrared spectrophotometry (2.2.24). In the range 740 cm1 to 760 cm1 using scale
expansion, any absorption band at 758 1 cm1 may indicate the presence of tremolite or of chlorite.
If the absorption band remains after ignition of the substance at 850C for at least 30 min, it
indicates the presence of the tremolite. In the range 600 cm1 to 650 cm-1 using scale expansion, any
absorption band or shoulder may indicate the presence of serpentines. Examine the substance
prepared as discs using potassium bromide R.
B. Examine by X-ray diffraction employing the following conditions:
Cu K monochromatic 40 kV radiation, 24 mA to 30 mA
incident slit: 1,
detection slit: 0.2,
goniometer speed: 1/10 2 /min,
scanning range: 10 to 13 2 and 24 to 26 2 ,
the sample is not oriented.
Place the sample on the sample holder; pack and smooth its surface with a polished glass
microscope slide.
Record the diffractograms.
The presence of amphiboles is detected by a diffraction peak at 10.5 0.1 2 , the presence of
serpentines is detected by diffraction peaks at 24.3 0.1 2 to 12.1 0.1 2 .
If, by one of the two methods, amphiboles and/or serpentine are detected, examine by a suitable method of
optical microscopy to determine the asbestos character.
Examined by optical microscopy, the presence of asbestos is shown if the following criteria are met:
a range of length to width ratios of 20:1 to 100:1, or higher for fibres longer than 5 m,
capability of splitting into very thin fibrils,
and if two or more of the following four criteria are met:
parallel fibres occurring in bundles,
fibre bundles displaying frayed ends,
fibres in the form of thin needles,
matted masses of individual fibres and/or fibres showing curvature.
CHARACTERS
A light, homogeneous, white or almost white powder, greasy to the touch (non abrasive), practically
insoluble in water, in alcohol and in dilute solutions of acids and alkali hydroxides.
IDENTIFICATION
A. Examine by infrared absorption spectrophotometry (2.2.24). The spectrum shows absorption
bands at 3677 2 cm1, at 1018 2 cm1 and at 669 2 cm1. Examine the substance as discs
prepared using potassium bromide R.
B. In a platinum crucible, melt a mixture of 0.2 g of anhydrous sodium carbonate R and 2.0 g of
39-4
potassium carbonate R. To the melted mass add 0.1 g of the substance to be examined and heat until
the mixture is completely melted. Allow to cool and transfer the melted mass into an evaporating dish
with 50 ml of hot water R. Add hydrochloric acid R until effervescence ceases. Add 10 ml of hydrochloric acid R and evaporate to dryness on a water-bath. Allow to cool. Add 20 ml of water R, heat to
boiling and filter. (The residue is used for identification test C). To 5 ml of the filtrate add 1 ml of
ammonia R and 1 ml of ammonium chloride solution R and filter. To the filtrate add 1 ml of disodium
hydrogen phosphate solution R. A white, crystalline precipitate is formed.
C. The residue obtained in identification test B gives the reaction of silicates (2.3.1).
TESTS
Solution S1 Weigh 10.0 g of the substance to be examined into a conical flask fitted with a reflux
condenser, add 50 ml of 0.5M hydrochloric acid gradually while stirring and heat on a water-bath for
30 min. Allow to cool. Transfer the mixture to a beaker and allow the undissolved material to settle.
Filter the supernatant through medium-speed filter paper into a 100 ml volumetric flask, retaining as
much as possible of the insoluble material in the beaker. Wash the residue and the beaker with three
quantities, each of 10 ml, of hot water R. Wash the filter with 15 ml of hot water R, allow the filtrate
to cool and dilute to 100.0 ml with the same solvent.
Solution S2 Weigh 0.5 g of the substance to be examined in a 100 ml polytetrafluoroethylene dish,
add 5 ml of hydrochloric acid R, 5 ml of lead-free nitric acid R, and 5 ml of perchloric acid R. Stir gently
then add 35 ml of hydrofluoric acid R and evaporate slowly to dryness on a hot plate. To the residue,
add 5 ml of hydrochloric acid R, cover with a watch-glass, heat to boiling and allow to cool. Rinse the
watch-glass and the dish with water R. Transfer into a volumetric flask containing 5 ml of a 25.34 g/l
solution of caesium chloride R, rinse the dish with water R and dilute to 50.0 ml with the same solvent.
pH (2.2.3). The pH of the filtrate obtained in the test for water-soluble substances, is 7.0 to
9.0. Read the pH 1 min after inserting the electrode.
Water-soluble substances To 10.0 g add 50 ml of carbon dioxide-free water R, heat to boiling and
maintain boiling under a reflux condenser for 30 min. Allow to cool, filter through a medium-speed
filter paper and dilute to 50.0 ml with carbon dioxide-free water R. Take 25.0 ml of the filtrate,
evaporate to dryness and heat at 105C for 1 h. The residue weighs not more than 10 mg (0.2 per
cent).
Aluminium Not more than 2.0 per cent of aluminium, determined by atomic absorption spectrometry (Method I, 2.2.23).
Test solution. To 5.0 ml of solution S2 add 10 ml of a 25.34 g/l solution of caesium chloride R, 10.0 ml
of hydrochloric acid R and dilute to 100.0 ml with water R.
Reference solutions. Into four identical volumetric flasks, each containing 10.0 ml of hydrochloric acid R
and 10 ml of a 25.34 g/l solution of caesium chloride R, introduce respectively 5.0 ml, 10.0 ml,
15.0 ml and 20.0 ml of aluminium standard solution (100 ppm Al) R and dilute to 100.0 ml with
water R.
Measure the absorbance at 309.3 nm, using an aluminium hollow-cathode lamp as the radiation
source and a nitrous oxide-acetylene flame.
Calcium Not more than 0.9 per cent of calcium, determined by atomic absorption spectrometry
(Method I, 2.2.23).
Test solution. To 5.0 ml of solution S2 add 10.0 ml of hydrochloric acid R, 10 ml of lanthanum chloride
solution R and dilute to 100.0 ml with water R.
and 10 ml of lanthanum chloride solution R, introduce respectively 1.0 ml, 2.0 ml, 3.0 ml and 4.0 ml of
calcium standard solution (100 ppm Ca) R1 and dilute to 100.0 ml with water R.
Measure the absorbance at 422.7 nm using a calcium hollow-cathode lamp as the radiation source
and a nitrous oxide-acetylene flame.
Iron Not more than 0.25 per cent of iron, determined by atomic absorption spectrometry (Method I,
2.2.23).
Test solution. To 2.5 ml of solution S1, add 50.0 ml of 0.5M hydrochloric acid and dilute to 100.0 ml
with water R.
Reference solutions. Into four identical volumetric flasks, each containing 50.0 ml of 0.5M hydrochloric
acid, introduce respectively 2.0 ml, 2.5 ml, 3.0 ml and 4.0 ml of iron standard solution (250 ppm Fe) R
and dilute to 100.0 ml with water R.
Measure the absorbance at 248.3 nm using an iron hollow-cathode lamp as the radiation source
and an air-acetylene flame. Make a correction using a deuterium lamp.
Magnesium 17.0 per cent to 19.5 per cent of magnesium, determined by atomic absorption spectrometry (Method I, 2.2.23).
Test solution. Dilute 0.5 ml of solution S2 to 100.0 ml with water R. To 4.0 ml of the solution, add
10.0 ml of hydrochloric acid R, 10 ml of lanthanum chloride solution R and dilute to 100.0 ml with
water R.
39-5
and 10 ml of lanthanum chloride solution R, introduce respectively 2.5 ml, 3.0 ml, 4.0 ml and 5.0 ml of
magnesium standard solution (10 ppm Mg) R1 and dilute to 100.0 ml with water R.
Measure the absorbance at 285.2 nm using a magnesium hollow-cathode lamp as the radiation
source and an air-acetylene flame.
Lead Not more than 10 ppm of lead, determined by atomic absorption spectrometry (Method I,
2.2.23).
Test solution. Use solution Sl.
Reference solutions. Into four identical volumetric flasks, each containing 50.0 ml of 0.5M hydrochloric
acid, introduce respectively 5.0 ml, 7.5 ml, 10.0 ml and 12.5 ml of lead standard solution (10 ppm
Pb) R1 and dilute to 100.0 ml with water R.
Measure the absorbance at 217.0 nm using a lead hollow-cathode lamp as the radiation source and
Loss on ignition Not more than 7.0 per cent, determined on 1.00 g by ignition to constant weight
at 1050C to 1100C.
Microbial contamination If intended for topical administration, the total viable aerobic count
(2.6.12) is not more than a total of 102 aerobic bacteria and fungi per gram.
If intended for oral administration, the total viable aerobic count (2.6.12) is not more than a total of
103 aerobic bacteria and not more than 102 fungi per gram.
LABELLING
The label states, where applicable, that the substance is suitable for oral or topical administration.
__________________________________________________________________________________________________________ Ph Eur
39-6
Tamoxifen Citrate
O
NMe2
H3C
CH 2COOH
,HO
COOH
CH 2COOH
C26H29NO,C6H8O7
563.6
54965-24-1
Tamoxifen Citrate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1046].
Action and use Antioestrogen.
Preparation
Tamoxifen Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Tamoxifen citrate contains not less than 99.0 per cent and not more than the equivalent of 101.0 per
cent of (Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]ethyldimethylamine citrate, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, slightly soluble in water, soluble in methanol, slightly
soluble in acetone.
IDENTIFICATION
A. Dissolve 20 mg in methanol R and dilute to 50.0 ml with the same solvent. Dilute 5.0 ml of the
obtained with tamoxifen citrate CRS. Examine the substances prepared as discs. If the spectra
obtained in the solid state show differences, dissolve the substance to be examined and the reference
substance separately in acetone R, evaporate to dryness and record new spectra using the residues.
the same solvent.
Reference solution (a). Dissolve 10 mg of tamoxifen citrate CRS in methanol R and dilute to 10 ml with
the same solvent.
Reference solution (b). Dissolve 10 mg of tamoxifen citrate CRS and 10 mg of clomifene citrate CRS in
10 volumes of triethylamine R and 90 volumes of toluene R. Allow the plate to dry in air and examine
in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution
is similar in position and size to the principal spot in the chromatogram obtained with reference
D. Dissolve about 10 mg in 4 ml of pyridine R with shaking and add 2 ml of acetic anhydride R. A
yellow colour is produced. Heat on a water-bath for 2 min. A pink to red colour is produced.
TESTS
E-isomer and related substances Examine by liquid chromatography (2.2.29). Prepare the solutions
39-7
immediately before use and protect from light.
Reference solution (a). Dissolve 15.0 mg of tamoxifen citrate for performance test CRS in the mobile
60 volumes of water R containing 0.9 g/l of sodium dihydrogen phosphate R and 4.8 g/l of
N,N-dimethyloctylamine R, adjusted to pH 3.0 with phosphoric acid R,
Equilibrate the column with the mobile phase at a flow rate of 1.2 ml per minute for about 30 min.
Adjust the sensitivity of the system so that the height of the peak due to the E-isomer in the chromatogram obtained with 10 l of reference solution (a) is not less than 40 per cent of the full scale of
the recorder. The test is not valid unless the chromatogram obtained resembles that of the specimen
chromatogram provided with the tamoxifen citrate for performance test CRS in that the resolution
between the peaks due to the E-isomer and to tamoxifen impurity F is at least three and there is baseline separation of the peak due to tamoxifen impurity F from the following peak of the principal
component.
Inject separately 10 l of the test solution and 10 l of reference solution (b). Continue the chromatography for twice the retention time of the tamoxifen peak. In the chromatogram obtained with the
test solution: the area of any peak, apart from the principal peak, is not greater than 0.3 times that of
the principal peak in the chromatogram obtained with reference solution (b) (0.3 per cent); the sum
of the areas of all the peaks, apart from the principal peak and any peak due to the E-isomer, is not
greater than 0.5 times the area of the principal peak in the chromatogram of reference solution (b)
(0.5 per cent). Disregard any peak with a retention time of less than 2.5 min and any peak with an
area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference
65C for 4 h.
ASSAY
Dissolve 0.400 g in 75 ml of anhydrous acetic acid R. Titrate with 0.1M perchloric acid using 0.1 ml of
naphtholbenzein solution R as indicator.
STORAGE
IMPURITIES
O
NMe2
CH3
A. (E)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]ethyldimethylamine,
O
H3C
OH
NMe2
B. 2-[4-(1-hydroxy-1,2-diphenylbutyl)phenoxy]ethyldimethylamine,
39-8
O
NMe2
H
C. 2-[4-(1,2-diphenylvinyl)phenoxy]ethyldimethylamine,
O
NMe2
H3C
D. 2-[4-(1,2-diphenylprop-1-enyl)phenoxy]ethyldimethylamine,
NMe2
O
H3C
E. 2-[2-(1,2-diphenylbut-1-enyl)phenoxy]ethyldimethylamine,
O
NHMe
H3C
F. (Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]ethylmethylamine,
O
O
NMe2
H3C
G. 1-(4-dimethylaminoethoxyphenyl)-2-phenylbutan-1-one.
__________________________________________________________________________________________________________ Ph Eur
39-9
Tannic Acid
1401-55-4
Tannic Acid complies with the requirements of the 3rd edition of the European Pharmacopoeia [1477]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Tannic acid is a mixture of esters of glucose with gallic acid and 3-galloylgallic acid.
CHARACTERS
A yellowish-white or slightly brown amorphous light powder or shiny plates, very soluble in water,
freely soluble in acetone, in alcohol and in glycerol (85 per cent), practically insoluble in methylene
chloride.
IDENTIFICATION
A. Dilute 0.1 ml of solution S (see Tests) to 5 ml with water R. Add 0.1 ml of ferric chloride solution R1. A blackish-blue colour is produced which becomes green on the addition of 1 ml of dilute
sulphuric acid R.
B. To 1 ml of solution S, add 3 ml of a 1 g/l solution of gelatin R. The mixture becomes turbid and a
flocculent precipitate is formed.
C. Dilute 0.1 ml of solution S to 5 ml with water R. Add 0.3 ml of barium hydroxide solution R. A
greenish-blue precipitate is formed.
TESTS
Dextrins, gum, salts, sugars To 2 ml of solution S, add 2 ml of alcohol R. The solution is clear.
Add 1 ml of ether R. The solution remains clear for at least 10 min.
Resins To 5 ml of solution S, add 5 ml of water R. The mixture remains clear for at least 15 min
(2.2.1).
to 105C.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
39-10
Tapioca Starch
Cassava Starch
Definition Tapioca Starch is obtained from the rhizomes of Manihot utilissima Pohl.
Characteristics Very fine powder which creaks when pressed between the fingers.
Practically insoluble in cold water and in ethanol (96%).
Identification
A. Principally simple granules, subspherical, muller-shaped or rounded polyhedral; smaller granules 5
to 10 m, larger granules 20 to 35 m in diameter; hilum, central, punctate, linear or triradiate;
striations, faint, concentric; compound granules, few, of two to three unequal components.
B. Heat to boiling a suspension of 1 g in 50 ml of water for 1 minute and cool. A thin, cloudy
mucilage is formed.
C. Mix 0.05 ml of iodine solution R1 with 1 ml of the mucilage obtained in test B. A dark blue colour
is produced which disappears on heating and reappears on cooling.
Acidity Add 10 g of the starch to 100 ml of ethanol (70%) previously neutralised to 0.5 ml of phenolphthalein solution, shake for 1 hour, filter and titrate 50 ml of the filtrate with 0.1M sodium hydroxide
VS. Not more than 2.0 ml is required to change the colour of the solution.
weight. Use 1 g.
Sulphated ash Not more than 0.6%, Appendix IX A, Method II. Use 1 g.
Storage Tapioca Starch should be kept in an airtight container.
Microbial contamination 1.0 g is free from Escherichia coli, Appendix XVI B1.
Wheat Starch or, in tropical and sub-tropical countries where these are not available, Tapioca Starch
may be supplied or used.
39-11
Tar
Definition Tar is bituminous liquid obtained from the wood of various trees of the family Pinaceae
by destructive distillation and is known in commerce as Stockholm Tar.
Characteristics Dark brown or nearly black, semi-liquid; denser than water; odour, characteristic
and empyreumatic.
Soluble in chloroform, in ethanol (90%), in ether and in fixed and volatile oils.
Identification
A. The aqueous liquid obtained by shaking 1 g with 20 ml of water for 5 minutes is acidic to litmus
paper.
B. Carefully add 0.5 g to 10 ml of petroleum spirit (boiling range, 40 to 60) and allow to stand for 30
minutes. When examined in daylight no fluorescence is produced.
Action and use Used in treatment of psoriasis.
39-12
Coal Tar
Definition Coal Tar is a product obtained from bituminous coal by destructive distillation at about
1000.
Characteristics A nearly black, viscous liquid; odour, strong, penetrating and characteristic. On
exposure to air, the viscosity gradually increases. It burns in air with a luminous, sooty flame. It has a
weight per ml of about 1.15 g.
Slightly soluble in water; partly soluble in chloroform, in ethanol, in ether and in volatile oils.
Identification
A. A saturated solution is alkaline to litmus solution.
B. Carefully add 0.5 g to 10 ml of petroleum spirit (boiling range, 40 to 60) and allow to stand for 30
minutes. When examined in daylight, the supernatant liquid has a blue fluorescence which becomes
more intense when viewed under ultraviolet light (365 nm).
Action and use Used in treatment of psoriasis.
Preparations
Coal Tar and Salicylic Acid Ointment
Coal Tar Solution
Strong Coal Tar Solution
Zinc and Coal Tar Paste
39-13
Tartaric Acid
H
OH
COOH
HOOC
H
C4H6O6
OH
150.1
87-69-4
Tartaric Acid complies with the requirements of the 3rd edition of the European Pharmacopoeia [0460].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Tartaric acid contains not less than 99.5 per cent and not more than the equivalent of 101.0 per cent
of (2R,3R)-2,3-dihydroxybutane dioic acid, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder or colourless crystals, very soluble in water, freely soluble
in alcohol.
IDENTIFICATION
A. Solution S (see Tests) is strongly acid (2.2.4).
B. It gives the reactions of tartrates (2.3.1).
TESTS
solvent. The specific optical rotation is +12.0 to +12.8.
Oxalic acid Dissolve 0.80 g in 4 ml of water R. Add 3 ml of hydrochloric acid R and 1 g of zinc R in
granules and boil for 1 min. Allow to stand for 2 min. Collect the liquid in a test-tube containing
0.25 ml of a 10 g/l solution of phenylhydrazine hydrochloride R and heat to boiling. Cool rapidly,
transfer to a graduated cylinder and add an equal volume of hydrochloric acid R and 0.25 ml of a
50 g/l solution of potassium ferricyanide R. Shake and allow to stand for 30 min. Any pink colour in
the solution is not more intense than that in a standard prepared at the same time in the same
manner using 4 ml of a 0.1 g/l solution of oxalic acid R (350 ppm calculated as anhydrous oxalic
acid).
Calcium (2.4.3). To 5 ml of solution S add 10 ml of a 50 g/l solution of sodium acetate R in distilled
water R. The solution complies with the limit test for calcium (200 ppm).
100C to 105C.
ASSAY
Dissolve 0.650 g in 25 ml of water R. Titrate with 1M sodium hydroxide using 0.5 ml of phenolphthalein
solution R as indicator, until a pink colour is obtained.
__________________________________________________________________________________________________________ Ph Eur
39-14
Temazepam
Me
O
N
OH
N
Cl
and enantiomer
C16H13ClN2O2
300.7
846-50-4
Temazepam complies with the requirements of the 3rd edition of the European Pharmacopoeia [0954]. These
Action and use Sedative and hypnotic.
Preparations
Temazepam Oral Solution
Temazepam Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Temazepam contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of (RS)-7-chloro-2,3-dihydro-3-hydroxy-1-methyl-5-phenyl-1H-1,4-benzodiazepin-2-one, calculated
CHARACTERS
A white or almost white, crystalline powder, practically insoluble in water, freely soluble in methylene
chloride, sparingly soluble in alcohol.
IDENTIFICATION
B. Prepare the solutions immediately before use and carry out the identification test protected from light.
Dissolve 40.0 mg in alcohol R and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml to 50.0 ml
with alcohol R. Examined between 220 nm and 350 nm (2.2.25), the solution shows an absorption
maximum at 230 nm and a shoulder at about 250 nm and may show a broad absorption maximum
at about 315 nm and a minor inflexion at 275 nm. The specific absorbance at the maximum at
230 nm is 1040 to 1140.
obtained with temazepam CRS. Examine the substances prepared as discs.
D. Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable silica gel
the same solvent.
Reference solution (a). Dissolve 10 mg of temazepam CRS in alcohol R and dilute to 10 ml with the
same solvent.
Reference solution (b). Dissolve 10 mg of temazepam CRS and 10 mg of flunitrazepam CRS in alcohol R
10 volumes of diethylamine R and 90 volumes of ether R. Dry the plate in a current of air and examine
39-15
TESTS
Related substances Prepare the solutions immediately before use and carry out the test protected from light.
Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating substance.
Reference solution (a). Dilute 0.5 ml of the test solution to 100 ml with a mixture of equal volumes of
Reference solution (b). Dilute 2 ml of reference solution (a) to 5 ml with a mixture of equal volumes of
volumes of methanol R and 98 volumes of methylene chloride R. Allow the plate to dry in air and
examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with the test solution,
reference solution (a) (0.5 per cent) and at most one such spot is more intense than the spot in the
100C to 105C for 4 h.
ASSAY
Dissolve 0.250 g in 50 ml of nitroethane R. Titrate with 0.1M perchloric acid, determining the endpoint potentiometrically (2.2.20).
STORAGE
IMPURITIES
NHMe
Cl
A. 5-chloro-2-methylaminobenzophenone.
__________________________________________________________________________________________________________ Ph Eur
39-16
Tenoxicam
O
O
S
NMe
H
N
S
OH
C13H11N3O4S2
N
337.4
59804-37-4
Tenoxicam complies with the requirements of the 3rd edition of the European Pharmacopoeia [1156]. These
Preparations
Tenoxicam Injection
Tenoxicam Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Tenoxicam contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of 4-hydroxy-2-methyl-N-(pyridin-2-yl)-2H-thieno[2,3-e]1,2-thiazine-3-carboxamide 1,1-dioxide,
CHARACTERS
A yellow, crystalline powder, practically insoluble in water, sparingly soluble in methylene chloride,
very slightly soluble in ethanol. It dissolves in solutions of acids and alkalis.
IDENTIFICATION
with tenoxicam CRS. If the spectra obtained in the solid state show differences, dissolve the substance
to be examined and the reference substance separately in the minimum quantity of methylene
chloride R, evaporate to dryness on a water-bath and record new spectra using the residues.
TESTS
Appearance of solution Dissolve 0.10 g in 20 ml of methylene chloride R. The solution is clear
(2.2.1).
coating substance.
Test solution. Dissolve 0.4 g in a mixture of 4 volumes of concentrated ammonia R and 96 volumes of
Reference solution (a). Dilute 1 ml of the test solution to 20 ml with a mixture of 4 volumes of concentrated ammonia R and 96 volumes of methanol R. Dilute 1 ml of the solution to 20 ml with the same
Reference solution (b). Dissolve 20 mg of tenoxicam CRS and 20 mg of salicylic acid CRS in a mixture
of 4 volumes of concentrated ammonia R and 96 volumes of methanol R and dilute to 5 ml with the
same mixture of solvents.
Reference solution (c). Dissolve 20 mg of pyridin-2-amine R in a mixture of 4 volumes of concentrated
ammonia R and 96 volumes of methanol R and dilute to 5 ml with the same mixture of solvents.
Dilute 2 ml of the solution to 50 ml with the same mixture of solvents.
Apply separately to the plate 10 l of each solution. Develop over a path of 15 cm with a mixture of 5
volumes of anhydrous formic acid R, 5 volumes of methanol R, 20 volumes of acetone R and 70 volumes
of methylene chloride R. Allow the plate to dry in air. Examine in ultraviolet light at 254 nm. Any spot
corresponding to pyridin-2-amine in the chromatogram obtained with the test solution is not more
intense than the spot in the chromatogram obtained with reference solution (c) (0.2 per cent). Any
spot in the chromatogram obtained with the test solution apart from the principal spot and any spot
corresponding to pyridin-2-amine, is not more intense than the spot in the chromatogram obtained
39-17
ASSAY
1 ml of 0.1M perchloric acid is equivalent to 33.74 mg of C13H11N3O4S2.
STORAGE
IMPURITIES
H2N
N
A. pyridin-2-amine,
O
O
S
NMe
S
OH
COOCH3
B. methyl 4-hydroxy-2-methyl-2H-thieno[2,3-e]-1,2-thiazine-3-carboxylate 1,1-dioxide.

__________________________________________________________________________________________________________ Ph Eur
39-18
Terbutaline Sulphate
H
OH
NHBut
HO
,H2SO4
OH
and enantiomer
(C12H19NO3)2,H2SO4
548.7
23031-32-5
Terbutaline Sulphate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Terbutaline Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Terbutaline sulphate contains not less than 98.0 per cent and not more than the equivalent of
101.0 per cent of bis[(1RS)-1-(3,5-dihydroxyphenyl)-2-[(1,1-dimethylethyl)amino]ethanol] sulphate,
CHARACTERS
A white or almost white, crystalline powder, freely soluble in water, slightly soluble in alcohol.
IDENTIFICATION
obtained with terbutaline sulphate CRS. If the spectra obtained in the solid state show differences,
dissolve the substance to be examined and the reference substance separately in aldehyde-free
methanol R, evaporate to dryness and record new spectra using the residues.
B. 5 ml of solution S (see Tests) gives reaction (a) of sulphates (2.3.1).
TESTS
Appearance of solution Solution S is clear (2.2.1). The absorbance (2.2.25) of solution S measured
at 400 nm in a 2 cm cell is not greater than 0.11.
Acidity To 10 ml of solution S add 0.05 ml of methyl red solution R. Not more than 1.2 ml of 0.01M
sodium hydroxide is required to change the colour of the indicator to yellow.
+0.10.
Reference solution (a). Dissolve 7.5 mg of terbutaline impurity C CRS and 22.5 mg of terbutaline sulphate
sodium hexanesulphonate R in 770 ml of 0.050M ammonium formate solution prepared as follows:
dissolve 3.15 g of ammonium formate R in about 980 ml of water R; adjust to pH 3.0 by adding
about 8 ml of anhydrous formic acid R and dilute to 1000 ml with water R; then add 230 ml of
methanol R,
as detector a spectrophotometer set a 276 nm.
39-19
obtained with reference solution (b) is at least 50 per cent of the full scale of the recorder.
Inject 20 l of reference solution (a). When the chromatograms are recorded under the prescribed
conditions, the retention times are: terbutaline impurity C, about 9 min and terbutaline sulphate,
about 11 min. The test is not valid unless the resolution between the two peaks corresponding to
terbutaline sulphate and terbutaline impurity C is at least 2.0. If necessary adjust the composition of
the mobile phase. Decrease the content of methanol to increase the retention time.
solution (b). Continue the chromatography for 2.5 times the retention time of terbutaline sulphate.
In the chromatogram obtained with the test solution, the area of any peak corresponding to
terbutaline impurity C is not greater than twice that of the corresponding peak in the chromatogram
obtained with reference solution (a) (0.2 per cent); the area of any other peak, apart from the
principal peak, is not greater than that of the principal peak obtained with reference solution (b)
(0.2 per cent); the sum of the areas of all the peaks, apart from the principal peak and the peak
corresponding to terbutaline impurity C, is not greater than twice the area of the principal peak
obtained with reference solution (b) (0.4 per cent). Disregard any peak due to the mobile phase and
any peak with an area less than 0.1 times the area of the principal peak obtained with reference
solution (b).
100C to 105C for 3 h.
ASSAY
Dissolve 0.400 g in 70 ml of anhydrous acetic acid R with heating. Titrate with 0.1M perchloric acid,
STORAGE
Store in an well-closed container.
IMPURITIES
HO
COOH
OH
A. 3,5-dihydroxybenzoic acid (-resorcylic acid),

H OH
HO
and enantiomer
N
Bu t
OH
B. (4RS)-2-(1,1-dimethylethyl)-1,2,3,4-tetrahydroisoquinoline-4,6,8-triol,
O
NHBu t
HO
OH
C. 1-(3,5-dihydroxyphenyl)-2-[(1,1-dimethylethyl)amino]ethanone.
__________________________________________________________________________________________________________ Ph Eur
39-20
Terconazole
N
Cl
N
O
O
O
Cl
H
N
Me
N
and enantiomer
Me
C26H31Cl2N5O3
532.5
67915-31-5
Terconazole complies with the requirements of the 3rd edition of the European Pharmacopoeia [1046]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Terconazole contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of 1-[4-[[(2RS,4SR)-2-(2,4-dichlorophenyl)-2-[(1H-1,2,4-triazol-l-yl)methyl]-1,3-dioxolan4-yl]methoxy]phenyl]-4-(1-methylethyl)piperazine, calculated with reference to the dried substance.
CHARACTERS
A white or almost white powder, practically insoluble in water, freely soluble in methylene chloride,
soluble in acetone, sparingly soluble in alcohol.
IDENTIFICATION
obtained with terconazole CRS. If the spectra obtained in the solid state show differences, dissolve the
substance to be examined and the reference substance separately in the minimum volume of
acetone R, evaporate to dryness in a current of air and record new spectra using the residues.
B. Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable octadecylsilyl silica gel.
the same solvent.
Reference solution (a). Dissolve 30 mg of terconazole CRS in methanol R and dilute to 5 ml with the
same solvent.
Reference solution (b). Dissolve 30 mg of terconazole CRS and 30 mg of ketoconazole CRS in methanol R
10 cm using a mixture of 20 volumes of ammonium acetate solution R, 40 volumes of dioxan R and 40
volumes of methanol R. Dry the plate in a current of warm air for 15 min and expose it to iodine
vapour until the spots appear. Examine in daylight. The principal spot in the chromatogram obtained
reference solution (b) shows two clearly separated spots.
C. To 30 mg in a porcelain crucible add 0.3 g of anhydrous sodium carbonate R. Heat over an open
TESTS
Optical rotation (2.2.7). Dissolve 1.0 g in methylene chloride R and dilute to 10 ml with the same
solvent. The angle of optical rotation is 0.10 to +0.10.
39-21
Reference solution (a). Dissolve 2.5 mg of terconazole CRS and 2.0 mg of ketoconazole CRS in
methanol R and dilute to 100.0 ml with the same solvent.
a stainless steel column 0.1 m long and 4.6 mm in internal diameter packed with base-deactivated
as mobile phase at a flow rate of 2 ml per minute:
Mobile phase A. A 3.4 g/l solution of tetrabutylammonium hydrogen sulphate R,
Interval
(min)
Mobile phase A
(per cent V/V)

(per cent V/V)
010
1015
1520
9550
50
95
550
50
5
20 = 0
95
Linear gradient
Isocratic elution
Switch to initial
eluent composition
Re-start gradient

Equilibrate the column for at least 30 min with acetonitrile R at a flow rate of 2 ml per minute and
then equilibrate at the initial elution composition for at least 5 min.
conditions, the retention times are: ketoconazole about 6 min and terconazole about 7.5 min. The test
is not valid unless the resolution between the peaks corresponding to ketoconazole and terconazole is
at least 13. If necessary, adjust the concentration of acetonitrile in the mobile phase or adjust the time
programme for the linear gradient elution.
the principal peak, is not greater than that of the principal peak in the chromatogram obtained with
reference solution (b) (0.5 per cent). Disregard any peak obtained with the blank run and any peak
100C to 105C.
ASSAY
ethyl ketone R. Titrate with 0.1M perchloric acid determining the end-point potentiometrically at the
second point of inflexion (2.2.20).
STORAGE
IMPURITIES
N
N
Cl
N
O
H
N
e
N
and enantiomer
Me
A. 1-[4-[[(2RS,4RS)2-(2,4-dichlorophenyl)-2-[(1H-1,2,4-triazol-1-yl)methyl]-1,3-dioxolan-4yl]methoxy]phenyl]-4-(1-methylethyl)piperazine,
39-22
N
N
Cl
N
O
H
N
e
and enantiomer
Me
B. 1-[4-[[(2RS,4SR)2-(2,4-dichlorophenyl)-2-[(4H-1,2,4-triazol-4-yl)methyl]-1,3-dioxolan-4yl]methoxy]phenyl]-4-(1-methylethyl)piperazine.
__________________________________________________________________________________________________________ Ph Eur
39-23
Terfenadine
OH
N
H
OH
But
and enantiomer
C32H41NO2
471.7
50679-08-8
Terfenadine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0955]. These
Preparations
Terfenadine Oral Suspension
Terfenadine Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Terfenadine contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent
of (RS)-1-[4-(1,1-dimethylethyl)phenyl]-4-[4-(hydroxydiphenyl-methyl)piperidin-1-yl]butan-1-ol,
CHARACTERS
A white, crystalline powder, very slightly soluble in water and in dilute hydrochloric acid, freely
soluble in methylene chloride, soluble in methanol.
IDENTIFICATION
B. Dissolve 50.0 mg in methanol R and dilute to 100.0 ml with the same solvent. Examined between
230 nm and 350 nm (2.2.25), the solution shows an absorption maximum at 259 nm and shoulders
at 253 nm and 270 nm. The specific absorbance at the maximum is 13.5 to 14.9.
obtained with terfenadine CRS.
D. Examine by thin-layer chromatography (2.2.27), using silica gel HF254 R as the coating substance.
Reference solution. Dissolve 50 mg of terfenadine CRS in methylene chloride R and dilute to 10 ml with
the same solvent.
solution is similar in position and size to the principal spot in the chromatogram obtained with the
reference solution.
TESTS
Reference solution (b). Dissolve 15 mg of terfenadine impurity A CRS in the mobile phase and dilute to
39-24
10.0 ml with the mobile phase. To 5.0 ml of this solution, add 5.0 ml of the test solution and dilute
Reference solution (c). Dilute 10.0 ml of reference solution (a) to 25.0 ml with the mobile phase.
Reference solution (d). Dissolve 0.1 g of potassium iodide R in the mobile phase and dilute to 100 ml
with mobile phase.
as mobile phase at a flow rate of 1 ml per minute a mixture prepared as follows: dilute 600 ml of
acetonitrile R to 1 litre with diethylammonium phosphate buffer solution pH 6.0 R,
as the detector a spectrophotometer set at 217 nm,
a loop injector.
Inject 20 l of each solution and continue the chromatography for five times the retention time of
terfenadine. The test is not valid unless, in the chromatogram obtained with reference solution (b),
the resolution between the peaks corresponding to terfenadine and terfenadine impurity A is greater
than 5.0 and the mass distribution ratio of the peak corresponding to terfenadine is greater than 2.0.
Determine the mass distribution ratio using, as unretained compound, potassium iodide R. In the
chromatogram obtained with the test solution, the area of any peak apart from the principal peak is
not greater than that of the peak in the chromatogram obtained with reference solution (c) (0.2 per
cent) and the sum of the areas of all the peaks apart from the principal peak is not greater than the
area of the peak in the chromatogram obtained with reference solution (a) (0.5 per cent). Disregard
any peak with an area less than 2.5 per cent of that of the peak in the chromatogram obtained with
reference solution (c).
under a pressure not exceeding 0.5 kPa.
ASSAY
STORAGE
IMPURITIES
O
N
HO
Ph Ph
But
A. 1-[4-(1,1-dimethylethyl)phenyl]-4-[4-(hydroxydiphenylmethyl)piperidin-1-yl]butan-1-one
(ketone),
OH
Ph Ph
But
B. 1-[4-(1,1-dimethylethyl)phenyl]-4-[4-(diphenylmethyl)piperidin-1-yl]butan-1-ol,
O
N
OH
HO
Ph Ph
But
C. 1-[4-(1,1-dimethylethyl)-phenyl]-4-(hydroxydiphenylmethyl)piperidin-1-yl]-butan-1-ol Noxide,
39-25
OH
Ph Ph
But
D. 1-[4-(1,1-dimethylethyl)phenyl]-4-[4-(diphenylmethylene)piperidin-1-yl]butan-1-ol,
OH
N
HOOC
But
E. 1-[4-[4-(1,1-dimethylethyl)phenyl]-4-hydroxybutyl]piperidine-4-carboxylic acid,
N
Ph Ph
But
F. 1-[4-[4-(1,1-dimethylethyl)phenyl]but-3-enyl]-4-(diphenylmethylene)piperidine,
N
HO
Ph Ph
But
G. 1-[4-[4-(1,1-dimethylethyl)phenyl]but-3-enyl]piperidin-4-yl](diphenyl)methanol,
N
HO
Ph Ph
But
H. 1-[4-[4-(1,1-dimethylethyl)phenyl]butyl]piperidin-4-yl](diphenyl)methanol,
NH
HO
Ph Ph
I. diphenyl(piperidin-4-yl)methanol,
N
OH
EtOOC
But
J. ethyl 1-[4-[4-(1,1-dimethylethyl)phenyl]-4-hydroxybutyl]piperidine-4-carboxylate.
__________________________________________________________________________________________________________ Ph Eur
39-26
Terpineol
Me
OH
Me
C10H18O
Me
154.3
98-55-5
Definition Terpineol is a mixture of structural isomers in which -terpineol predominates.

Characteristics A colourless, slightly viscous, liquid which may deposit crystals; odour, pleasant
and characteristic.
Very slightly soluble in water; freely soluble in ethanol (70%); soluble in ether.
Low-boiling substances Not more than 4.0% v/v distils below 214, Appendix V C.
39-27
Testosterone
Me
H
OH
Me
H
H
O
C19H28O2
288.4
58-22-0
Testosterone complies with the requirements of the 3rd edition of the European Pharmacopoeia [1373]. These
Preparation
Testosterone Implants
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Testosterone contains not less than 97.0 per cent and not more than the equivalent of 103.0 per cent
of 17-hydroxyandrost-4-en-3-one, calculated with reference to the dried substance.
CHARACTERS
A white crystalline powder, or colourless or yellowish-white crystals, practically insoluble in water,
freely soluble in alcohol and in methylene chloride, practically insoluble in fatty oils.
IDENTIFICATION
obtained with testosterone CRS.
TESTS
substance.
plate R.
Reference solution (a). Dilute 1 ml of test solution (a) to 100 ml with methylene chloride R.
Reference solution (b). Dissolve 12.5 mg of testosterone CRS in methylene chloride R and dilute to 5 ml
Reference solution (c). Dissolve 10 mg of testosterone impurity A CRS in methylene chloride R and dilute
to 10 ml with the same solvent. Dilute 1 ml of the solution to 5 ml with test solution (a).
Apply to the plate 5 l of each solution. Develop over a path of 15 cm using a mixture of equal
volumes of ethyl acetate R and toluene R. Allow the plate to dry in air. Examine in ultraviolet light at
is not more intense than the principal spot in the chromatogram obtained with reference solution (a)
(1 per cent). The test is not valid unless the chromatogram obtained with reference solution (c)
100C to 105C for 2 h.
ASSAY
Dissolve 50.0 mg in alcohol R and dilute to 100.0 ml with the same solvent. Dilute 2.0 ml to
100.0 ml with alcohol R. Measure the absorbance (2.2.25) at the maximum at 241 nm.
39-28
STORAGE
IMPURITIES
Me O
Me
H
H
A. androst-4-ene-3,17-dione (androstenedione),
Me O
Me
H
H
EtO
B. 3-ethoxyandrosta-3,5-dien-17-one (androstenedione enolether).

__________________________________________________________________________________________________________ Ph Eur
39-29
Testosterone Decanoate
Me
H
OCO[CH2] 8CH3
Me
H
H
O
C29H46O3
442.7
5721-91-5
Definition Testosterone Decanoate is 3-oxo-androst-4-en-17-yl decanoate. It contains not less

than 97.0% and not more than 103.0% of C29H46O3, calculated with reference to the dried
substance.
Characteristics White to creamy white crystals or crystalline powder.
Practically insoluble in water; very soluble in chloroform and in ethanol (96%).
Identification
testosterone decanoate (RS 330).
B. Carry out the method for thin-layer chromatography, Appendix III A, using a plate precoated with
silica gel F254 the surface of which has been modified with chemically-bonded octadecylsilyl groups
(Whatman KC18F plates are suitable) and a mixture of 60 volumes of propan-2-ol, 40 volumes of
three solutions in a mixture of 9 volumes of chloroform and 1 volume of methanol containing (1)
0.05% w/v of the substance being examined, (2) 0.05% w/v of testosterone decanoate BPCRS and (3)
0.05% w/v each of testosterone decanoate BPCRS, testosterone enanthate EPCRS and testosterone
isocaproate BPCRS. After removal of the plate, allow it to dry in air until the solvent has evaporated
and heat at 100 for 10 minutes. Allow to cool and examine under ultraviolet light (254 nm). The
principal spot in the chromatogram obtained with solution (1) corresponds to that in the chromatogram obtained with solution (2). Spray the plate with ethanolic sulphuric acid (20%), heat at 120 for
10 minutes and allow to cool. The principal spot in the chromatogram obtained with solution (1) is
green and corresponds in position and size to the spot in the chromatogram obtained with solution
(2). The test is not valid unless the chromatogram obtained with solution (3) shows three distinct
principal spots by each method of visualisation.
Free decanoic acid Dissolve 0.65 g in 10 ml of ethanol (96%), previously neutralised to bromothymol
blue solution R3, and titrate immediately with 0.01M sodium hydroxide VS using bromothymol blue
solution R3 as indicator. Not more than 0.6 ml of 0.01M sodium hydroxide VS is required to change
the colour of the solution.
gel G as the coating substance and a mixture of 80 volumes of toluene and 20 volumes of ethyl acetate
as the mobile phase. Apply separately to the plate 5 l of each of two solutions in a mixture of 9
volumes of chloroform and 1 volume of methanol containing (1) 2.0% w/v of the substance being
examined and (2) 0.020% w/v of testosterone. After removal of the plate, allow it to dry in air until the
odour of solvent is no longer detectable, heat at 110 for 10 minutes and spray the hot plate with
ethanolic sulphuric acid (20%) and heat at 110 for a further 10 minutes. Any secondary spot in the
Loss on drying When dried to constant weight over phosphorus pentoxide at a pressure not exceeding
0.7 kPa, loses not more than 0.5% of its weight. Use 1 g.
maximum at 241 nm.
Storage Testosterone Decanoate should be kept in a well-closed container, protected from light and
39-30
Testosterone Enantate
Me
H
OCO[CH2]5CH3
Me
H
H
O
C26H40O3
400.6
315-37-7
Testosterone Enantate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Anabolic steroid; androgen.
When testosterone enanthate is prescribed or demanded, Testosterone Enantate shall be dispensed or
supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Testosterone enantate contains not less than 97.0 per cent and not more than the equivalent of
103.0 per cent of 3-oxoandrost-4-en-17-yl heptanoate, calculated with reference to the dried
substance.
CHARACTERS
A white or yellowish-white, crystalline powder, practically insoluble in water, very soluble in ethanol
and in ether, freely soluble in fatty oils.
IDENTIFICATION
obtained with testosterone enantate CRS.
C. Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable octadecylsilyl silica gel with a fluorescent indicator having an optimal intensity at 254 nm.
Reference solution (a). Dissolve 5 mg of testosterone enantate CRS in a mixture of 1 volume of
solvents.
Reference solution (b). Dissolve 5 mg of testosterone enantate CRS, 5 mg of testosterone decanoate CRS
and 5 mg of testosterone isocaproate CRS in a mixture of 1 volume of methanol R and 9 volumes of
methylene chloride R and dilute to 10 ml with the same mixture of solvents.
20 volumes of water R, 40 volumes of acetonitrile R and 60 volumes of 2-propanol R. Allow the plate
to dry in air and heat at 100C for 10 min. Allow to cool and examine in ultraviolet light at 254 nm.
to the principal spot in the chromatogram obtained with reference solution (a). Spray the plate with
alcoholic solution of sulphuric acid R. Heat at 120C for 10 min. Allow to cool and examine in daylight.
The principal spot in the chromatogram obtained with the test solution is green and is similar in
position and size to the principal spot in the chromatogram obtained with reference solution (a). The
test is not valid unless the chromatogram obtained with reference solution (b) shows three clearly
separated principal spots by each method of visualisation.
D. To about 25 mg add 2 ml of a 10 g/l solution of potassium hydroxide R in methanol R and boil
under a reflux condenser for 1 h. Cool. Add 10 ml of water R. Acidify with dilute hydrochloric acid R
until blue litmus paper R turns red. Filter and wash the precipitate with a small quantity of water R.
The residue, after drying at 60C at a pressure not exceeding 0.7 kPa for 3 h, melts (2.2.14) at 150C
to 153C.
39-31
TESTS
substance.
coating substance.
methanol R and 9 volumes of methylene chloride R.
Reference solution (b). Dissolve 20 mg of testosterone propionate CRS in a mixture of 1 volume of
methanol R and 9 volumes of methylene chloride R, add 1 ml of the test solution and dilute to 100 ml
20 volumes of ethyl acetate R and 80 volumes of toluene R. Allow the plate to dry in air. Spray with
alcoholic solution of sulphuric acid R and heat at 120C for 10 min. Any spot in the chromatogram
obtained with the test solution, apart from the principal spot, is not more intense than the principal
spot in the chromatogram obtained with reference solution (a) (1.0 per cent). The test is not valid
unless the chromatogram obtained with reference solution (b) shows two clearly separated principal
spots.
Testosterone caproate Examine by liquid chromatography (2.2.29).
Reference solution (a). Dissolve 10.0 mg of testosterone caproate CRS in the mobile phase and dilute to
20.0 ml with the mobile phase. Dilute 1.0 ml to 50.0 ml with the mobile phase.
Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. To 1 ml of
this solution add 1 ml of reference solution (a).
as mobile phase at a flow rate of 1.0 ml per minute a mixture of 30 volumes of water R and 70
volumes of acetonitrile R,
Inject 20 l of reference solution (b). When using a recorder, adjust the sensitivity of the system so
that the height of the two peaks in the chromatogram obtained with reference solution (b) is at least
50 per cent of the full scale of the recorder. The test is not valid unless in the chromatogram obtained
the peak corresponding to testosterone caproate has a retention time of about 0.7 relative to the
second peak, which corresponds to testosterone enantate.
Inject 20 l of the test solution and 20 l of reference solution (a). In the chromatogram obtained
with the test solution, the area of any peak corresponding to testosterone caproate is not greater than
the area of the peak in the chromatogram obtained with reference solution (a) (1.0 per cent).
Heptanoic acid Dissolve 0.50 g of the substance to be examined in 10 ml of alcohol R previously
neutralised to bromothymol blue solution R3. Titrate immediately with 0.01M sodium hydroxide. Not
more than 0.6 ml of 0.01M sodium hydroxide is required to change the colour of the indicator to blue
(0.16 per cent).
desiccator over diphosphorus pentoxide R at a pressure not exceeding 0.7 kPa.
ASSAY
Dissolve 50.0 mg in ethanol R and dilute to 100.0 ml with the same solvent. Dilute 2.0 ml to
100.0 ml with ethanol R. Measure the absorbance (2.2.25) at the maximum at 241 nm.
STORAGE
Store in a well-closed container, protected from light at a temperature of 2C to 8C.
IMPURITIES
H3C[CH2] 5COOH
A. heptanoic acid,
39-32
Me
H
OCO[CH2] 4CH3
Me
H
H
H
O
B. 3-oxoandrost-4-en-17-yl hexanoate (testosterone caproate),

Me
H
OCO[CH2] 5CH3
Me
H
H
C. 3-oxo-5-androstan-17-yl heptanoate,
Me H
OH
Me
H
H
D. 17-hydroxyandrost-4-en-3-one (testosterone).
__________________________________________________________________________________________________________ Ph Eur
39-33
Testosterone Isocaproate
Me
H
OCOCH2CH2CHMe 2
Me
H
H
O
C25H38O3
386.6
15262-86-9
Definition Testosterone Isocaproate is 3-oxo-androst-4-en-17-yl 4-methylpentanoate. It contains

not less than 97.0% and not more than 103.0% of C25H38O3, calculated with reference to the dried
substance.
Characteristics White to creamy white crystals or crystalline powder.
Practically insoluble in water; very soluble in chloroform and in ethanol (96%).
Identification
testosterone isocaproate (RS 331).
B. Carry out the method for thin-layer chromatography, Appendix III A, using a plate precoated with
silica gel F254 the surface of which has been modified with chemically-bonded octadecylsilyl groups
(Whatman KC18F plates are suitable) and a mixture of 60 volumes of propan-2-ol, 40 volumes of
three solutions in a mixture of 9 volumes of chloroform and 1 volume of methanol containing (1)
0.05% w/v of the substance being examined, (2) 0.05% w/v of testosterone isocaproate BPCRS and (3)
0.05% w/v each of testosterone decanoate BPCRS, testosterone enanthate EPCRS and testosterone
isocaproate BPCRS. After removal of the plate, allow it to dry in air until the solvent has evaporated
and heat at 100 for 10 minutes. Allow to cool and examine under ultraviolet light (254 nm). The
principal spot in the chromatogram obtained with solution (1) corresponds to that in the chromatogram obtained with solution (2). Spray the plate with ethanolic sulphuric acid (20%), heat at 120 for
10 minutes and allow to cool. The principal spot in the chromatogram obtained with solution (1) is
green and corresponds in position and size to the spot in the chromatogram obtained with solution
(2). The test is not valid unless the chromatogram obtained with solution (3) shows three distinct
principal spots by each method of visualisation.
Free caproic acid Dissolve 0.44 g in 10 ml of ethanol (96%), previously neutralised to bromothymol
blue solution R3, and titrate immediately with 0.01M sodium hydroxide VS, using bromothymol blue
solution R3 as indicator. Not more than 0.6 ml of 0.01M sodium hydroxide VS is required.
gel G as the coating substance and a mixture of 80 volumes of toluene and 20 volumes of ethyl acetate
as the mobile phase. Apply separately to the plate 5 l of each of two solutions in a mixture of 9
volumes of chloroform and 1 volume of methanol containing (1) 2.0% w/v of the substance being
examined and (2) 0.020% w/v of testosterone. After removal of the plate, allow it to dry in air until the
odour of solvent is no longer detectable, heat at 110 for 10 minutes and spray the hot plate with
ethanolic sulphuric acid (20%) and heat at 110 for a further 10 minutes. Any secondary spot in the
Loss on drying When dried to constant weight over phosphorus pentoxide at a pressure not exceeding
0.7 kPa, loses not more than 0.5% of its weight. Use 1 g.
maximum at 241 nm.
Storage Testosterone Isocaproate should be kept in a well-closed container, protected from light and
39-34
Testosterone Propionate
Me
H
OCOEt
Me
H
H
O
C22H32O3
344.5
57-85-2
Testosterone Propionate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Anabolic steroid; androgen.
Preparation
Testosterone Propionate Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Testosterone propionate contains not less than 97.0 per cent and not more than the equivalent of
103.0 per cent of 3-oxoandrost-4-en-17-yl propionate, calculated with reference to the dried
substance.
CHARACTERS
A white or almost white powder or colourless crystals, practically insoluble in water, freely soluble in
acetone, in alcohol and in methanol, soluble in fatty oils.
IDENTIFICATION
obtained with testosterone propionate CRS.
254 nm. The principal spot in each of the chromatograms obtained with test solution (b) and test
solution (c) is similar in position and size to the principal spot in the chromatogram obtained with the
corresponding reference solution. Spray the plate with alcoholic solution of sulphuric acid R. Heat at
120C for 15 min and allow to cool. Examine in daylight and in ultraviolet light at 365 nm. The
principal spot in each of the chromatograms obtained with test solution (b) and test solution (c) is
similar in position, colour in daylight, fluorescence in ultraviolet light at 365 nm and size to the
principal spot in the chromatogram obtained with the corresponding reference solution. The principal spots in the chromatograms obtained with test solution (b) and reference solution (b) have Rf
values distinctly smaller than those of the principal spots in the chromatograms obtained with test
solution (c) and reference solution (c).
TESTS
substance.
Related substances Examine by thin-layer chromatography (2.2.27), using as coating substance a
suitable silica gel with a fluorescent indicator having an optimal intensity at 254 nm.
Test solution (b). In a 25 ml volumetric flask, dissolve 50 mg of the substance to be examined in 6 ml
of methanol R and add 2 ml of 1M sodium hydroxide. Place a small funnel in the neck of the flask to act
as a condenser and heat on a water-bath for 5 min. Cool under running water, add 2.0 ml of 1M
hydrochloric acid and dilute to 25.0 ml with methanol R.
Test solution (c). Dilute 1.0 ml of test solution (a) to 25.0 ml with chloroform R.
Reference solution (a). Dilute 1.0 ml of test solution (a) to 10.0 ml with chloroform R. Dilute 1.0 ml of
this solution to 10.0 ml with chloroform R.
39-35
Reference solution (b). In a 25 ml volumetric flask, dissolve 50 mg of testosterone propionate CRS in 6 ml
of methanol R and add 2 ml of 1M sodium hydroxide. Place a small funnel in the neck of the flask to act
as a condenser and heat on a water-bath for 5 min. Cool under running water, add 2.0 ml of 1M
hydrochloric acid and dilute to 25.0 ml with methanol R.
Reference solution (c). Dissolve 20 mg of testosterone propionate CRS in chloroform R and dilute to
Reference solution (d). Dissolve 10 mg of testosterone acetate CRS in chloroform R and dilute to 10.0 ml
Reference solution (e). To 5.0 ml of reference solution (d) add 1.0 ml of test solution (a) and dilute to
15.0 ml with chloroform R.
Apply separately to the plate 5 l of test solutions (b) and (c) and reference solutions (b) and (c) and
2 l of test solution (a) and reference solutions (a), (d) and (e). Develop over a path of 15 cm using a
mixture of 1 volume of anhydrous acetic acid R, 30 volumes of light petroleum R and 70 volumes of
butyl acetate R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Any spot
corresponding to testosterone acetate in the chromatogram obtained with test solution (a) is not
more intense than the spot in the chromatogram obtained with reference solution (d) (2.0 per cent).
Any spot in the chromatogram obtained with test solution (a) apart from the principal spot and the
spot corresponding to testosterone acetate is not more intense than the spot in the chromatogram
obtained with reference solution (a) (1.0 per cent). The test is not valid unless the chromatogram
obtained with reference solution (e) shows two clearly separated spots.
100C to 105C for 2 h.
ASSAY
Dissolve 25.0 mg in ethanol R and dilute to 250.0 ml with the same solvent. Dilute 10.0 ml of the
STORAGE
__________________________________________________________________________________________________________ Ph Eur
39-36
Tetracaine Hydrochloride / Amethocaine Hydrochloride

1/01
O
O
NMe2
,HCl
Bu n HN
C15H25ClN2O2
300.8
136-47-0
Tetracaine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Tetracaine Eye Drops/Amethocaine Eye Drops
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Tetracaine hydrochloride contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of 2-dimethylaminoethyl 4-butylaminobenzoate hydrochloride, calculated with
CHARACTERS
A white, crystalline powder, slightly hygroscopic, freely soluble in water, soluble in alcohol.
It melts at about 148C or it may occur in either of two other crystalline forms which melt
respectively at about 134C and 139C. Mixtures of these forms melt within the range 134C to
147C.
IDENTIFICATION
obtained with tetracaine hydrochloride CRS.
B. To 10 ml of solution S (see Tests) add 1 ml of ammonium thiocyanate solution R. A white, crystalline precipitate is formed which, after recrystallisation from water R and drying at 80C for 2 h, melts
(2.2.14) at about 131C.
C. To about 5 mg add 0.5 ml of fuming nitric acid R. Evaporate to dryness on a water-bath, allow to
cool and dissolve the residue in 5 ml of acetone R. Add 1 ml of 0.1M alcoholic potassium hydroxide. A
violet colour develops.
D. Solution S gives reaction (a) of chlorides (2.3.1).
TESTS
plate R. Carry out a preliminary development over a path of 12 cm using a mixture of 4 volumes of
glacial acetic acid R, 16 volumes of hexane R and 80 volumes of dibutyl ether R. Remove the plate and
dry it in a current of warm air for a few minutes. Allow the plate to cool before use.
same solvent.
Reference solution. Dissolve 50 mg of 4-aminobenzoic acid R in water R and dilute to 100 ml with the
39-37
of glacial acetic acid R, 16 volumes of hexane R and 80 volumes of dibutyl ether R. Dry the plate at
100C to 105C for 10 min and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with the test solution, apart from the principal spot, is not more intense than the spot
in the chromatogram obtained with the reference solution (0.05 per cent). The principal spot in the
chromatogram obtained with the test solution remains at the starting point.
100C to 105C.
ASSAY
Dissolve 0.250 g in 50 ml of alcohol R and add 5.0 ml of 0.01M hydrochloric acid. Carry out a potentiometric titration (2.2.20), using 0.1M sodium hydroxide. Read the volume added between the 2
STORAGE
__________________________________________________________________________________________________________ Ph Eur
39-38
Tetracosactide
1/01
SerTyrSerMetGluHisPheArgTrpGlyLysProValGlyLysLys
ArgArgProValLysValTyrPro
C136H210N40O31S
2933
16960-16-0
Tetracosactide complies with the requirements of the 3rd edition of the European Pharmacopoeia [0644].
Action and use Corticotrophic peptide.
Preparations
Tetracosactide Injection
Tetracosactide Zinc Injection
When tetracosactrin is prescribed or demanded, tetracosactide shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Tetracosactide is a synthetic tetracosapeptide in which the sequence of amino acids is the same as
that of the first twenty-four residues of human corticotropin. It is available as an acetate and contains
water. It increases the rate at which corticoid hormones are secreted by the adrenal glands. The
potency is not less than 800 International Units per milligram, calculated with reference to the
anhydrous, acetic acid-free substance.
CHARACTERS
A white or yellow, amorphous powder, sparingly soluble in water.
IDENTIFICATION
A. It increases the amount of corticosterone produced by isolated rat adrenal cells in the conditions of
the assay.
B. Examine by electrophoresis (2.2.31) and thin-layer chromatography (2.2.27) to obtain a twodimensional separation using two plates with cellulose for chromatography R1 as the coating substance.
Test solution. Dissolve 1 mg of the substance to be examined in 0.2 ml of a 15.4 g/l solution of
ammonium acetate R adjusted to pH 8.2 with dilute ammonia R2. Add 10 l of a 2 g/l solution of
trypsin R, maintain the mixture at 37C to 38C for 40 min, heat on a water-bath for 3 min and add
5 l of glacial acetic acid R. Evaporate to dryness at 40C at a pressure not exceeding 3 kPa, dry the
glassy residue at 40C for 1 h and dissolve in 0.1 ml of glacial acetic acid R. Dry the solution from the
frozen state, dissolve the residue in 0.1 ml of water R and dry again from the frozen state. Dry the
final residue at 45C for 1 h at a pressure not exceeding 3 kPa and dissolve in 50 l of water R.
Reference solution. Prepare at the same time and in the same manner as the test solution, using
tetracosactide CRS instead of the substance to be examined.
Spray the plates with the electrolyte solution which consists of a solution containing 0.2 per cent V/V
of glacial acetic acid R and 0.2 per cent V/V of pyridine R. Place the filter paper tongues to connect the
plates with the appropriate compartment of each trough so that each tongue covers an area 1.5 cm
wide at one end of the plate. Close the tank and allow to stand for 30 min. Apply the solutions on the
anodic side. Apply to the first plate, at a point about 2.5 cm from each of two adjacent edges, 4 l of
the test solution. Apply to the second plate, at a similar position, 4 l of the reference solution. Apply
to both plates a potential of 280 V for a plate 200 mm long and allow electrophoresis to proceed for
90 min. Allow the plates to dry in air for 30 min and then dry in a current of air at 30C for 30 min.
Carry out a second separation on each plate by thin-layer chromatography. Develop at right angles to
the direction of electrophoresis over a path of 15 cm using a mixture of 8 volumes of glacial acetic
acid R, 24 volumes of pyridine R, 30 volumes of water R and 38 volumes of butanol R. Dry the plates
in a current of air and spray with ninhydrin solution R1. The principal spots in the chromatogram
obtained with the test solution are similar in position to those in the chromatogram obtained with the
reference solution, but their intensity may differ.
TESTS
Specific optical rotation (2.2.7). Dissolve 10.0 mg in 1.0 ml of a mixture of 1 volume of glacial
acetic acid R and 99 volumes of water R. The specific optical rotation is 99 to 109, calculated with
reference to the anhydrous, acetic acid-free substance.
Absorbance (2.2.25). Dissolve 1.0 mg in 0.1M hydrochloric acid and dilute to 5.0 ml with the same
acid. Examined between 240 nm and 280 nm, the solution shows an absorption maximum at
39-39
276 nm. The absorbance at the maximum is 0.51 to 0.61, calculated with reference to the anhydrous, acetic acid-free substance. The ratio of the absorbance at the maximum at 276 nm to the
absorbance at 248 nm is 2.4 to 2.9.
acids:
Lysine
Threonine
Alanine
Leucine
Histidine
Serine
Valine
Tyrosine
Arginine
Glutamic acid Methionine
Phenylalanine
Aspartic acid
Proline
Isoleucine
together with half the equimolar amount of L-cystine. For the validation of the method, an appropriate internal standard, such as DL-norleucine R, is used.
Test solution. Place 1.0 mg of the substance to be examined in a rigorously cleaned hard-glass tube,
100 mm long and 6 mm in internal diameter. Add a suitable amount of a 50 per cent V/V solution of
hydrochloric acid R. Immerse the tube in a freezing mixture at 5C, reduce the pressure to below
133 Pa and seal. Heat at 110C to 115C for 16 h. Cool, open the tube, transfer the contents to a
10 ml flask with the aid of five quantities, each of 0.2 ml, of water R and evaporate to dryness over
potassium hydroxide R under reduced pressure. Take up the residue in water R and evaporate to
dryness over potassium hydroxide R under reduced pressure; repeat these operations once. Take up the
residue in a buffer solution suitable for the amino-acid analyser used and dilute to a suitable volume
with the same buffer solution. Apply a suitable volume to the amino-acid analyser.
acids, taking that for valine to be equivalent to three. The values fall within the following limits: lysine
3.5 to 4.7; histidine 0.9 to 1.1; arginine 2.7 to 3.3; serine 1.1 to 2.2; glutamic acid 0.9 to 1.1; proline
2.5 to 3.5; glycine 1.8 to 2.2; methionine 0.9 to 1.1; tyrosine 1.7 to 2.2; phenylalanine 0.9 to 1.1.
Not more than traces of other amino acids are present, with the exception of tryptophan.
Related peptides
A. Examine by liquid chromatography (2.2.29). Use degassed solvents.
Test solution. Dissolve 1.0 mg of the substance to be examined in 1 ml of water R.
Reference solution (a). Dissolve 1.0 mg of the substance to be examined in 1 ml of a 1 per cent V/V
solution of glacial acetic acid R and add 50 l of a mixture of 1 volume of strong hydrogen peroxide
solution R and 999 volumes of water R. Allow to stand for 2 h.
Reference solution (b). Dissolve 1.0 mg of tetracosactide CRS in 1 ml of water R.
as mobile phase at a flow rate of 2.0 ml/min, a mixture of 365 ml of acetonitrile R, 10.0 ml of
glacial acetic acid R and 10.0 g of ammonium sulphate R, diluted to 2000 ml with water R,
Inject 20 l of each solution ensuring that the syringe used to inject the test solution is not
contaminated with peroxide. The chromatogram obtained with reference solution (a) shows a peak
due to tetracosactide corresponding to the principal peak in the chromatogram obtained with the test
solution and a peak with a lower retention time, due to tetracosactide sulphoxide, of significantly
greater area than any corresponding peak in the chromatogram obtained with the test solution. The
test is not valid unless in the chromatogram obtained with reference solution (a), the resolution
between the peaks corresponding to tetracosactide and tetracosactide sulphoxide is at least 7. In the
chromatogram obtained with the test solution, the area of the peak corresponding to tetracosactide
sulphoxide is not greater than 4 per cent of the sum of the areas of all the peaks, disregarding any
peaks due to the solvent and the mobile phase.
B. Examine by thin-layer chromatography (2.2.27), using cellulose for chromatography R as the coating
substance.
Test solution. Dissolve 3.0 mg of the substance to be examined in 1.5 ml of a mixture of equal
volumes of dilute acetic acid R and water R.
Reference solution (a). Dilute 0.5 ml of the test solution to 10 ml with a mixture of equal volumes of
dilute acetic acid R and water R.
Reference solution (b). Dilute 5 ml of reference solution (a) to 10 ml with a mixture of equal volumes
of dilute acetic acid R and water R.
Reference solution (c). To 0.5 ml of the test solution add 50 l of a mixture of 1 volume of strong
hydrogen peroxide solution R and 999 volumes of water R. Allow to stand for 2 h.
Apply to the plate as 1 cm bands 10 l of each solution. Develop over a path of 15 cm using a
39-40
mixture of 4 volumes of glacial acetic acid R, 24 volumes of pyridine R, 30 volumes of water R and 42
volumes of butanol R. Dry the plate in a current of air and spray with ninhydrin solution R1. In the
chromatogram obtained with reference solution (c), the band corresponding in position to the
principal band in the chromatogram obtained with the test solution is reduced in intensity, and a
prominent band, due to tetracosactide sulphoxide, of lower Rf is present. In the chromatogram
obtained with the test solution, any band, apart from the principal band and any band due to
tetracosactide sulphoxide, is not more intense than the band in the chromatogram obtained with
reference solution (a) (5.0 per cent) and at most one such band is more intense than the band in the
Peptide Not less than 85.0 per cent of C136H210N40O31S, calculated with reference to the anhydrous, acetic acid-free substance.
Examine the chromatograms obtained in test A for related peptides. Calculate the content of
C136H210N40O31S from the peak heights or areas in the chromatograms obtained with the test
solution and reference solution (b), and the declared content of C136H210N40O31S in tetracosactide
CRS.
Acetic acid (2.5.34). 8.0 per cent to 13.0 per cent.
ASSAY
The potency of tetracosactide is estimated by comparing in given conditions its activity in increasing
the amount of corticosterone produced by isolated rat adrenal cells with that of the International
Reference Preparation of tetracosactide or a reference preparation calibrated in International Units.
The International Unit is the activity contained in a stated amount of the International Reference
Preparation which consists of a quantity of synthetic tetracosactide with mannitol. The equivalence in
International Units of the International Reference Preparation is stated by the World Health
Organisation.
Use siliconised glassware and rinse well with water R before use. Kill four male rats, each weighing
between 200 g and 400 g, by exsanguination. Remove the adrenal glands, carefully free them of
adhering fat and immerse in solution B maintained at 4C. Cut each gland into four equal pieces and
transfer to a suitable plastic stirring apparatus (see Fig. 06441) containing 5 ml of solution C
maintained at 37C. Disperse the adrenal cells by stirring the mixture at 500 r/min. After 20 min,
remove the supernatant liquid, cool to 4C, add 5 ml of solution C and repeat the dispersal
procedure. Repeat the operation a further three times. Combine the five supernatant liquids, centrifuge at 4C in a polyethylene test-tube for 30 min after slow acceleration to 100 g. Suspend the
resulting pellet in 8 ml of solution D and centrifuge for 30 min at 100 g. Again suspend the residue in
8 ml of solution D and filter the mixture through nylon gauze with 100 m pores into a polyethylene
beaker and add a suitable volume of solution D to the filtrate (a total volume of 65 ml to 105 ml has
been found suitable). Maintain the resulting suspension at 4C.
39-41
Fig. 06441 Stirring Apparatus

Dimensions in millimetres
A pulley and stirring paddle,
B bearing,
C incubation medium.
Prepare four independent dilutions, with two-fold dose intervals, from solutions of suitable concentrations of the substance to be examined and of the reference preparation using solution E as diluent.
Pipette 0.1 ml of each dilution into each of four polystyrene test-tubes and add 1.0 ml of the cell
suspension prepared above to each tube. Incubate at 37C for 2 h and then cool to 4C. Transfer
1 ml of the contents of each tube to a glass tube containing 1.4 ml of methylene chloride R, mix in a
vortex mixer for 10 s and centrifuge at 3000 g for 5 min. Transfer 1 ml of each of the methylene
chloride layers, avoiding taking up aqueous phase, to a glass tube containing 0.6 ml of a mixture of
15 volumes of alcohol R and 35 volumes of sulphuric acid R. Mix in a vortex mixer for 10 s and
centrifuge at 1500 g for 5 min. Allow each tube to stand for 30 min. Examine by fluorimetry (2.2.21),
irradiating the lower layer with an excitant beam of suitable wavelength such as 436 nm or 470 nm
and measuring the fluorescence at the maximum between 530 nm and 545 nm. If the solutions are
not transferred to spectrophotometric measurement cells, select glass tubes that give fluorescence
values for a standard corticosterone which do not differ from each other by more than 5 per cent.
Calculate the result of the assay by the usual statistical methods using the linear portion of the log
dose-response curve.
Solution A
Sodium chloride R
6.60 g
Potassium chloride R
0.353 g
Sodium hydrogen carbonate R
0.840 g
Potassium dihydrogen phosphate R
0.161 g
Magnesium sulphate R
0.291 g
Calcium chloride R
0.373 g
2-[4-(2-Hydroxyethyl)piperazin-1-yl]ethanesulphonic acid R
4.77 g
Dissolve the above ingredients in about 950 ml of water R, adjust to pH 7.4 with 1M sodium hydroxide
and add 60 mg of benzylpenicillin sodium R and a quantity of streptomycin sulphate R equivalent to
39-42
100 mg of streptomycin. Dilute to 1000 ml with water R.
Solution B
Add 2 g/l of glucose R to solution A.
Solution C
To solution B add 1 g/l of a preparation of collagenase obtained from Clostridium histolyticum and of a
grade suitable for the preparation of dispersed cells.
Solution D
Add 5 g/l of bovine albumin R to solution B.
Solution E
A 9 g/l sterile solution of sodium chloride R containing 1 g/l of bovine albumin R and adjusted to pH 2.0
STORAGE
Store under nitrogen, protected from light, at a temperature between 2C and 8C.
LABELLING
The label states:
the potency in International Units per milligram,
the peptide content per container,
the storage conditions.
__________________________________________________________________________________________________________ Ph Eur
39-43
Tetracycline
OH
OH
O
OH
CONH2
OH
Me
OH
C22H24N2O8
444.4
NMe2
60-54-8 (anhydrous)
Tetracycline complies with the requirements of the 3rd edition of the European Pharmacopoeia [0211]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Tetracycline contains not less than 88.0 per cent and not more than the equivalent of 100.5 per cent
of (4S,4aS, 5aS,6S,12aS)-4-dimethylamino-1,4,4a,5,5a,6,11,12a-octahydro-3,6,10,12,12apentahydroxy-6-methyl-1,11-dioxonaphthacene-2-carboxamide, calculated with reference to the
dried substance.
CHARACTERS
A yellow, crystalline powder, very slightly soluble in water, soluble in alcohol and in methanol, sparingly soluble in acetone, practically insoluble in ether. It dissolves in dilute acid and alkaline solutions.
IDENTIFICATION
1 h.
the same solvent.
Reference solution (a). Dissolve 5 mg of tetracycline hydrochloride CRS in methanol R and dilute to
Reference solution (b). Dissolve 5 mg of tetracycline hydrochloride CRS, 5 mg of chlortetracycline hydrochloride CRS and 5 mg of doxycycline hyclate CRS in methanol R and dilute to 10 ml with the same
solvent.
obtained with reference solution (b) shows three clearly separated spots.
B. To about 2 mg add 5 ml of sulphuric acid R. A violet-red colour develops. Add the solution to
C. Dissolve about 10 mg in a mixture of 1 ml of dilute nitric acid R and 5 ml of water R. Shake and
add 1 ml of silver nitrate solution R2. Any opalescence in the solution is not more intense than that in a
mixture of 1 ml of dilute nitric acid R, 5 ml of water R and 1 ml of silver nitrate solution R2.
TESTS
pH (2.2.3). Suspend 0.1 g in 10 ml of carbon dioxide-free water R. The pH of the suspension is 3.5 to
6.0.
dried substance.
Related substances Examine by liquid chromatography (2.2.29), as prescribed under Assay. Inject
39-44
reference solution (g). The test is not valid unless the peak corresponding to anhydrotetracycline has
a signal- to-noise ratio of at least three. Inject the test solution and reference solution (f). In the
chromatogram obtained with the test solution: the area of any peak, corresponding to 4-epitetracycline or anhydrotetracycline or 4-epianhydrotetracycline is not greater than the area of the
corresponding peak in the chromatogram obtained with reference solution (f) (5.0 per cent, 0.5 per
cent and 1.0 per cent respectively); the area of any peak appearing on the tail of the principal peak is
not greater than 0.4 times the area of the peak corresponding to 4-epitetracycline in the chromatogram obtained with reference solution (f) (2.0 per cent).
at 100C to 105C.
ASSAY
Reference solution (a). Dissolve 25.0 mg of tetracycline hydrochloride CRS in 0.01M hydrochloric acid and
Reference solution (b). Dissolve 12.5 mg of 4-epitetracycline hydrochloride CRS in 0.01M hydrochloric acid
Reference solution (c). Dissolve 10.0 mg of anhydrotetracycline hydrochloride CRS in 0.01M hydrochloric
acid and dilute to 100.0 ml with the same acid.
Reference solution (d). Dissolve 10.0 mg of 4-epianhydrotetracycline hydrochloride CRS in 0.01M hydrochloric acid and dilute to 50.0 ml with the same acid.
Reference solution (e). Mix 1.0 ml of reference solution (a), 2.0 ml of reference solution (b) and 5.0 ml
of reference solution (d) and dilute to 25.0 ml with 0.01M hydrochloric acid.
Reference solution (f). Mix 40.0 ml of reference solution (b), 20.0 ml of reference solution (c) and
5.0 ml of reference solution (d) and dilute to 200.0 ml with 0.01M hydrochloric acid.
Reference solution (g). Dilute 1.0 ml of reference solution (c) to 50.0 ml with 0.01M hydrochloric acid.
a column 0.25 m long and 4.6 mm in internal diameter packed with styrene-divinylbenzene copolymer R (8 m to 10 m),
as mobile phase at a flow rate of 1.0 ml per minute a solution prepared as follows: weigh 80.0 g
water R. Add 100 ml of a 35 g/l solution of dipotassium hydrogen phosphate R adjusted to pH 9.0
with dilute phosphoric acid solution R, 200 ml of a 10 g/l solution of tetrabutylammonium hydrogen
sulphate R adjusted to pH 9.0 with dilute sodium hydroxide solution R and 10 ml of a 40 g/l solution of sodium edetate R adjusted to pH 9.0 with dilute sodium hydroxide solution R; dilute to
1000.0 ml with water R,
maintaining the column at 60C. Inject reference solution (e). Adjust the sensitivity of the detector
so that the height of the peaks is not less than 50 per cent of full scale of the recorder. The test is not
valid unless the resolution between the first peak (4-epitetracycline) and the second peak
(tetracycline) is at least 2.5, and the resolution between the second peak and the third peak (4-epianhydrotetracycline) is at least 8.0. Adjust the concentration of 2-methyl-2-propanol in the mobile
phase if necessary. The test is not valid unless the symmetry factor for the second peak is at most
1.25. Inject reference solution (a) six times. The test is not valid unless the relative standard deviation
of the peak area for tetracycline is at most 1.0 per cent. If necessary, adjust the integrator parameters.
Inject alternately the test solution and reference solution (a).
Calculate the percentage content of tetracycline.
STORAGE
39-45
IMPURITIES
OH
OH O HO
O
COR3
H
Me OH
H
R1
OH
R2
A. R1 = N(CH3)2, R2 = H, R3 = NH2: 4-epitetracycline,

B. R1 = H, R2 = N(CH3)2, R3 = CH3: 2-acetyl-2-dicarboxamidotetracycline,
OH O HO
OH
O
CONH2
H
Me
R1
OH
R2
C. R1 = H, R2 = N(CH3)2: anhydrotetracycline,
D. R1 = N(CH3)2, R2 = H: 4-epianhydrotetracycline.
__________________________________________________________________________________________________________ Ph Eur
39-46
Tetracycline Hydrochloride
OH
OH
O
OH
CONH2
,HCl
OH
Me
H
OH
C22H24N2O8,HCl
NMe2
480.9
64-75-5
Tetracycline Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparations
Tetracycline Capsules
Tetracycline Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Tetracycline hydrochloride contains not less than 95.0 per cent and not more than the equivalent of
100.5 per cent of (4S,4aS,5aS,6S,12aS)-4-dimethylamino-1,4,4a,5,5a,6,11, 12a-octahydro3,6,10,12,12a-pentahydroxy-6-methyl-1,11-dioxonaphthacene-2-carboxamide hydrochloride, calculated with reference to the dried substance.
CHARACTERS
A yellow, crystalline powder, soluble in water, slightly soluble in alcohol, practically insoluble in
acetone and in ether. It dissolves in solutions of alkali hydroxides and carbonates. Solutions in water
become turbid on standing, owing to the precipitation of tetracycline.
IDENTIFICATION
1 h.
the same solvent.
Reference solution (a). Dissolve 5 mg of tetracycline hydrochloride CRS in methanol R and dilute to
Reference solution (b). Dissolve 5 mg of tetracycline hydrochloride CRS, 5 mg of chlortetracycline hydrochloride CRS and 5 mg of doxycycline hyclate CRS in methanol R and dilute to 10 ml with the same
solvent.
B. To about 2 mg add 5 ml of sulphuric acid R. A violet-red colour develops. Add the solution to
TESTS
2.8.
dried substance.
39-47
Related substances Examine by liquid chromatography (2.2.29), as prescribed under Assay. Inject
reference solution (g). The test is not valid unless the peak corresponding to anhydrotetracycline has
a signal- to-noise ratio of at least three. Inject the test solution and reference solution (f). In the
chromatogram obtained with the test solution: the area of any peak corresponding to 4-epitetracycline, anhydrotetracycline or 4-epianhydrotetracycline is not greater than the area of the corresponding peak in the chromatogram obtained with reference solution (f) (3.0 per cent, 0.5 per cent
and 0.5 per cent respectively); the area of any peak appearing on the tail of the principal peak is not
greater than 50.0 per cent of the area of the peak corresponding to 4-epitetracycline in the chromatogram obtained with reference solution (f) (1.5 per cent).
ASSAY
Reference solution (a). Dissolve 25.0 mg of tetracycline hydrochloride CRS in 0.01M hydrochloric acid and
Reference solution (b). Dissolve 15.0 mg of 4-epitetracycline hydrochloride CRS in 0.01M hydrochloric acid
Reference solution (c). Dissolve 10.0 mg of anhydrotetracycline hydrochloride CRS in 0.01M hydrochloric
acid and dilute to 100.0 ml with the same acid.
Reference solution (d). Dissolve 10.0 mg of 4-epianhydrotetracycline hydrochloride CRS in 0.01M hydrochloric acid and dilute to 50.0 ml with the same acid.
Reference solution (e). Mix 1.0 ml of reference solution (a), 2.0 ml of reference solution (b) and 5.0 ml
of reference solution (d) and dilute to 25.0 ml with 0.01M hydrochloric acid.
Reference solution (f). Mix 20.0 ml of reference solution (b), 10.0 ml of reference solution (c) and
5.0 ml of reference solution (d) and dilute to 200.0 ml using 0.01M hydrochloric acid.
Reference solution (g). Dilute 1.0 ml of reference solution (c) to 50.0 ml with 0.01M hydrochloric acid.
a column 0.25 m long and 4.6 mm in internal diameter packed with styrene-divinylbenzene copolymer R (8 m to 10 m),
as mobile phase at a flow rate of 1.0 ml per minute a solution prepared as follows: weigh 80.0 g
water R; add 100 ml of a 35 g/l solution of dipotassium hydrogen phosphate R adjusted to pH 9.0
with dilute phosphoric acid R, 200 ml of a 10 g/l solution of tetrabutylammonium hydrogen
sulphate R adjusted to pH 9.0 with dilute sodium hydroxide solution R and 10 ml of a 40 g/l solution of sodium edetate R adjusted to pH 9.0 with dilute sodium hydroxide solution R; dilute to
1000.0 ml with water R,
as detector, a spectrophotometer set at 254 nm,
maintaining the column at 60C. Inject reference solution (e). Adjust the sensitivity of the detector
so that the height of the peaks is not less than 50 per cent of the full scale of the recorder. The test is
not valid unless the resolution between the first peak (4-epitetracycline) and the second peak
(tetracycline) is at least 2.5, and the resolution between the second peak and the third peak (4-epianhydrotetracycline) is at least 8.0. Adjust the concentration of 2-methyl-2-propanol in the mobile
phase if necessary. The test is not valid unless the symmetry factor for the second peak is at most
1.25. Inject reference solution (a) six times. The test is not valid unless the relative standard deviation
of the peak area for tetracycline is at most 1.0 per cent. If necessary, adjust the integrator parameters.
Inject alternately the test solution and reference solution (a).
Calculate the percentage content of tetracycline hydrochloride.
STORAGE
39-48
LABELLING
The label states:
IMPURITIES
OH
OH O HO
O
COR3
H
Me OH
H
R1
OH
R2
A. R1 = N(CH3)2, R2 = H, R3 = NH2: 4-epitetracycline,

B. R1 = H, R2 = N(CH3)2, R3 = CH3: 2-acetyl-2-dicarboxamidotetracycline,
OH O HO
OH
O
CONH2
H
Me
R1
OH
R2
C. R1 = H, R2 = N(CH3)2: anhydrotetracycline,
D. R1 = N(CH3)2, R2 = H: 4-epianhydrotetracycline.
__________________________________________________________________________________________________________ Ph Eur
39-49
Theobroma Oil
Cocoa Butter
Definition Theobroma Oil is the solid fat obtained from the roasted seeds of Theobroma cacao L.
Characteristics A yellowish white, solid fat; odour, slight, agreeable and resembling that of cocoa.
Somewhat brittle.
Freely soluble in chloroform, in ether and in petroleum spirit (boiling range, 40 to 60); slightly soluble
in ethanol (96%).
Iodine value 35 to 40 (iodine monochloride method), Appendix X E.
Melting point 31 to 34, Appendix V A, Method IV. Prepare the substance being examined in the
following manner. Melt about 30 g in an oven at a temperature of 55 to 60 and filter through a
suitable dry filter paper, maintaining the temperature between 53 and 60. Cool with occasional
stirring until the temperature falls to between 32 and 34, stir continuously with a mechanical stirrer
until the first signs of cloudiness appear and continue to stir by hand until the substance has the
consistence of a paste. Immediately transfer to a vessel previously kept at a temperature of 15 to 22
and allow to stand at this temperature for 24 hours before carrying out the test.
Refractive index At 40, 1.456 to 1.458, Appendix V E.
Saponification value 188 to 196, Appendix X G.
Storage Theobroma Oil should be stored at a temperature not exceeding 25.
Action and use Suppository basis.
39-50
Theobromine
O
HN
O
Me
Me
C7H8N4O2
180.2
83-67-0
Theobromine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0298]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Theobromine contains not less than 99.0 per cent and not more than the equivalent of 101.0 per
cent of 3,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione, calculated with reference to the dried
substance.
CHARACTERS
A white powder, very slightly soluble in water and in ethanol, slightly soluble in ammonia, practically
insoluble in ether. It dissolves in dilute solutions of alkali hydroxides and in mineral acids.
IDENTIFICATION
obtained with theobromine CRS.
B. Dissolve about 20 mg in 2 ml of dilute ammonia R1, warming slightly, and cool. Add 2 ml of silver
nitrate solution R2. The solution remains clear. Boil the solution for a few minutes. A white, crystalline
C. It gives the reaction of xanthines (2.3.1).
TESTS
Acidity To 0.4 g add 20 ml of boiling water R and boil for 1 min. Allow to cool and filter. Add
0.05 ml of bromothymol blue solution R1. The solution is yellow or yellowish-green. Not more than
0.2 ml of 0.01M sodium hydroxide is required to change the colour of the indicator to blue.
coating substance.
Test solution. To 0.2 g of the finely powdered substance to be examined add 10 ml of a mixture of 4
volumes of methanol R and 6 volumes of chloroform R. Heat under a reflux condenser on a water-bath
for 15 min, shaking occasionally. Cool and filter.
Reference solution. Dissolve 5 mg of theobromine CRS in a mixture of 4 volumes of methanol R and 6
volumes of chloroform R and dilute to 50 ml with the same mixture of solvents.
10 volumes of concentrated ammonia R, 30 volumes of acetone R, 30 volumes of chloroform R and 40
volumes of butanol R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Any
100C to 105C.
ASSAY
Dissolve 0.150 g in 125 ml of boiling water R, cool to 50C to 60C and add 25 ml of 0.1M silver
nitrate. Using 1 ml of phenolphthalein solution R as indicator, titrate with 0.1M sodium hydroxide until a
pink colour is obtained.
__________________________________________________________________________________________________________ Ph Eur
39-51
Theophylline
O
H
N
MeN
O
Me
C7H8N4O2
180.2
58-55-9
Theophylline complies with the requirements of the 3rd edition of the European Pharmacopoeia [0299]. These
Preparation
Aminophylline Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Theophylline contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of 1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder, slightly soluble in water, sparingly soluble in ethanol. It dissolves in
solutions of alkali hydroxides, in ammonia and in mineral acids.
IDENTIFICATION
A. Melting point (2.2.14): 270C to 274C, determined after drying at 100C to 105C.
obtained with theophylline CRS.
C. Heat 10 mg with 1.0 ml of a 360 g/l solution of potassium hydroxide R in a water-bath at 90C for
3 min, then add 1.0 ml of diazotised sulphanilic acid solution R. A red colour slowly develops. Carry
out a blank test.
D. It complies with the test for loss on drying (see Tests).
E. It gives the reaction of xanthines (2.3.1).
TESTS
Solution S Dissolve 0.5 g with heating in carbon dioxide-free water R, cool and dilute to 75 ml with
the same solvent.
Acidity To 50 ml of solution S add 0.1 ml of methyl red solution R. The solution is red. Not more
Test solution. Dissolve 0.2 g of the substance to be examined in a mixture of 4 volumes of methanol R
Reference solution. Dilute 0.5 ml of the test solution to 100 ml with a mixture of 4 volumes of
10 volumes of concentrated ammonia R, 30 volumes of acetone R, 30 volumes of chloroform R and 40
volumes of butanol R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Any
100C to 105C.
39-52
ASSAY
Dissolve 0.150 g in 100 ml of water R, add 20 ml of 0.1M silver nitrate and shake. Add 1 ml of
bromothymol blue solution R1. Titrate with 0.1M sodium hydroxide.
__________________________________________________________________________________________________________ Ph Eur
39-53
Theophylline Hydrate
C7H8N4O2,H2O
198.2
5967-84-0
Theophylline Hydrate complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Theophylline Monohydrate [0302]. These requirements are reproduced after the heading Definition below.
Preparation
Aminophylline Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Theophylline monohydrate contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of 1,3-dimethyl-3,7dihydro-1H-purine-2,6-dione, calculated with reference to the
CHARACTERS
It has the characters described in the monograph on Theophylline (299).
IDENTIFICATION
It complies with the identification tests prescribed in the monograph on Theophylline (299) except for
identification test D, which is replaced by that shown below; dry the substance to be examined at
100C to 105C before carrying out identification test B.
D. It complies with the test for the determination of water (see Tests).
TESTS
It complies with the tests prescribed in the monograph on Theophylline (299) with the exception of
the test for loss on drying which is replaced by the following test.
Water (2.5.12). 8.0 per cent to 9.5 per cent, determined on 1.00 g by the semi-micro determination
of water.
ASSAY
Carry out the assay prescribed in the monograph on Theophylline (299) using 0.160 g.
__________________________________________________________________________________________________________ Ph Eur
39-54
Thiamine Hydrochloride
1/01
Me
NH2
CH2CH2OH
+
N
Cl ,HCl
S
Me
C12H17ClN4OS,HCl
337.3
67-03-8
Thiamine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Component of Vitamin B.
Preparations
Thiamine Injection
Thiamine Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Thiamine hydrochloride contains not less than 98.5 per cent and not more than the equivalent of
101.5 per cent of 3-[(4-amino-2-methylpyrimidin-5-yl)methyl]-5-(2-hydroxyethyl)-4methylthiazolium chloride hydrochloride, calculated with reference to the anhydrous substance.
CHARACTERS
A white or almost white, crystalline powder or colourless crystals, freely soluble in water, soluble in
glycerol, slightly soluble in alcohol.
IDENTIFICATION
obtained with thiamine hydrochloride CRS. If the spectra obtained show differences, dissolve the
substance to be examined and the reference substance separately in water R, evaporate to dryness and
record new spectra using the residues.
B. Dissolve about 20 mg in 10 ml of water R, add 1 ml of dilute acetic acid R and 1.6 ml of 1M sodium
hydroxide. Heat on a water-bath for 30 min and allow to cool. Add 5 ml of dilute sodium hydroxide
solution R, 10 ml of potassium ferricyanide solution R and 10 ml of butanol R and shake vigorously for
2 min. The alcoholic layer shows an intense light-blue fluorescence, especially in ultraviolet light at
365 nm. Repeat the test using 0.9 ml of 1M sodium hydroxide and 0.2 g of sodium sulphite R instead of
1.6 ml of 1M sodium hydroxide. Practically no fluorescence is seen.
TESTS
Appearance of solution Dilute 2.5 ml of solution S to 5 ml with water R. The solution is clear
(2.2.1) and not more intensely coloured than reference solution Y7 or GY7 (Method II, 2.2.2).
pH (2.2.3). Dilute 2.5 ml of solution S to 10 ml with carbon dioxide-free water R. The pH of the
Nitrates To 0.4 ml of solution S add 1.6 ml of water R and 2 ml of sulphuric acid R and cool.
Superimpose 2 ml of an 80 g/l solution of ferrous sulphate R prepared immediately before use with
carbon dioxide-free water R. No brown ring is produced at the junction of the two layers.
of water.
39-55
ASSAY
1 ml of 0.1M sodium hydroxide is equivalent to 16.86 mg of C12H18Cl2N4OS.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
39-56
Thiamine Nitrate
Me
NH2
CH 2CH2OH
+
N
NO3
S
Me
C12H17N5O4S
327.4
532-43-4
Thiamine Nitrate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0531].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Thiamine nitrate contains not less than 98.0 per cent and not more than the equivalent of 102.0 per
cent of 3-[(4-amino-2-methylpyrimidin-5-yl)methyl]-5-(2-hydroxyethyl)-4-methylthiazolium nitrate,
CHARACTERS
A white or almost white, crystalline powder or small, colourless crystals, sparingly soluble in water,
slightly soluble in alcohol and in methanol, freely soluble in boiling water.
IDENTIFICATION
obtained with thiamine nitrate CRS.
B. Dissolve about 20 mg in 10 ml of water R, add 1 ml of dilute acetic acid R and 1.6 ml of 1M sodium
hydroxide, heat on a water-bath for 30 min and allow to cool. Add 5 ml of dilute sodium hydroxide
solution R, 10 ml of potassium ferricyanide solution R and 10 ml of butanol R and shake vigorously for
2 min. The upper (alcoholic) layer shows an intense light-blue fluorescence in ultraviolet light at
365 nm. Repeat the test using 0.9 ml of 1M sodium hydroxide and 0.2 g of sodium sulphite R instead of
1.6 ml of 1M sodium hydroxide. Practically no fluorescence is produced.
C. About 5 mg gives the reaction of nitrates (2.3.1).
TESTS
105C.
ASSAY
with 0.1M perchloric acid determining the end-point potentiometrically (2.2.20). Carry out a blank
titration.
1.0 ml of 0.1M perchloric acid is equivalent to 16.37 mg of C12H17N5O4S.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
39-57
Thiamphenicol
H
OH
OH
H
NH
MeO2S
O
C12H15Cl2NO5S
356.2
CHCl2
15318-45-3
Thiamphenicol complies with the requirements of the 3rd edition of the European Pharmacopoeia [0109].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Thiamphenicol contains not less than 98.0 per cent and not more than the equivalent of 100.5 per
cent of 2,2-dichloro-N-[(1R,2R)-2-hydroxy-1-hydroxymethyl-2-(4-methylsulphonylphenyl)ethyl]acetamide, calculated with reference to the dried substance.
CHARACTERS
A fine, white or yellowish-white, crystalline powder or crystals, slightly soluble in water, in ether and
in ethyl acetate, very soluble in dimethylacetamide, freely soluble in acetonitrile and in dimethylformamide, soluble in methanol, sparingly soluble in acetone and in ethanol.
A solution in ethanol is dextrorotatory and a solution in dimethylformamide is laevorotatory.
IDENTIFICATION
obtained with thiamphenicol CRS. Dry the substances at 100C to 105C for 2 h and examine in the
form of discs prepared using potassium bromide R.
the same solvent.
Reference solution. Dissolve 0.1 g of thiamphenicol CRS in methanol R and dilute to 10 ml with the
same solvent.
volumes of methanol R and 97 volumes of ethyl acetate R. Allow the plate to dry in air and examine in
ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is
similar in position and size to the spot in the chromatogram obtained with the reference solution.
C. To 50 mg in a porcelain crucible add 0.5 g of anhydrous sodium carbonate R. Heat over an open
TESTS
Acidity or alkalinity Shake 0.1 g with 20 ml of carbon dioxide-free water R and add 0.1 ml of
bromothymol blue solution R1. Not more than 0.1 ml of 0.02M hydrochloric acid or 0.02M sodium
hydroxide is required to change the colour of the indicator.
Specific optical rotation (2.2.7). Dissolve 1.25 g in dimethylformamide R and dilute to 25.0 ml with
the same solvent. The specific optical rotation is 21 to 24, calculated with reference to the dried
substance.
Melting point (2.2.14). 163C to 167C.
Absorbance (2.2.25). Dissolve 20 mg in water R, heating to about 40C, and dilute to 100.0 ml with
the same solvent. Examined between 240 nm and 300 nm, the solution shows two absorption
maxima, at 266 nm and 273 nm. The specific absorbances at these maxima are 25 to 28 and 21.5 to
23.5, respectively. Dilute 2.5 ml of the solution to 50.0 ml with water R. Examined between 200 nm
and 240 nm, the solution shows an absorption maximum at 224 nm. The specific absorbance at this
maximum is 370 to 400.
Chlorides (2.4.4). Shake 0.5 g with 30 ml of water R for 5 min and filter. 15 ml of the filtrate
39-58
100C to 105C.
ASSAY
Dissolve 0.300 g in 30 ml of alcohol R, add 20 ml of a 500 g/l solution of potassium hydroxide R, mix
and heat under a reflux condenser for 4 h. Cool, add 100 ml of water R, neutralise with dilute nitric
acid R and add 5 ml in excess. Titrate with 0.1M silver nitrate, determining the end-point potentiometrically (2.2.20), using a silver indicator electrode and a mercurous sulphate reference electrode or
any other appropriate electrode. Carry out a blank test.
1 ml of 0.1M silver nitrate is equivalent to 17.81 mg of C12H15Cl2NO5S.
STORAGE
Store in a well-closed container, protected from light and moisture.
__________________________________________________________________________________________________________ Ph Eur
39-59
Thiomersal
Thimerosal
S
Hg
Et
COONa
C9H9HgNaO2S
404.8
54-64-8
Definition Thiomersal is the sodium salt of (2-carboxyphenylthio)ethylmercury. It contains not less

than 97.0% and not more than 101.0% of C9H9HgNaO2S, calculated with reference to the dried
substance.
Characteristics A light, cream, crystalline powder; odour, slight and characteristic.
Freely soluble in water and in ethanol (96%); practically insoluble in ether.
Identification
A. Dissolve 0.1 g in 10 ml of water and add 2 ml of silver nitrate solution. A pale yellow precipitate is
produced.
B. Dissolve 0.5 g in 10 ml of water and add 2 ml of 2M hydrochloric acid. A white precipitate is
produced which, after washing with water and drying over phosphorus pentoxide at a pressure not
exceeding 0.7 kPa, has a melting point of about 110, Appendix V A.
Acidity or alkalinity pH of a 1.0% w/v solution, 6.0 to 8.0, Appendix V L.
Ether-soluble matter Shake 0.5 g with 20 ml of ether for 10 minutes, filter and evaporate the
filtrate to dryness. The residue, after drying over phosphorus pentoxide at a pressure not exceeding
0.7 kPa for 24 hours, weighs not more than 3 mg.
Inorganic mercury compounds Not more than 0.70% when determined by the following method.
Protect the solutions from light throughout the procedure. Label five 10-ml graduated flasks A, B, C,
D and E. Place 5 ml of a 0.1% w/v solution of the substance being examined in each of flasks A, B, C
and D. To each of flasks C and D add 0.5 ml of a solution of mercury(II) chloride containing 95 g
per ml. Add sufficient water to flasks A and C to produce 10 ml and add sufficient of a freshly
prepared 33.2% w/v solution of potassium iodide to flasks B and D to produce 10 ml. Place 5 ml of the
potassium iodide solution in flask E and add sufficient water to produce 10 ml. Measure the absorbances of each of the solutions (Aa, Ab, Ac, Ad, Ae) at 323 nm, Appendix II B, using water in the reference cell throughout.
Calculate the content of inorganic mercury compounds, expressed as Hg, in the substance being
examined from the expression
0.7388 Ct ( A b Aa Ae )
1000 Cs ( Ad + Aa A b Ac )
where 0.7388 = the ratio of the atomic weight of mercury to the molecular weight of HgCl2,
Ct = concentration of HgCl2 in g per ml and
Cs = the concentration of the substance being examined in % w/v.
Assay Place 0.5 g in a 100-ml Kjeldahl flask, add 5 ml of sulphuric acid and heat gently until charring
occurs; continue to heat and add hydrogen peroxide solution (100 vol), dropwise, until the mixture is
colourless. Dilute with water, evaporate until slight fuming occurs, dilute to 10 ml with water, cool
and titrate with 0.1M ammonium thiocyanate VS using ammonium iron(III) sulphate solution R2 as
indicator. Each ml of 0.1M ammonium thiocyanate VS is equivalent to 20.24 mg of C9H9HgNaO2S.
Storage Thiomersal should be protected from light.
39-60
Thiopental Sodium
Thiopental Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Thiopental Sodium and Sodium Carbonate [0212]. These requirements are reproduced after the heading
Definition below.
Action and use General anaesthetic.
Preparation
Thiopental Injection
When thiopentone sodium is prescribed or demanded, Thiopental Sodium shall be dispensed or
supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Thiopental sodium and sodium carbonate is a mixture of the sodium derivative of 5-ethyl-5-(1methylbutyl)-2-thioxo-1H,5H-pyrimidine-4,6-dione (C11H17N2NaO2S; Mr 264.3) and anhydrous
sodium carbonate, containing the equivalent of not less than 84.0 per cent and not more than
87.0 per cent of thiopental and not less than 10.2 per cent and not more than 11.2 per cent of Na,
both calculated with reference to the dried substance.
CHARACTERS
A yellowish-white powder, hygroscopic, freely soluble in water, partly soluble in ethanol, practically
insoluble in ether.
IDENTIFICATION
A. Acidify 10 ml of solution S (see Tests) with dilute hydrochloric acid R. An effervescence is
produced. Shake with 20 ml of ether R. Separate the ether layer, wash with 10 ml of water R, dry over
anhydrous sodium sulphate R and filter. Evaporate the filtrate to dryness and dry the residue at 100C
to 105C (test residue). Determine the melting point (2.2.14) of the test residue. Mix equal parts of
the test residue and thiopental CRS and determine the melting point of the mixture. The difference
between the melting points (which are about 160C) is not greater than 2C.
B. Examine by infrared absorption spectrophotometry (2.2.24), comparing the test residue (see
identification test A) with the spectrum obtained with thiopental CRS.
same solvent.
Reference solution. Dissolve 85 mg of thiopental CRS in 10 ml of dilute sodium hydroxide solution R and
dilute to 100 ml with water R.
chloroform R. Examine immediately in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution.
E. It gives reaction (a) of sodium (2.3.1).
TESTS
coating substance.
same solvent. Disregard any slight residue.
chloroform R. Examine immediately in ultraviolet light at 254 nm. Any spot in the chromatogram
39-61
chromatogram obtained with the reference solution (0.5 per cent). Disregard any spot at the startingpoint.
Chlorides (2.4.4). To 5 ml of solution S add 35 ml of water R and 10 ml of dilute nitric acid R. Shake
with three quantities, each of 25 ml, of ether R and discard the ether layers. Eliminate the ether from
the aqueous layer by heating on a water-bath. 15 ml of the aqueous layer complies with the limit test
for chlorides (330 ppm).
100C for 4 h.
ASSAY
Sodium Dissolve 0.400 g in 30 ml of water R. Add 0.1 ml of methyl red solution R and titrate with
0.1M hydrochloric acid until a red colour is obtained. Boil gently for 2 min. Cool and, if necessary,
continue the titration with 0.1M hydrochloric acid until the red colour is again obtained.
1 ml of 0.1M hydrochloric acid is equivalent to 2.299 mg of Na.
Thiopental Dissolve 0.150 g in 5 ml of water R. Add 2 ml of dilute sulphuric acid R and shake with
four quantities, each of 10 ml, of chloroform R. Combine the chloroform layers, filter and evaporate
the filtrate to dryness on a water-bath. Dissolve the residue in 30 ml of previously neutralised
dimethylformamide R and add 0.1 ml of a 2 g/l solution of thymol blue R in methanol R. Titrate immediately with 0.1M lithium methoxide until a blue colour is obtained. Protect the solution from
atmospheric carbon dioxide during the titration.
1 ml of 0.1M lithium methoxide is equivalent to 24.23 mg of C11H18N2O2S.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
40-1
Thioridazine
Me
N
SMe
S
and enantiomer
C21H26N2S2
370.6
50-52-2
Definition Thioridazine is (RS)-10-[2-(1-methyl-2-piperidyl)ethyl]-2-methylthiophenothiazine. It

contains not less than 99.0% and not more than 101.0% of C21H26N2S2, calculated with reference to
Characteristics A white or almost white, finely crystalline powder.
Practically insoluble in water; very soluble in chloroform; freely soluble in absolute ethanol and in
ether.
Identification
A. The infrared absorption spectrum, Appendix II A, is concordant with reference spectrum 1 of
thioridazine (RS 335). Examine the material as a dispersion in potassium bromide.
B. Complies with the test for identification of phenothiazines, Appendix III A, using as solution (2) a
0.2% w/v solution of thioridazine hydrochloride EPCRS in chloroform.
C. Dissolve about 20 mg in a mixture of 2 ml of water and 0.2 ml of 1M sulphuric acid and add 1 ml
of a 5% w/v solution of silver nitrate. No precipitate is produced (distinction from thioridazine hydrochoride).
Colour of solution A 5.0% w/v solution in methanol is not more intensely coloured than reference
solution Y6, BY6 or GY6, Appendix IV B, Method II.
Heavy metals 1.0 g complies with limit test C for heavy metals, Appendix VII. Use 2 ml of lead
from light, using silica gel G as the coating substance and a mixture of 1 volume of 13.5M ammonia,
25 volumes of propan-2-ol and 74 volumes of chloroform as the mobile phase. Carry out all operations
as rapidly as possible and apply solution (1) to the plate as the last solution. Apply separately to the
plate 5 l of each of three solutions of the substance to be examined in a mixture of 2 volumes of
13.5M ammonia and 98 volumes of methanol containing (1) 2.0% w/v, (2) 0.0040% w/v and (3)
0.0020% w/v. After removal of the plate, allow it to dry in air and spray with a mixture of 10 volumes
of 2M acetic acid and 1 volume of potassium iodobismuthate solution and then with freshly prepared
hydrogen peroxide solution (10 vol) and immediately cover the plate with a glass plate of the same size.
Any secondary spot in the chromatogram obtained with solution (1) is not more intense than the
principal spot in the chromatogram obtained with solution (2) and not more than two such spots are
more intense than the spot in the chromatogram obtained with solution (3). The test is not valid
unless the spot in the chromatogram obtained with solution (3) is clearly visible.
Loss on drying When dried to constant weight at 50 at a pressure of 2.0 kPa, loses not more than
0.5% of its weight. Use 0.5 g.
Sulphated ash Not more than 0.1%, Appendix IX A, Method II. Use 1 g.
Assay Dissolve 0.3 g in 60 ml of glacial acetic acid and carry out Method I for non-aqueous titration,
equivalent to 37.06 mg of C21H26N2S2.
Storage Thioridazine should be kept in a well-closed container and protected from light.
Preparation
Thioridazine Oral Suspension
40-2
Thioridazine Hydrochloride
Me
N
SMe
,HCl
S
and enantiomer
C21H26N2S2,HCl
407.0
130-61-0
Thioridazine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparations
Thioridazine Oral Solution
Thioridazine Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Thioridazine hydrochloride contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of (RS)-10-[2-(1-methyl-2-piperidyl)ethyl]-2-(methylthio)phenothiazine hydrochloride, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, freely soluble in water and in methanol, soluble in
IDENTIFICATION
obtained with thioridazine hydrochloride CRS.
B. It complies with the identification of phenothiazines by thin-layer chromatography (2.3.3).
C. 0.2 g gives reaction (b) of chlorides (2.3.1).
TESTS
Carry out all operations protected from light.
The solution is clear (2.2.1) and not more intensely coloured than intensity 6 of the range of reference solutions of the most appropriate colour (Method II, 2.2.2).
5.2.
coating substance.
Carry out all operations as rapidly as possible, protected from light. Apply the test solution last.
Test solution. Dissolve 0.2 g in a mixture of 2 volumes of concentrated ammonia R and 98 volumes of
Reference solution (a). Dilute 2 ml of the test solution to 100 ml with a mixture of 2 volumes of
concentrated ammonia R and 98 volumes of methanol R. Dilute 10 ml of this solution to 100 ml with a
mixture of 2 volumes of concentrated ammonia R and 98 volumes of methanol R.
Reference solution (b). Dilute 1 ml of the test solution to 100 ml with a mixture of 2 volumes of concentrated ammonia R and 98 volumes of methanol R. Dilute 10 ml of this solution to 100 ml with a
mixture of 2 volumes of concentrated ammonia R and 98 volumes of methanol R.
volume of concentrated ammonia R, 25 volumes of 2-propanol R and 74 volumes of chloroform R. Allow
40-3
the plate to dry in air. Spray first with a freshly prepared mixture of 1 volume of potassium
iodobismuthate solution R and 10 volumes of dilute acetic acid R and then with freshly prepared dilute
hydrogen peroxide solution R. Cover the plate immediately with a glass plate of the same size. Any spot
intense than the spot in the chromatogram obtained with reference solution (a) (0.2 per cent) and at
most two such spots are more intense than the spot in the chromatogram obtained with the reference
solution (b) (0.1 per cent). The test is not valid unless the spot in the chromatogram obtained with
reference solution (b) is clearly visible.
100C to 105C for 4 h.
ASSAY
1 ml of 0.1M perchloric acid is equivalent to 40.70 mg of C21H27ClN2S2.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
40-4
Thiotepa
S
N
N
C6H12N3PS
189.2
52-24-4
Definition Thiotepa is phosphorothioic tri(ethyleneamide). It contains not less than 97.0% and not
more than 102.0% of C6H12N3PS, calculated with reference to the anhydrous substance.
Characteristics Fine, white crystalline flakes; odourless or almost odourless.
Freely soluble in water, in chloroform and in ethanol (96%).
Identification
thiotepa (RS 337).
B. Burn 20 mg by the method for oxygen-flask combustion, Appendix VIII C, using 5 ml of 1.25M
sodium hydroxide as the absorbing liquid. When the process is complete, dilute to 25 ml with water.
To 5 ml of the resulting solution add 0.1 ml of hydrogen peroxide solution (100 vol) and 1 ml of 1M
hydrochloric acid, mix and add 0.05 ml of barium chloride solution. The solution becomes turbid.
C. To 2 ml of the solution obtained in test B add 40 ml of water and 4 ml of ammonium molybdate
sulphuric acid solution, mix, add 0.1 g of L-ascorbic acid and boil for 1 minute. A blue colour is
produced.
Clarity of solution A 2.0% w/v solution is clear, Appendix IV A.
following freshly prepared solutions. Solutions (1) and (2) are solutions of the substance being examined in water containing 0.350% w/v and 0.00035% w/v, respectively. For solution (3) dissolve 10 mg
of the substance being examined in 2 ml of methanol in a ground-glass-stoppered tube, add 50 l of a
0.1% v/v solution of orthophosphoric acid, stopper the tube and heat in a water bath at 65 for 50
seconds (generation of methoxythiotepa). Allow the solution to cool and add 1 ml of methanol. For
solution (4) dissolve 15 mg of the substance being examined in 10 ml of water, add 1 g of sodium
chloride, boil in a water bath for 10 minutes and cool (generation of chloro-adduct).
(15 cm 4.6 mm) packed with stationary phase C (5 m) (Nucleosil C18 is suitable), (b) 15 volumes
of acetonitrile and 85 volumes of 0.1M mixed phosphate buffer pH 7.0 as the mobile phase with a flow
rate of 1 ml per minute and (c) a detection wavelength of 215 nm.
The chromatogram obtained with solution (3) shows a peak corresponding to methoxythiotepa
with a retention time relative to thiotepa of about 1.3 and the chromatogram obtained with solution
(4) shows a peak due to the chloro-adduct with a retention time relative to thiotepa of about 3.75.
The test is not valid unless the resolution factor between the two principal peaks in the chromatogram
obtained with solution (3) is at least 3.
For solution (1) allow the chromatography to proceed for 4 times the retention time of the principal peak. In the chromatogram obtained with solution (1) the area of any peak corresponding to the
chloro-adduct (identified from the peak in the chromatogram obtained with solution (4)) is not
(0.15%), the area of any other secondary peak is not greater than the area of the principal peak in the
chromatogram obtained with solution (2) (0.1%) and the sum of the areas of all the secondary peaks is
not greater than twice the area of the principal peak in the chromatogram obtained with solution (2)
(0.2%).
Water Not more than 0.5% w/w, Appendix IX C. Cool the reagents and titration vessel in ice
throughout the procedure and use 1.2 g. Complete the procedure as quickly as possible.
Assay Transfer 0.2 g to an iodine flask with the aid of 50 ml of a 20% w/v solution of sodium
thiosulphate and titrate immediately with 0.1M hydrochloric acid VS, using 0.05 ml of methyl orange
solution as indicator, until a faint red colour persists for 10 seconds. Stopper the flask, allow to stand
for 30 minutes and titrate with 0.1M sodium hydroxide VS using phenolphthalein solution R1 as
indicator. Subtract the volume of 0.1M sodium hydroxide VS used from the volume of 0.1M hydrochloric acid VS used. Repeat the operation without the substance being examined. The difference
between the titrations represents the amount of hydrochloric acid required. Each ml of 0.1M hydrochloric acid VS is equivalent to 6.307 mg of C6H12N3PS.
Storage Thiotepa should be kept in a well-closed container and stored at a temperature of 2 to 8.
At higher temperatures it polymerises and becomes inactive.
40-5
Preparation
Thiotepa Injection
IMPURITIES
A. chloro-adduct
B. hydroxythiotepa (A and B).
40-6
Threonine
H
H3C
H
NH2
COOH
OH
C4H9NO3
119.1
72-19-5
Threonine complies with the requirements of the 3rd edition of the European Pharmacopoeia [1049]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Threonine contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of
(2S,3R)-2-amino-3-hydroxybutanoic acid, calculated with reference to the dried substance.
PRODUCTION
When Threonine is produced by a process involving fermentation steps, it complies with the requirements of the monograph on Products of fermentation (1468).
CHARACTERS
A white, crystalline powder or colourless crystals, soluble in water, practically insoluble in alcohol and
in ether.
IDENTIFICATION
obtained with threonine CRS. Examine the substances prepared as discs.
D. Mix 1 ml of a 2 g/l solution of the substance to be examined with 1 ml of a 20 g/l solution of
sodium periodate R. Add 0.2 ml of piperidine R and 0.1 ml of a 25 g/l solution of sodium nitroprusside R.
A blue colour develops that changes to yellow after a few minutes.
TESTS
substance.
gel plate R.
Reference solution (a). Dissolve 10 mg of threonine CRS in a 1 per cent V/V solution of hydrochloric
acid R and dilute to 50 ml with the same acid solution.
Reference solution (c). Dissolve 10 mg of threonine CRS and 10 mg of proline CRS in a 1 per cent V/V
solution of hydrochloric acid R and dilute to 25 ml with the same acid solution.
Apply separately to the plate 5 l of each solution. Allow the plate to dry in air. Develop over a path
of 15 cm using a mixture of 20 volumes of glacial acetic acid R, 20 volumes of water R and 60 volumes
of butanol R. Allow the plate to dry in air, spray with ninhydrin solution R and heat at 100C to 105C
40-7
100C to 105C.
ASSAY
STORAGE
__________________________________________________________________________________________________________ Ph Eur
40-8
Thyme
Thyme complies with the requirements of the 3rd edition of the European Pharmacopoeia [0865]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Thyme consists of the whole leaves and flowers separated from the previously dried stems of Thymus
vulgaris L. or Thymus zygis L. or a mixture of both species. It contains not less than 12 ml/kg of
essential oil and not less than 0.5 per cent m/m of volatile phenols, expressed as thymol (C10H14O;
Mr 150.2), both calculated with reference to the anhydrous drug.
CHARACTERS
Thyme has a strong aromatic odour reminiscent of thymol.
IDENTIFICATION
A. The leaf of Thymus vulgaris is usually 4 mm to 12 mm long and up to 3 mm wide: it is sessile or
has a very short petiole. The lamina is tough, entire, lanceolate to ovate, covered on both surfaces by
a grey to greenish-grey indumentum; the edges are markedly rolled up towards the abaxial surface.
The midrib is depressed on the adaxial surface and is very prominent on the abaxial surface. The
calyx is green, often with violet spots and is tubular; at the end are two lips of which the upper one is
bent back and at the end has three lobes, the lower is longer and has two hairy teeth. After flowering,
the calyx tube is closed by a crown of long, stiff hairs. The corolla, about twice as long as the calyx, is
usually brownish in the dry state and is slightly bilabiate.
The leaf of Thymus zygis is usually 1.7 mm to 6.5 mm long and 0.4 mm to 1.2 mm wide; it is
acicular to linear-lanceolate and the edges are markedly rolled towards the abaxial surface. Both
surfaces of the lamina are green to greenish-grey and the midrib is sometimes violet; the edges, in
particular at the base, have long, white hairs. The dried flowers are very similar to those of Thymus
vulgaris.
B. Reduce to a powder (355). The powder of the two species is greyish-green to greenish-brown.
Examine under a microscope using chloral hydrate solution R. The epidermises of the leaves have cells
with anticlinal walls which are sinuous and beaded and the stomata are of the diacytic type (2.8.3);
numerous secretory trichomes made up of twelve secretory cells, the cuticle of which is generally
raised by the secretion to form a globular to ovoid bladder-like covering; the glandular trichomes
have a unicellular stalk and a globular to ovoid head; the covering trichomes of the adaxial surface are
common to both species; they have warty walls and are shaped as pointed teeth; the warty covering
trichomes of the abaxial surface are of many types: unicellular, straight or slightly curved, and
bicellular or tricellular, and often elbow-shaped (Thymus vulgaris); bicellular or tricellular, more or
less straight (Thymus zygis). Fragments of calyx are covered by numerous, uniseriate trichomes with
five or six cells and with a weakly striated cuticle. Fragments of the corolla have numerous uniseriate
covering trichomes, often collapsed, and secretory trichomes with generally twelve cells. Pollen grains
are relatively rare, spherical and smooth with six germinal slit-like pores, measuring about 35 m in
diameter. The powder of Thymus zygis also contains numerous thick bundles of fibres from the main
veins and from fragments of stems.
C. Examine by thin-layer chromatography (2.2.27), using a suitable silica gel with a fluorescent
indicator having an optimal intensity at 254 nm as the coating substance.
Test solution. To 1.0 g of the powdered drug (355) add 5 ml of methylene chloride R and shake for
3 min, filter through about 2 g of anhydrous sodium sulphate R. Use the filtrate as the test solution.
Reference solution. Dissolve 5 mg of thymol R and 10 l of carvacrol R in 10 ml of methylene chloride R.
Apply separately to the plate as bands, 20 l of each solution. Develop twice over a path of 12 cm
using methylene chloride R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm.
Mark the quenching zones. The chromatograms obtained with the reference solution and the test
solution show in the central part a quenching zone due to thymol. The chromatogram obtained with
the test solution shows slightly above the zone due to thymol a prominent quenching zone and other
quenching zones in the lower third of the chromatogram. Spray with anisaldehyde solution R using
10 ml for a plate 200 mm square and heat at 100C to 105C for 10 min. The chromatogram
obtained with the reference solution shows in the central part a brownish-pink zone corresponding to
thymol and, immediately below it, a pale violet zone corresponding to carvacrol. The chromatogram
obtained with the test solution shows these two zones in the central part of the plate; they are more or
less prominent, depending upon the species examined. Between these two zones and the starting-line
are four zones of similar intensity; in order of decreasing Rf value these bands are: pink, violet
(cineole and linalol), greyish-brown (borneol) and violet-blue. Near the solvent front, an intense
violet-red to greyish-violet band is visible. Other bands are also present adjacent to the starting-line.
40-9
TESTS
Foreign matter (2.8.2). Not more than 10 per cent of stem. Stems must not be more than 1 mm in
diameter and 15 mm in length. Leaves with long trichomes at their base and with weakly pubescent
other parts are not allowed (Thymus serpyllum L.).
Water (2.2.13). Not more than 10.0 per cent, determined by distillation on 20.0 g of powdered drug
(355).
ASSAY
Essential oil Carry out the determination of essential oils in vegetable drugs (2.8.12). Use 30.0 g of
the drug, a 1000 ml round-bottomed flask and 400 ml of water R as the distillation liquid. Distil at a
rate of 2 ml/min to 3 ml/min for 2 h without xylene R in the graduated tube.
Phenols Taking care that as little water as possible is transferred, transfer the essential oil obtained in
the assay of essential oil to a 50 ml volumetric flask with the aid of small portions of alcohol (90 per
cent V/V) R rinsing the graduated tube of the apparatus with the same solvent and dilute to 50.0 ml
with the same solvent. To 5.0 ml of the solution add 40 ml of alcohol (90 per cent V/V) R and dilute
to 100.0 ml with water R. Place 5.0 ml of this solution in a separating funnel and add 45 ml of
water R, 0.5 ml of dilute ammonia R2 and 1 ml of a 20 g/l solution of aminopyrazolone R. Mix and add
4 ml of a freshly prepared 20 g/l solution of potassium ferricyanide R and mix again. Allow to stand for
5 min, add 25 ml of methylene chloride R and shake. Separate the methylene chloride layer and filter
through a plug of absorbent cotton moistened with methylene chloride R into a 100 ml volumetric
flask. Shake the aqueous layer with two quantities, each of 25 ml, and with 10 ml of methylene
chloride R, filter the methylene chloride layers through the plug of absorbent cotton. Rinse the plug
with methylene chloride R and dilute to 100.0 ml with the same solvent. Measure the absorbance
(2.2.25) at 450 nm using methylene chloride R as the compensation liquid.
Calculate the percentage content of phenols, expressed as thymol, taking the specific absorbance to
be 805.
STORAGE
Store in a well-closed container, protected from light and moisture.
__________________________________________________________________________________________________________ Ph Eur
40-10
Thyme Oil
Thyme Oil complies with the requirements of the 3rd edition of the European Pharmacopoeia [1374]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Thyme oil is obtained by steam distillation from the fresh flowering aerial parts of Thymus vulgaris L.,
T. zygis Loefl. ex L. or a mixture of the two species.
CHARACTERS
A clear, yellow or very dark reddish-brown, mobile liquid with a characteristic aromatic, spicy odour,
reminiscent of thymol, miscible with ethanol, with ether and with petroleum ether.
IDENTIFICATION
substance.
Test solution. Dissolve 0.2 g of the substance to be examined in pentane R and dilute to 10 ml with the
same solvent.
Reference solution. Dissolve 0.15 g of thymol R, 25 l of terpinen-4-ol R and 40 l of linalol R in
pentane R and dilute to 10 ml with the same solvent.
Spray with anisaldehyde solution R. Heat the plate at 100C to 105C for 5 min to 10 min while
observing. Examine in daylight. The chromatogram obtained with the test solution shows three zones
similar in position and colour to those in the chromatogram obtained with the reference solution: a
violet zone corresponding to terpinen-4-ol, a violet zone corresponding to linalol and a brownish-pink
zone corresponding to thymol and immediately below it a pale violet zone corresponding to
carvacrol. It also shows a large violet zone at the solvent front (hydrocarbons).
of the principal peaks in the chromatogram obtained with the test solution are similar to those of the
peaks in the chromatogram obtained with the reference solution.
TESTS
Reference solution. Dissolve 0.15 g of -myrcene R, 0.1 g of -terpinene R, 0.1 g of p-cymene R, 0.1 g of
linalol R, 0.2 g of terpinen-4-ol R, 0.2 g of thymol R and 0.05 g of carvacrol R in 5 ml of hexane R.
a fused-silica capillary column 25 m to 60 m long and about 0.3 mm in internal diameter coated
with macrogol 20,000 R,
helium for chromatography R as the carrier gas,
of 3C per min to 180C and maintaining at 180C; maintaining the temperature of the injection
port at about 200C and that of the detector at 220C.
prescribed conditions, the components elute in the order indicated in the composition of the reference solution. Record the retention times of these substances.
The test is not valid unless: the number of theoretical plates calculated from the p-cymene peak at
80C is at least 30,000; the resolution between the peaks corresponding to thymol and carvacrol is at
least 1.5.
Inject about 0.2 l of the test solution. Using the retention times determined from the chromatogram obtained with the reference solution, locate the components of the reference solution on the
chromatogram obtained with the test solution. Disregard the peak due to hexane.
Determine the percentage content of the components of the normalisation procedure.
40-11
The percentages range between the following values:
-Myrcene
-Terpinene
p-Cymene
Linalol
Terpinen-4-ol
Thymol
Carvacrol

15.0 percent to 28.0 per cent
STORAGE
1. -myrcene
5. terpinen-4-ol
2. -terpinene 6. thymol
3. p-cymene
7. carvacrol
4. linalol
Fig. 13741 Type chromatogram for thyme oil (chromatographic profile test)
__________________________________________________________________________________________________________ Ph Eur
40-12
Thymol
Me
OH
Me
C10H14O
Me
150.2
89-83-8
Thymol complies with the requirements of the 3rd edition of the European Pharmacopoeia [0791]. These
Action and use Antimicrobial preservative; antiseptic.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Thymol is 5-methyl-2-(methylethyl)phenol.
CHARACTERS
Colourless crystals, very slightly soluble in water, very soluble in alcohol and in ether, freely soluble in
essential oils and in fatty oils, sparingly soluble in glycerol. It dissolves in dilute solutions of alkali
hydroxides.
IDENTIFICATION
obtained with thymol CRS.
C. Dissolve 0.2 g with heating in 2 ml of dilute sodium hydroxide solution R and add 0.2 ml of chloroform R. Heat on a water-bath. A violet colour develops.
D. Dissolve about 2 mg in 1 ml of anhydrous acetic acid R. Add 0.15 ml of sulphuric acid R and
0.05 ml of nitric acid R. A bluish-green colour develops.
TESTS
Appearance of solution Dissolve 1.0 g in 10 ml of dilute sodium hydroxide solution R. The solution is
not more opalescent than reference suspension IV (2.2.1) and not more intensely coloured than
reference solution R6 (Method II, 2.2.2).
Acidity To 1.0 g in a 100 ml glass-stoppered conical flask add 20 ml of water R. Boil until dissolution is complete, cool and stopper the flask. Shake vigorously for 1 min. Add a few crystals of the
substance to be examined to initiate crystallisation. Shake vigorously for 1 min and filter. To 5 ml of
the filtrate, add 0.05 ml of methyl red solution R and 0.05 ml of 0.01M sodium hydroxide. The solution
is yellow.
Test solution. Dissolve 0.100 g in alcohol R and dilute to 10.0 ml with the same solvent.
Reference solution (a). Dilute 1 ml of the test solution to 100 ml with alcohol R.
Reference solution (c). Dilute 5 ml of reference solution (b) to 10 ml with alcohol R.
a glass or steel column 4 m long and 2 mm in internal diameter packed with diatomaceous earth
for gas chromatography R, impregnated with a mixture suitable for the separation of free fatty
acids,
Inject separately 1 l of each solution and, after 2 min, increase the temperature of the column to
40-13
240C at a rate of 8C per minute and maintain at this temperature for 15 min. The test is not valid
unless the peak in the chromatogram obtained with reference solution (b) has a signal-to-noise ratio
not less than five. In the chromatogram obtained with the test solution, the sum of the areas of the
peaks, apart from the principal peak, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (a) (1.0 per cent). Disregard any peak with an area less
than that of the principal peak in the chromatogram obtained with reference solution (c).
Residue on evaporation Evaporate 2.00 g on a water-bath and heat in an oven at 100C to 105C
for 1 h. The residue weighs not more than 1.0 mg (0.05 per cent).
STORAGE
__________________________________________________________________________________________________________ Ph Eur
40-14
Tiabendazole
H
N
N
C10H7N3S
S
N
201.2
148-79-8
Tiabendazole complies with the requirements of the 3rd edition of the European Pharmacopoeia [0866]. These
Preparation
Tiabendazole Tablets
When thiabendazole is prescribed or demanded, Tiabendazole shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Tiabendazole contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent
of 2-(1,3-thiazol-4-yl)-1H-benzimidazole, calculated with reference to the anhydrous substance.
CHARACTERS
A white or almost white, crystalline powder, practically insoluble in water, slightly soluble in alcohol,
in ether and in methylene chloride. It dissolves in dilute mineral acids. It melts at about 300C.
IDENTIFICATION
A. Dissolve 25 mg in 0.1M hydrochloric acid and dilute to 100.0 ml with the same acid. Dilute 2.0 ml
(2.2.25), the solution shows two absorption maxima, at 243 nm and 302 nm. The ratio of the
absorbance measured at the maximum at 302 nm to that measured at the maximum at 243 nm is 1.8
to 2.1.
obtained with tiabendazole CRS. Examine the substances prepared as discs.
D. Dissolve about 5 mg in 0.1M hydrochloric acid and dilute to 5 ml with the same acid. Add 3 mg of
p-phenylenediamine dihydrochloride R and shake until dissolved. Add 0.1 g of zinc powder R, mix, allow
to stand for 2 min and add 5 ml of ferric ammonium sulphate solution R2. A bluish-violet colour
develops.
TESTS
coating substance.
Reference solution (a). Dissolve 20 mg of tiabendazole CRS in methanol R and dilute to 20 ml with the
same solvent.
2.5 volumes of water R, 10 volumes of acetone R, 25 volumes of glacial acetic acid R and 62.5 volumes
of toluene R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Any spot in the
such spot is more intense than the spot in the chromatogram obtained with reference solution (c)
(0.4 per cent).
40-15
o-Phenylenediamine To 5.0 g in a flask fitted with a ground-glass stopper, add 25 ml of a mixture
of 1 volume of methanol R and 2 volumes of water R. Shake for 3 min. Filter through a sintered-glass
filter (16) under reduced pressure. To 10 ml of the filtrate add 0.5 ml of hydrochloric acid R and
0.5 ml of acetylacetone R and shake until the solution is clear. The solution is not more intensely
coloured than reference solution R7 (Method I, 2.2.2) (10 ppm).
of water.
ASSAY
Dissolve 0.150 g in 30 ml of glacial acetic acid R. Titrate with 0.1M perchloric acid, determining the
1 ml of 0.1M perchloric acid is equivalent to 20.12 mg of C10H7N3S.
STORAGE
IMPURITIES
NH2
NH2
A. o-phenylenediamine.
__________________________________________________________________________________________________________ Ph Eur
40-16
Tiapride Hydrochloride
1/01
O
S
e
N
H
NEt2
,HCl
OMe
C15H25ClN2O4S
364.9
51012-33-0
Tiapride Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Tiapride hydrochloride contains not less than 98.5 per cent and not more than the equivalent of
101.0 per cent of N-[2-(diethylamino)ethyl]-2-methoxy-5-(methylsulphonyl)benzamide hydrochloride, calculated with reference to the dried substance.
CHARACTERS
A white or almost white crystalline powder, very soluble in water, soluble in methanol, slightly
soluble in ethanol.
IDENTIFICATION
obtained with tiapride hydrochloride CRS. Examine the substances prepared as discs.
B. Solution S (see Tests) gives reaction (a) of chlorides (2.3.1).
TESTS
Appearance of solution Solution S is clear (2.2.1). The absorbance (2.2.25) of solution S measured
at 450 nm is not more than 0.030.
Impurity C Examine by thin-layer chromatography (2.2.27), using a TLC silica gel G plate R.
the same solvent.
Reference solution. Dissolve 20.0 mg of metoclopramide impurity E CRS (tiapride impurity C) in
methanol R and dilute to 50 ml with the same solvent. Dilute 2.0 ml of this solution to 20 ml with
methanol R.
Apply to the plate 10 l of the test solution and 10 l of the reference solution. Develop over a path
of 12 cm using a mixture of 2 volumes of concentrated ammonia R, 10 volumes of dioxan R, 14
volumes of methanol R and 90 volumes of methylene chloride R. Allow the plate to dry in air. Spray
with a 2 g/l solution of ninhydrin R in butanol R and heat at 100C for 15 min. Any spot corresponding to impurity C in the chromatogram obtained with the test solution is not more intense than the
Reference solution (b). Dissolve 5.0 mg of tiapride hydrochloride CRS and 5.0 mg of tiapride N-oxide
potassium dihydrogen phosphate R and 0.08 g of sodium octanesulphonate R in 780 ml of water R,
adjust the pH to 2.7 using phosphoric acid R and dilute to 800 ml with water R; add 150 ml of
40-17
methanol R and 50 ml of acetonitrile R and mix,
Inject 10 l of reference solution (b). The test is not valid unless the resolution between the peaks
corresponding to tiapride (retention time of about 9 min) and tiapride N-oxide (retention time of
about 13 min) is at least 4.0.
Inject 10 l of the test solution. Continue the chromatography for 3 times the retention time of
tiapride. In the chromatogram obtained with the test solution: the area of any peak, apart from the
reference solution (a) (0.1 per cent) and the sum of the areas of any such peaks is not greater than 3
times the area of the principal peak in the chromatogram obtained with reference solution (a)
(0.3 per cent). Disregard any peak with an area less than half the area of the principal peak in the
chromatogram obtained with reference solution (a) (0.05 per cent).
Heavy metals (2.4.8). 1.0 g complies with limit test A for heavy metals (20 ppm). Prepare the
100C to 105C.
ASSAY
1 ml of 0.1M perchloric acid is equivalent to 36.49 mg of C15H25ClN2O4S.
IMPURITIES
O O
S
COOR
OMe
A. R = CH3: methyl 2-methoxy-5-(methylsulphonyl)benzoate,

B. R = H: 2-methoxy-5-(methylsulphonyl)benzoic acid,
H2N
NEt2
C. N,N-diethylethane-1,2-diamine.
__________________________________________________________________________________________________________ Ph Eur
40-18
Tiaprofenic Acid
H
Me
S
COOH
and enantiomer
C14H12O3S
260.3
33005-95-7
Tiaprofenic Acid complies with the requirements of the 3rd edition of the European Pharmacopoeia [1157].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Tiaprofenic acid contains not less than 99.0 per cent and not more than the equivalent of 101.0 per
cent of (RS)-2-(5-benzoyl-2-thienyl)propanoic acid, calculated with reference to the dried substance.
CHARACTERS
in alcohol and in methylene chloride.
IDENTIFICATION
B. Dissolve 25.0 mg in ethanolic hydrochloric acid R and dilute to 50.0 ml with the same solvent.
Dilute 1.0 ml of the solution to 50.0 ml with ethanolic hydrochloric acid R. Examined between 220 nm
and 350 nm (2.2.25), the solution shows a shoulder at 262 nm and an absorption maximum at
obtained with tiaprofenic acid CRS.
D. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel F254 plate R.
Reference solution (a). Dissolve 10 mg of tiaprofenic acid CRS in methylene chloride R and dilute to
Reference solution (b). Dissolve 10 mg of ketoprofen CRS in methylene chloride R and dilute to 10 ml
with the same solvent. Dilute 1 ml of the solution to 2 ml with reference solution (a).
1 volume of acetic acid R, 20 volumes of methylene chloride R and 80 volumes of acetone R. Allow the
obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a). The identification is not valid unless the chromatogram
obtained with reference solution (b) shows two clearly separated principal spots.
TESTS
2.2.2).
Optical rotation (2.2.7). Dissolve 0.50 g in ethyl acetate R and dilute to 10.0 ml with the same
solvent. The angle of optical rotation is 0.10 to +0.10.
40-19
Reference solution (c). Dissolve 10.0 mg of tiaprofenic acid impurity C CRS in the mobile phase and
dilute to 100.0 ml with the mobile phase. Dilute 1.0 ml of the solution to 50.0 ml with the mobile
phase.
Reference solution (d). Dilute 1.0 ml of reference solution (a) to 2.0 ml with reference solution (c).
as mobile phase at a flow rate of 1 ml per minute a mixture of 0.25 volumes of water R, 20
volumes of glacial acetic acid R, 500 volumes of hexane R and 500 volumes of methylene
chloride R. Add the water to the acetic acid, then hexane and methylene chloride. Sonicate the
mixture for 2 min. Do not degas with helium during analysis,
Inject 20 l of reference solution (d). When the chromatograms are recorded in the prescribed
conditions the retention times relative to tiaprofenic acid are: tiaprofenic acid impurity A, 0.19;
tiaprofenic acid impurity B, 0.43; tiaprofenic acid impurity C, 0.86.
Adjust the sensitivity of the system so that the heights of the principal peaks in the chromatogram
obtained with reference solution (d) are at least 50 per cent of the full scale of the recorder. The test
is not valid unless in the chromatogram obtained, the resolution between the peaks corresponding to
tiaprofenic acid and tiaprofenic acid impurity C is at least 3.0.
Inject 20 l of the test solution, 20 l of reference solution (a), 20 l of reference solution (b) and
20 l of reference solution (c). Continue the chromatography of the test solution for twice the retention time of tiaprofenic acid. In the chromatogram obtained with the test solution, the area of any
peak corresponding to impurity C is not greater than the area of the corresponding peak in the
chromatogram obtained with reference solution (c) (0.2 per cent); the area of any peak, apart from
the principal peak and any peak corresponding to impurity C, is not greater than the area of the
the areas of all the peaks, apart from the principal peak and any peak corresponding to impurity C, is
not greater than 1.5 times the area of the principal peak in the chromatogram obtained with reference
solution (a) (0.3 per cent). Disregard any peak with an area less than half the area of the principal
peak in the chromatogram obtained with the reference solution (b).
Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.000 g by drying at 60C in
an oven at a pressure not exceeding 0.7 kPa for 3 h.
ASSAY
Dissolve 0.250 g in 25 ml of alcohol R. Add 25 ml of water R and 0.5 ml of phenolphthalein solution R.
Titrate with 0.1M sodium hydroxide.
1 ml of 0.1M sodium hydroxide is equivalent to 26.03 mg of C14H12O3S.
STORAGE
IMPURITIES
O
S
CH3
A. (5-ethyl-2-thienyl)phenylmethanone,
O
O
S
CH3
B. (5-acetyl-2-thienyl)phenylmethanone,
O
S
COOH
and enantiomer
H Me
C. (RS)-2-(5-benzoyl-3-thienyl)propanoic acid,
40-20
COOH
D. benzoic acid,
H Me
and enantiomer
COOH
E. (R,S) 2-(2-thienyl)propanoic acid,

O
S
Br
F. (5-bromo-2-thienyl)phenylmethanone.
__________________________________________________________________________________________________________ Ph Eur
40-21
Ticarcillin Sodium
H
O
COONa
H
COONa
Me
N
*
S
H
Me
O
and epimer at C*
C15H14N2Na2O6S2
428.4
4697-14-7
Ticarcillin Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia [0956].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ticarcillin sodium contains not less than 89.0 per cent and not more than the equivalent of 100.5 per
cent of disodium (2S,5R,6R)-6-[[(2RS)-2-carboxylato-2-(thiophen-3-yl)acetyl]-amino]-3,3dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate, calculated with reference to the
PRODUCTION
If manufactured by a process that may leave residues of 2-ethylhexanoic acid in the substance, it
complies with the following test:
2-Ethylhexanoic acid Not more than 0.5 per cent m/m, determined by gas chromatography
(2.2.28), using a suitable, validated method.
CHARACTERS
A white or slightly yellow powder, hygroscopic, freely soluble in water, soluble in methanol,
IDENTIFICATION
Second identification B, C, D.
obtained with ticarcillin sodium CRS.
substance.
the same solvent.
Reference solution (a). Dissolve 25 mg of ticarcillin sodium CRS in methanol R and dilute to 5 ml with
the same solvent.
Reference solution (b). Dissolve 25 mg of carbenicillin sodium CRS and 25 mg of ticarcillin sodium CRS
in methanol R and dilute to 5 ml with the same solvent.
glacial acetic acid R. Allow the plate to dry in a current of hot air and expose it to iodine vapour. The
principal spot in the chromatogram obtained with the test solution is similar in position, colour and
size to the principal spot in the chromatogram obtained with reference solution (a). The test is not
valid unless the chromatogram obtained with reference solution (b) shows two clearly separated
spots.
C. Place about 2 mg in a test-tube about 15 cm long and 15 mm in diameter. Moisten with 0.05 ml
of water R and add 2 ml of sulphuric acid-formaldehyde reagent R. Mix the contents of the tube by
swirling; the solution is brown. Place the test-tube in a water-bath for 1 min; a dark reddish-brown
colour develops.
40-22
TESTS
substance.
Reference solution (a). Dissolve 20.0 mg of ticarcillin impurity A CRS in mobile phase A and dilute to
100.0 ml with the same mobile phase. Dilute 5.0 ml of this solution to 50.0 ml with mobile phase A.
Reference solution (b). Dilute 1 ml of the test solution to 50 ml with mobile phase A.
Mobile phase A. A 1.3 g/l solution of ammonium phosphate R adjusted to pH 7.0 with phosphoric
acid R,
Mobile phase B. A mixture of equal volumes of mobile phase A and methanol R,
Time
(min)
Mobile phase A
(per cent V/V)

(per cent V/V)
030
3040
4045
10030
30
100
070
70
0
Linear gradient
Isocratic
Equilibration
as detector a spectrophotometer at 220 nm.

two principal peaks are at least 50 per cent of the full scale of the recorder. The test is not valid unless
the resolution between the two principal peaks (diastereoisomers) is at least 2.0. Inject 20 l of the
test solution and 20 l of reference solution (a). In the chromatogram obtained with the test solution:
the area of any peak corresponding to ticarcillin impurity A is not greater than twice the area of the
principal peak in the chromatogram obtained with reference solution (a) (4 per cent); and the area of
any peak apart from the two principal peaks and any peak corresponding to ticarcillin impurity A, is
not greater than 1.25 times the area of the principal peak in the chromatogram obtained with the
Dimethylaniline Not more than 20 ppm, determined by gas chromatography (2.2.28), using
naphthalene R as the internal standard.
Internal standard solution. Dissolve 50.0 mg of naphthalene R in cyclohexane R and dilute to 50.0 ml
with the same solvent. Dilute 5.0 ml of this solution to 100.0 ml with cyclohexane R.
Test solution. To 1.00 g of the substance to be examined in a ground-glass-stoppered tube add 5 ml of
1M sodium hydroxide and 1.0 ml of the internal standard solution. Stopper the tube and shake
vigorously for 1 min. Centrifuge if necessary and use the upper layer.
Reference solution. To 50.0 mg of dimethylaniline R add 2 ml of hydrochloric acid R and 20 ml of
water R. Shake to dissolve and dilute to 50.0 ml with water R. Dilute 5.0 ml of this solution to
250.0 ml with water R. To 1.0 ml of the latter solution in a ground-glass-stoppered tube add 5 ml of
1M sodium hydroxide and 1.0 ml of the internal standard solution. Stopper the tube and shake for
1 min. Centrifuge if necessary and use the upper layer.
a glass column 2 m long and 2 mm in internal diameter packed with silanised diatomaceous earth
for gas chromatography R impregnated with 3 per cent m/m of polymethylphenylsiloxane R,
detector at 150C. Inject 1 l of the test solution and 1 l of the reference solution.
without a further procedure for the removal of bacterial endotoxins, not more than 0.05 I.U. of
endotoxin per milligram.
40-23
ASSAY
100.0 ml with the same solvent. Dilute 10.0 ml of the solution to 50.0 ml with the mobile phase.
Reference solution. Dissolve 50.0 mg of ticarcillin sodium CRS in the mobile phase and dilute to
100.0 ml with the same solvent. Dilute 10.0 ml of the solution to 50.0 ml with the mobile phase.
a stainless steel column 0.25 m long and 4 mm in internal diameter packed with octadecyl silyl
as mobile phase at a flow rate of 1 ml/min a mixture of 20 volumes of methanol R and 80
volumes of a 1.3 g/l solution of ammonium phosphate R (adjusted to pH 7.0 with phosphoric
acid R),
Inject 20 l of the reference solution. Adjust the sensitivity of the system so that the heights of the
two principal peaks are at least 50 per cent of the full scale of the recorder. The test is not valid unless
the resolution between the two principal peaks is at least 2.5. Inject the reference solution six times.
The test is not valid unless the relative standard deviation of the two peak areas for ticarcillin is at
most 1.0 per cent. Inject alternately the test solution and the reference solution.
Calculate the percentage content of ticarcillin sodium as the sum of the two peaks.
STORAGE
Store in an airtight container, at a temperature of 2C to 8C. If the substance is sterile, store in a
LABELLING
The label states:
IMPURITIES
H
O
H
N
S
O
COOH
Me
N
S
Me
H H
A. (2S,5R,6R)-3,3-dimethyl-7-oxo-6-[[(thiophen-3-yl)acetyl]amino]-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid (decarboxyticarcillin),

S
COOH
B. (thiophen-3-yl)acetic acid,
COOH
S
COOH
C. 2-(thiophen-3-yl)propanedioic acid (3-thienylmalonic acid),
H COOH
COOH
HN
Me
H
Me
N
*S
*
*
O
COOH
D. (4S)-2-[carboxy[[2-carboxy-2-(thiophen-3-yl)acetyl]amino]methyl]-5,5-dimethylthiazolidine-4-carboxylic acid (penicilloic acids of ticarcillin),
COOH
H
N
*
O
HN
COOH
Me
*S
Me
E. (4S)-2-[[[2-carboxy-2-(thiophen-3-yl)acetyl]amino]methyl]-5,5-dimethylthiazolidine-4carboxylic acid (penilloic acids of ticarcillin),

__________________________________________________________________________________________________________ Ph Eur
40-24
Ticlopidine Hydrochloride
1/01
S
,HCl
N
Cl
C14H14ClNS,HCl
300.2
53885-35-1
Ticlopidine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Platelet aggregation inhibitor.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ticlopidine hydrochloride contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of 5-(2-chlorobenzyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine hydrochloride, calculated with reference to the anhydrous substance.
CHARACTERS
A white or almost white, crystalline powder, sparingly soluble in water and in ethanol, very slightly
soluble in ethyl acetate.
IDENTIFICATION
A. Dissolve 40 mg in water R and dilute to 100.0 ml with the same solvent (solution A). Dilute
5.0 ml of solution A to 100.0 ml with water R (solution B). Examined between 200 nm and 350 nm
(2.2.25), solution B shows two absorption maxima, at 214 nm and 232 nm. Examined between
250 nm and 350 nm, solution A shows two absorption maxima, at 268 nm and 275 nm. The ratio of
the absorbance measured at the maximum at 268 nm to that measured at the maximum at 275 nm is
1.1 to 1.2.
obtained with ticlopidine hydrochloride CRS. Examine the substances prepared as discs.
C. Mix about 6 mg of citric acid R and 0.3 ml of acetic anhydride R. Add about 5 mg of the substance
to be examined and heat in a water-bath at 80C. A red colour develops.
D. About 20 mg gives reaction (a) of chlorides (2.3.1).
TESTS
Appearance of solution Dissolve 0.5 g in a 1 per cent V/V solution of hydrochloric acid R and dilute
to 20 ml with the same acid solution. The solution is clear (2.2.1) and colourless (Method II, 2.2.2).
Test solution. Dissolve 0.250 g of the substance to be examined in a mixture of 20 volumes of mobile
phase B and 80 volumes of mobile phase A. Dilute to 50.0 ml with the same mixture of solvents.
Reference solution. Dissolve 5.0 mg of ticlopidine impurity F CRS in a mixture of 20 volumes of mobile
phase B and 80 volumes of mobile phase A. Add 1.00 ml of the test solution and dilute to 100.0 ml
with a mixture of 20 volumes of mobile phase B and 80 volumes of mobile phase A. Dilute 1.0 ml of
this solution to 10.0 ml with a mixture of 20 volumes of mobile phase B and 80 volumes of mobile
phase A.
a stainless steel column, 0.15 m long and 4.6 mm in internal diameter, packed with basedeactivated octadecylsilyl silica gel for chromatography R (5 m),
Mobile phase A. A 0.95 g/l solution of sodium pentanesulphonate monohydrate R, adjusted to pH
3.4 with a 50 per cent V/V solution of concentrated phosphoric acid R,
40-25
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
0 45
80 20
20 80
Linear gradient
45 50
20
80
Isocratic
50 55
20 80
80 20
Linear gradient

Inject 10 l of a mixture of 20 volumes of mobile phase B and 80 volumes of mobile phase A
(blank). Inject 10 l of the reference solution. When the chromatogram is recorded in the prescribed
conditions the retention time for ticlopidine is about 15 min (see Fig. 10501).
Adjust the sensitivity of the system so that, in the chromatogram obtained with the reference
solution, the height of the peak due to ticlopidine is at least 5 per cent of the full scale of the recorder.
The test is not valid unless in the chromatogram obtained with the reference solution: the resolution
between the peaks corresponding to ticlopidine and impurity F is at least 2.0 (modify the pH of
mobile phase A if necessary); the signal-to-noise ratio of the peak due to ticlopidine is at least 50.
with the test solution: the area of the peak corresponding to impurity F is not greater than half the
area of the corresponding peak in the chromatogram obtained with the reference solution (0.05 per
cent); the area of any peak, apart from the principal peak and the peak due to impurity F, is not
greater than half the area of the principal peak in the chromatogram obtained with the reference
solution (0.05 per cent); the sum of the areas of all the peaks apart from the peak corresponding to
ticlopidine, is not greater than the area of the peak corresponding to ticlopidine in the chromatogram
obtained with the reference solution (0.1 per cent). Disregard any peak with an area less than 0.1
times that of ticlopidine in the chromatogram obtained with the reference solution.
Formaldehyde Dissolve 0.200 g in 4.0 ml of water R. Add 0.4 ml of dilute sodium hydroxide solution R. Centrifuge, filter the supernatant liquid through cotton previously impregnated with water R
and dilute to 5.0 ml with water R. Transfer to a test-tube. Add 5.0 ml of acetylacetone reagent R1.
Place the test-tube in a water-bath at 40C for 40 min. The test solution is not more intensely
coloured than a standard prepared at the same time and in the same manner using 5.0 ml of a
0.8 ppm solution of formaldehyde (CH2O), obtained by dilution of formaldehyde standard solution
(5 ppm CH2O) R with water R (20 ppm). Examine the tubes down their vertical axis.
Heavy metals (2.4.8). Dissolve 2.0 g in a 85 per cent V/V solution of methanol R and dilute to
20.0 ml with the same solvent. 12 ml of the solution complies with limit test B for heavy metals
(10 ppm). Prepare the standard using 10 ml of lead standard solution (1 ppm Pb) R.
ASSAY
1 ml of 0.1M perchloric acid is equivalent to 30.02 mg of C14H15Cl2NS.
IMPURITIES
S
A. thieno[3,2-c]pyridine,
S
HN
O
B. 6,7-dihydrothieno[3,2-c]pyridin-4(5H)-one,
NH2
Cl
C. (2-chlorophenyl)methanamine,
40-26
S
N
D. 5-benzyl-4,5,6,7-tetrahydrothieno[3,2-c]pyridine,
S
N
Cl
E. 5-(2-chlorobenzyl)thieno[3,2-c]pyridinium,
Cl
F. 6-(2-chlorobenzyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine,
S
N
G. 5-(3-chlorobenzyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine,
l
S
N
H. 5-(4-chlorobenzyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine,
S
NH
Cl
I. N-(2-chlorobenzyl)-2-(thiophen-2-yl)ethanamine,
Cl
NH
N
H
Cl
J. N,N-bis(2-chlorobenzyl)ethane-1,2-diamine,
S
N
Cl
Cl
K. 2,8-bis(2-chlorobenzyl)-1,2,3,4,6,7,8,9-octahydrothieno[3,2-c:4,5-c]dipyridine (bisticlopidine),
S
N
Cl
L. 5-(2-chlorobenzyl)-6,7-dihydrothieno[3,2-c]pyridin-4(5H)-one.
40-27
14.783
mAU
100
Imp K
23.359
Imp L
Imp F
Imp G
Imp H
Imp I
Imp j
15.794
16.666
17.173
18.464
19.575
60
Imp D
Imp E
Imp A
Imp B
Imp C
80
4.361
10
15
20
30.559
6.937
20
11.753
12.754
3.124
6.193
40
25
30
35
40
Fig. 10501 Type chromatogram for the test for related substances.
__________________________________________________________________________________________________________ Ph Eur
45
min
40-28
Timolol Maleate
1/01
O
H
N
OH
COOH
COOH
NHBut ,
O
N
S
C13H24N4O3S,C4H4O4
432.5
26921-17-5
Timolol Maleate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0572].
Preparations
Timolol Eye Drops
Timolol Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Timolol maleate contains not less than 98.5 per cent and not more than the equivalent of 101.0 per
cent of (2S)-1-[(1,1-dimethylethyl)amino]-3-[[4-(morpholin-4-yl)-1,2,5-thiadiazol-3-yl]oxy]propan2-ol (Z)-butanedioate, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder or colourless crystals, soluble in water and in alcohol,
IDENTIFICATION
A. Dissolve 1.000 g in 1M hydrochloric acid and dilute to 10.0 ml with the same acid. The specific
optical rotation (2.2.7) is 5.7 to 6.2.
obtained with timolol maleate CRS.
C. Examine the chromatograms obtained in the test for related substances after exposure to iodine
vapour. The principal spot in the chromatogram obtained with test solution (b) is similar in position,
colour and size to the principal spot in the chromatogram obtained with reference solution (a).
D. Triturate 0.1 g with a mixture of 1 ml of dilute sodium hydroxide solution R and 3 ml of water R.
Shake with three quantities, each of 5 ml, of ether R. To 0.1 ml of the aqueous layer add a solution of
10 mg of resorcinol R in 3 ml of sulphuric acid R. Heat on a water-bath for 15 min. No violet-red
colour develops. Neutralise the remainder of the aqueous layer with dilute sulphuric acid R and add
1 ml of bromine water R. Heat on a water-bath for 15 min, then heat to boiling and cool. To 0.2 ml of
this solution add a solution of 10 mg of resorcinol R in 3 ml of sulphuric acid R. Heat on a water-bath
for 15 min; a violet-red colour develops. Add 0.2 ml of a 100 g/l solution of potassium bromide R and
heat for 5 min on a water-bath. The colour becomes violet-blue.
TESTS
Enantiomeric purity Examine by liquid chromatography (2.2.29). Carry out the test protected from
actinic light.
Test solution. Dissolve 30.0 mg of the substance to be examined in a mixture of 1 volume of methylene
chloride R and 3 volumes of 2-propanol R and dilute to 10.0 ml with the same mixture of solvents.
Reference solution (a). Dissolve 30 mg of timolol maleate CRS in a mixture of 1 volume of methylene
chloride R and 3 volumes of 2-propanol R and dilute to 10 ml with the same mixture of solvents.
Reference solution (b). Dissolve 15.0 mg of (R)-timolol maleate CRS in a mixture of 1 volume of
40-29
methylene chloride R and 3 volumes of 2-propanol R and dilute to 10.0 ml with the same mixture of
solvents. Dilute 1.0 ml of the solution to 50.0 ml with a mixture of 1 volume of methylene chloride R
and 3 volumes of 2-propanol R.
Reference solution (c). Dilute 1 ml of reference solution (a) to 100 ml with a mixture of 1 volume of
methylene chloride R and 3 volumes of 2-propanol R. Mix 1 ml of this solution with 1 ml of reference
solution (b).
a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with silica gel OD
for chiral separations R (5 m),
as mobile phase at a flow rate of 1 ml/min a mixture of 2 ml of diethylamine R, 40 ml of
2-propanol R and 960 ml of hexane R,
Under these conditions the peak corresponding to the (R)-isomer appears first.
Inject 5 l of each solution. The test is not valid unless: in the chromatogram obtained with reference-solution (c), the resolution between the peaks corresponding to the (R)-enantiomer and to the
(S)-enantiomer is at least 4.0; the retention times of the principal peaks (corresponding to the (S)enantiomer) in the chromatograms obtained with the test solution and reference solution (a) are
identical. In the chromatogram obtained with the test solution, the area of any peak corresponding to
the (R)-enantiomer is not greater than the area of the principal peak in the chromatogram obtained
with reference solution (b) (1 per cent).
Related substances Examine by thin-layer chromatography (2.2.27), using a TLC silica gel plate
GF254 R.
Reference solution (a). Dissolve 10 mg of timolol maleate CRS in methanol R and dilute to 10 ml with
the same solvent.
of concentrated ammonia R, 20 volumes of methanol R and 80 volumes of methylene chloride R. Allow
obtained with test solution (a), apart from the principal spot, is not more intense than the spot in the
chromatogram obtained with reference solution (b) (0.4 per cent). Disregard the spot remaining at
the starting point. Expose the plate to iodine vapour for 2 h. Any spot in the chromatogram obtained
with test solution (a), apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (b) (0.4 per cent). Disregard the spot remaining at the
starting point.
100C to 105C.
ASSAY
STORAGE
IMPURITIES
H OH
S
NHBu t
COOH
,
COOH
O
A (2R)-1-[(1,1-dimethylethyl)amino]-3-[[4-(morpholin-4-yl)-1,2,5-thiadiazol-3yl]oxy]propan-2-ol (Z)-butenedioate.
__________________________________________________________________________________________________________ Ph Eur
40-30
Tinidazole
NO2
O
S
CH3
N
Me
C8H13N3O4S
247.3
19387-91-8
Tinidazole complies with the requirements of the 3rd edition of the European Pharmacopoeia [1051]. These
Action and use Antiprotozoan; antibacterial.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Tinidazole contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent of
1-(2-ethylsulphonylethyl)-2-methyl-5-nitroimidazole, calculated with reference to the dried
substance.
CHARACTERS
An almost white or pale yellow, crystalline powder, practically insoluble in water, soluble in acetone
and in methylene chloride, sparingly soluble in methanol.
IDENTIFICATION
B. Dissolve 10.0 mg in methanol R and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml of the
obtained with tinidazole CRS. Examine the substances prepared as discs.
E. To about 10 mg add about 10 mg of zinc powder R, 0.3 ml of hydrochloric acid R and 1 ml of
water R. Heat in a water-bath for 5 min and cool. The solution gives the reaction of primary aromatic
amines (2.3.1).
TESTS
Appearance of solution Dissolve 1.0 g in acetone R and dilute to 20 ml with the same solvent. The
2.2.2).
coating substance.
Test solution (a). Dissolve 0.20 g of the substance to be examined in methanol R with the aid of
ultrasound and dilute to 10 ml with the same solvent.
Reference solution (a). Dissolve 20 mg of tinidazole CRS in methanol R and dilute to 10 ml with the
same solvent.
Reference solution (c). Dilute 4 ml of reference solution (b) to 10 ml with methanol R.
Reference solution (d). Dissolve 10 mg of 2-methyl-5-nitroimidazole R (impurity A) in methanol R and
Reference solution (e). Dissolve 10 mg of tinidazole impurity B CRS in methanol R and dilute to 100 ml
Heat the plate at 110C for 1 h and allow to cool.
Apply separately to the plate 10 l of each solution. Develop over a path of 15 cm using a mixture
40-31
of 25 volumes of butanol R and 75 volumes of ethyl acetate R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Any spots corresponding to tinidazole impurity A and tinidazole
impurity B in the chromatogram obtained with test solution (a) are not more intense than the corresponding spots in the chromatogram obtained with reference solutions (d) and (e) (0.5 per cent),
respectively. Any spot, apart from the principal spot and any spots corresponding to tinidazole impurity A and tinidazole impurity B in the chromatogram obtained with test solution (a), is not more
100C to 105C.
ASSAY
STORAGE
Store in an well-closed container, protected from light.
IMPURITIES
NO2
NH
N
Me
A. 2-methyl-5-nitroimidazole,
O2N
O O
S
CH3
N
Me
B. 1-(2-ethylsulphonylethyl)-2-methyl-4-nitroimidazole.
__________________________________________________________________________________________________________ Ph Eur
40-32
Tinzaparin Sodium
OSO3Na
O
OR
R2
O
COONa
O
O
R3
OH
OR
OH
O
NHR 1
OR
OH
OH
NHSO3Na
OSO3Na
n = 1 to 25, R = H or SO3Na, R1 = H or SO3Na or COCH3
R2 = H and R3 = CO2Na or R2 = CO2Na and R3 = H
Tinzaparin Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Tinzaparin sodium is the sodium salt of a low-molecular-mass heparin that is obtained by controlled
enzymatic depolymerisation of heparin from porcine intestinal mucosa using heparinase from
Flavobacterium heparinum. The majority of the components have a 2-O-sulpho-4-enepyranosuronic
acid structure at the non-reducing end and a 2-N,6-O-disulpho-D-glucosamine structure at the
reducing end of their chain.
Tinzaparin sodium complies with the monograph on Low-molecular-mass heparins (828) with the modifications and additional requirements below:
6500.
The degree of sulphatation is 1.8 to 2.5 per disaccharide unit.
milligram calculated with reference to the dried substance. The ratio of the anti-factor Xa activity to
anti-factor IIa activity is between 1.5 and 2.5.
IDENTIFICATION
Carry out identification test C as described in the monograph for Low-molecular-mass heparins (828).
The following requirements apply:
lower than 2000 is not more than 10.0 per cent. The mass percentage of chains between 2000 and
8000 ranges between 60.0 and 72.0 per cent. The mass percentage of chains above 8000 ranges
between 22.0 and 36.0 per cent.
TESTS
Appearance of solution. Dissolve 1.0 g in 10 ml of water R. The solution is clear (2.2.1) and not
more intensely coloured than intensity 5 of the range of reference solutions of the most appropriate
colour (Method II, 2.2.2)
Absorbance (2.2.25). Dissolve 50.0 mg in 100 ml of 0.01M hydrochloric acid. The specific absorbance, measured at 231 nm and calculated with reference to the dried substance, is 8.0 to 12.5.
__________________________________________________________________________________________________________ Ph Eur
40-33
Tioconazole
Cl
Cl
Cl
H
N
O
S
and enantioner
C16H13Cl3N2OS
387.7
65899-73-2
Definition Tioconazole is (RS)-1-[2,4-dichloro--(2-chloro-3-thenyloxy)phenethyl]imidazole. It

contains not less than 97.5% and not more than 101.5% of C16H13Cl3N2OS, calculated with
Characteristics A white or almost white crystalline powder.
Very slightly soluble in water; freely soluble in acetone, in ethanol, in ethyl acetate and in methanol.
spectrum of tioconazole (RS 393).
Optical rotation 0.1 to 0.1, Appendix V F, when measured in a 1% w/v solution in asbolute
ethanol.
following solutions in the mobile phase. Solutions (1), (2) and (3) contain 0.20% w/v, 0.00040% w/v
and 0.0010% w/v, respectively, of the substance being examined. Solution (4) contains 0.20% w/v of
tioconazole impurity standard BPCRS.
4.6 mm) packed with octadecylsilyl silica gel for chromatography (5 m) (Hypersil ODS is suitable), (b)
a mixture of 1 volume of 0.005M tetrabutylammonium dihydrogen orthophosphate adjusted to pH 7.4
with 2M ammonia and 4 volumes of methanol as the mobile phase with a flow rate of 1.0 ml per
minute and (c) a detection wavelength of 218 nm.
For solution (1) allow the chromatography to proceed for three times the retention time of the
principal peak.
The test is not valid unless the chromatogram obtained with solution (4) closely resembles the
reference chromatogram supplied with tioconazole impurity standard BPCRS.
In the chromatogram obtained with solution (1) the areas of any peaks corresponding to
tioconazole impurities A, B, C and D are not greater than the area of the principal peak in the
chromatogram obtained with solution (3) (0.5% of each). The area of any other secondary peak is not
greater than the area of the principal peak in the chromatogram obtained with solution (2) (0.2%).
The sum of the areas of any secondary peaks is not greater than five times the area of the peak in the
chromatogram obtained with solution (3) (2.5%).
the end-point potentiometrically. Each ml of 0.1M perchloric acid VS is equivalent to 38.77 mg of
C16H13Cl3N2OS.
Preparations
Tioconazole Cream
Tioconazole Nail Solution
IMPURITIES
Cl
Cl
S
A. 1-[2,4-dichloro--[(3-thenyl)oxy]phenethyl]imidazole,
40-34
Cl
Cl
Cl
N
S
l
B. 1-[2,4-dichloro--[2,5-dichloro-3-thenyl)oxy]phenethyl]imidazole,
Cl
Cl
Cl
N
S
Br
C. 1-[2,4-dichloro--[5-bromo-2-chloro-3-thenyl)oxy]phenethyl]imidazole,
Cl
l
HO
D. 1-(2,4-dichlorophenyl)-2-(1H-imidazole-1-yl)ethanol.
40-35
Tioguanine
S
H
N
HN
H2N
C5H5N5S
N
167.2
154-42-7
Definition Tioguanine is 2-aminopurine-6(1H)-thione. It contains not less than 98.0% and not
more than 101.0% of C5H5N5S, calculated with reference to the dried substance.
Characteristics A pale yellow, crystalline powder; odourless or almost odourless.
Practically insoluble in water, in ethanol (96%) and in chloroform. It dissolves in dilute solutions of
alkali hydroxides.
Identification
A. Heat a suitable quantity at 105 at a pressure not exceeding 0.7 kPa for 5 hours. The infrared
absorption spectrum of the residue, Appendix II A, is concordant with the reference spectrum of
tioguanine (RS 340).
hydrochloric acid exhibits two maxima, at 258 nm and 348 nm. The absorbance at 348 nm is about
1.24.
C. Mix 10 mg with 10 mg of sodium formate in a test tube and heat gently until melted. A gas is
evolved which turns lead acetate paper black.
Phosphate To 0.5 g add 2 ml of 5M sulphuric acid and heat on a water bath for 5 minutes. Add,
dropwise, 5 ml of nitric acid and continue heating until a clear solution is obtained. Cool and add
10 ml of water and 0.75 ml of a solution prepared by dissolving 8.3 g of ammonium molybdate in
40 ml of water and adding 33 ml of 5M sulphuric acid and sufficient water to produce 100 ml. Add
1.0 ml of strong aminohydroxynaphthalenesulphonic acid solution, mix and dilute to 25 ml with water.
Measure the absorbance of the resulting solution at 820 nm, Appendix II B, using in the reference cell
a solution prepared in the same manner but omitting the substance being examined. The absorbance
is not more than that obtained by treating 1.5 ml of phosphate standard solution (100 ppm PO4) in the
same manner, beginning at the words add 10 ml of water ....
Free sulphur Dissolve 50 mg in 5 ml of 1M sodium hydroxide. The solution is clear, Appendix IV A.
gel GF254 as the coating substance and a mixture of 90 volumes of methanol and 15 volumes of 13.5M
ammonia as the mobile phase. Apply separately to the plate 2 l of solution (1) and 5 l and 1 l of
solution (2). Solution (1) contains 1.0% w/v of the substance being examined in 0.1M sodium
hydroxide. Solution (2) contains 0.010% w/v of guanine in 0.1M sodium hydroxide. After removal of the
plate, allow it to dry in air and examine under ultraviolet light (254 nm). Any spot corresponding to
guanine in the chromatogram obtained with solution (1) is not more intense than the spot in the
chromatogram obtained with 5 l of solution (2). Any other secondary spot in the chromatogram
obtained with solution (1) is not more intense than the spot in the chromatogram obtained with 1 l
of solution (2).
Assay Carry out Method I for non-aqueous titration, Appendix VIII A, using a solution prepared by
dissolving 0.15 g in 15 ml of anhydrous formic acid, adding 50 ml of butan-2-one and determining the
end point potentiometrically. Each ml of 0.1M perchloric acid VS is equivalent to 16.72 mg of
C5H5N5S.
Storage Tioguanine should be kept in a well-closed container.
Preparation
Tioguanine Tablets
When thioguanine is prescribed or demanded, Tioguanine shall be dispensed or supplied.
40-36
Titanium Dioxide
TiO2
79.9
13463-67-7
Titanium Dioxide complies with the requirements of the 3rd edition of the European Pharmacopoeia [0150].
Action and use Protective; pharmaceutical aid.
Preparation
Titanium Ointment
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Titanium dioxide contains not less than 98.0 per cent and not more than the equivalent of 100.5 per
cent of TiO2.
CHARACTERS
A white or almost white powder, practically insoluble in water. It does not dissolve in dilute mineral
acids but dissolves slowly in hot concentrated sulphuric acid.
IDENTIFICATION
A. When strongly heated, it becomes pale yellow; the colour disappears on cooling.
B. To 5 ml of solution S2 (see Tests) add 0.1 ml of strong hydrogen peroxide solution R. An orange-red
colour appears.
C. To 5 ml of solution S2 add 0.5 g of zinc R in granules. After 45 min, the mixture has a violet-blue
colour.
TESTS
Solution S1 Shake 20.0 g with 30 ml of hydrochloric acid R for 1 min. Add 100 ml of distilled water R
and heat the mixture to boiling. Filter the hot mixture through a hardened filter paper until a clear
filtrate is obtained. Wash the filter with 60 ml of distilled water R and dilute the combined filtrate and
washings to 200 ml with distilled water R.
Solution S2 Mix 0.500 g (m g) with 5 g of anhydrous sodium sulphate R in a 300 ml long-necked
combustion flask. Add 10 ml of water R and mix. Add 10 ml of sulphuric acid R and boil vigorously,
with the usual precautions, until a clear solution is obtained. Cool, add slowly a cooled mixture of
30 ml of water R and 10 ml of sulphuric acid R, cool again and dilute to 100.0 ml with water R.
Appearance of solution Solution S2 is not more opalescent than reference suspension II (2.2.1)
and is colourless (Method II, 2.2.2).
Acidity or alkalinity Shake 5.0 g with 50 ml of carbon dioxide-free water R for 5 min. Centrifuge or
filter until a clear solution is obtained. To 10 ml of the solution add 0.1 ml of bromothymol blue solution R1. Not more than 1.0 ml of 0.01M hydrochloric acid or 0.01M sodium hydroxide is required to
Water-soluble substances To 10.0 g add a solution of 0.5 g of ammonium sulphate R in 150 ml of
water R and boil for 5 min. Cool, dilute to 200 ml with water R and filter until a clear solution is
obtained. Evaporate 100 ml of the solution to dryness in a tared evaporating dish and ignite. The
residue weighs not more than 25 mg (0.5 per cent).
Antimony To 10 ml of solution S2 add 10 ml of hydrochloric acid R and 10 ml of water R. Cool to
20C, if necessary, and add 0.15 ml of sodium nitrite solution R. After 5 min, add 5 ml of a 10 g/l
solution of hydroxylamine hydrochloride R and 10 ml of a freshly prepared 0.1 g/l solution of rhodamine
B R. Mix thoroughly after each addition. Shake vigorously with 10.0 ml of toluene R for 1 min. Allow
to separate and centrifuge for 2 min if necessary. Any pink colour in the toluene phase is not more
intense than that in the toluene phase of a standard prepared at the same time in the same manner
using a mixture of 5.0 ml of antimony standard solution (1 ppm Sb) R, 10 ml of hydrochloric acid R and
15 ml of a solution containing 0.5 g of anhydrous sodium sulphate R and 2 ml of sulphuric acid R
instead of the mixture of 10 ml of solution S2, 10 ml of hydrochloric acid R and 10 ml of water R
(100 ppm).
Arsenic (2.4.2). Place 0.50 g in a 250 ml round-bottomed flask, fitted with a thermometer, a funnel
with stopcock and a vapour-outlet tube connected to a flask containing 30 ml of water R. Add 50 ml
of water R, 0.5 g of hydrazine sulphate R, 0.5 g of potassium bromide R and 20 g of sodium chloride R.
Through the funnel, add dropwise 25 ml of sulphuric acid R, heat and maintain the temperature of
the liquid at 110C to 115C for 20 min. Collect the vapour in the flask containing 30 ml of water R.
Dilute to 50 ml with water R. 20 ml of the solution complies with limit test A for arsenic (5 ppm).
Barium To 10 ml of solution S1 add 1 ml of dilute sulphuric acid R. After 30 min, any opalescence in
40-37
the solution is not more intense than that in a mixture of 10 ml of solution S1 and 1 ml of distilled
water R.
Heavy metals (2.4.8). Dilute 10 ml of solution S1 to 20 ml with water R. 12 ml of the solution
Iron To 8 ml of solution S2 add 4 ml of water R. Mix and add 0.05 ml of bromine water R. Allow to
stand for 5 min and remove the excess of bromine with a current of air. Add 3 ml of potassium
thiocyanate solution R. Any colour in the solution is not more intense than that in a standard prepared
at the same time in the same manner using a mixture of 4 ml of iron standard solution (2 ppm Fe) R
and 8 ml of a 200 g/l solution of sulphuric acid R (200 ppm).
ASSAY
To 300 g of zinc R in granules (710) add 300 ml of a 20 g/l solution of mercuric nitrate R and 2 ml of
nitric acid R, shake for 10 min and wash with water R. Pack the amalgamated zinc into a glass tube
about 400 mm long and about 20 mm in diameter fitted with a tap and a filter plate. Pass through
the column 100 ml of dilute sulphuric acid R followed by 100 ml of water R, making sure that the
amalgam is always covered with liquid. Pass slowly at a rate of about 3 ml per minute through the
column a mixture of 100 ml of dilute sulphuric acid R and 100 ml of water R followed by 100 ml of
water R. Collect the eluate in a 500 ml conical flask containing 50.0 ml of a 150 g/l solution of ferric
ammonium sulphate R in a mixture of 1 volume of sulphuric acid R and 3 volumes of water R. Add
0.1 ml of ferroin R and titrate immediately with 0.1M ammonium and cerium nitrate until a greenish
colour is obtained (n1 ml). Pass slowly at a rate of about 3 ml per minute through the column a
mixture of 50 ml of dilute sulphuric acid R and 50 ml of water R, followed by 20.0 ml of solution S2, a
mixture of 50 ml of dilute sulphuric acid R and 50 ml of water R and finally 100 ml of water R. Collect
the eluate in a 500 ml conical flask containing 50.0 ml of a 150 g/l solution of ferric ammonium
sulphate R in a mixture of 1 volume of sulphuric acid R and 3 volumes of water R. Rinse the lower end
of the column with water R, add 0.1 ml of ferroin R and titrate immediately with 0.1M ammonium and
cerium nitrate until a greenish colour is obtained (n2 ml).
Calculate the percentage content of TiO2 from the expression:
3.99 ( n 2 n1)
m
m = mass in grams of the substance to be examined used for the preparation of solution S2.
__________________________________________________________________________________________________________ Ph Eur
40-38
Tobramycin
CH2OH
O
NH2
HO
H2NCH2
OH
O
O
O
HO
H2N
OH
NH2
NH2
C18H37N5O9
467.5
32986-56-4
Tobramycin complies with the requirements of the 3rd edition of the European Pharmacopoeia [0645]. These
Preparation
Tobramycin Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Tobramycin is O-3-amino-3-deoxy--D-glucopyranosyl-(16)-O-[2,6-diamino-2,3,6-trideoxy--Dribo-hexopyranosyl-(14)]-2-deoxy-D-streptamine, an antimicrobial substance produced by
Streptomyces tenebrarius or obtained by any other means. The potency is not less than 930 I.U. per
milligram, calculated with reference to the anhydrous, 2-methylpropanol-free substance.
PRODUCTION
It is produced by methods of manufacture designed to eliminate or minimise substances lowering
blood pressure.
CHARACTERS
A white or almost white powder, freely soluble in water, very slightly soluble in alcohol, practically
insoluble in ether.
IDENTIFICATION
A. The nuclear magnetic resonance spectrum (2.2.33) of a 100 g/l solution in deuterium oxide R is
concordant with that of a similar solution of tobramycin CRS.
B. Examine by thin-layer chromatography (2.2.27), using silica gel H R as the coating substance.
same solvent.
Reference solution (a). Dissolve 20 mg of tobramycin CRS in water R and dilute to 5 ml with the same
solvent.
Reference solution (b). Dissolve 4 mg of neomycin sulphate CRS and 4 mg of kanamycin sulphate CRS in
1 ml of reference solution (a).
10 volumes of chloroform R, 20 volumes of concentrated ammonia R and 30 volumes of methanol R.
Dry the plate in a current of warm air and spray with a mixture of equal volumes of a 2 g/l solution of
1,3-dihydroxynaphthalene R in alcohol R and a 460 g/l solution of sulphuric acid R. Heat at 105C for 5
min to 10 min. The principal spot in the chromatogram obtained with the test solution is similar in
position, colour and size to the principal spot in the chromatogram obtained with reference solution
(a). The identification is not valid unless the chromatogram obtained with reference solution (b)
shows three major spots which are clearly separated.
C. Dissolve about 5 mg in 5 ml of water R. Add 5 ml of a 1 g/l solution of ninhydrin R in alcohol R
and heat in a water-bath for 3 min. A violet-blue colour develops.
40-39
TESTS
11.0.
solvent. The specific optical rotation is +138 to +148, calculated with reference to the anhydrous,
2-methylpropanol-free substance.
coating substance.
Test solution. Dissolve 80 mg of the substance to be examined in a mixture of 1 volume of dilute
ammonia R2 and 99 volumes of water R and dilute to 10 ml with the same mixture of solvents.
Reference solution. Dilute 1 ml of the test solution to 100 ml with a mixture of 1 volume of dilute
ammonia R2 and 99 volumes of water R.
equal volumes of alcohol R, concentrated ammonia R and methyl ethyl ketone R. Allow the plate to dry in
air, heat at 110C for 10 min and spray the hot plate with a solution prepared immediately before use
by diluting strong sodium hypochlorite solution R with water R to contain 0.5 per cent of available
chlorine. Dry in a current of cold air until the area of the plate sprayed, below the line of application
gives at most a very faint blue colour with a drop of potassium iodide and starch solution R; avoid
prolonged exposure to cold air. Spray the plate with potassium iodide and starch solution R. Any spot in
the chromatogram obtained with the test solution, apart from the principal spot, is not more intense
than the principal spot in the chromatogram obtained with the reference solution (1.0 per cent).
2-Methylpropanol Not more than 1.0 per cent m/m, determined by gas chromatography (2.2.28)
using propanol R as the internal standard.
Internal standard solution. Dilute 2.0 ml of propanol R to 500.0 ml with water R.
Test solution (a). Dissolve 0.10 g of the substance to be examined in 1.0 ml of water R.
Test solution (b). Dissolve 0.20 g of the substance to be examined in 1.0 ml of the internal standard
solution and add 1.0 ml of water R.
Reference solution. To 0.500 g of 2-methylpropanol R add 1.0 ml of propanol R and dilute to 500.0 ml
with water R.
a column 1.5 m long and 4 mm in internal diameter packed with ethylvinylbenzenedivinylbenzene
copolymer R (150 m to 180 m),
maintaining the temperature of the column at 165C. Inject the selected volume of the test solutions
and of the reference solution.
Sterility (2.6.1). If intended for use in the manufacture of parenteral or ophthalmic dosage forms
without a further appropriate sterilisation procedure, it complies with the test for sterility.
ASSAY
STORAGE
Store in a well-closed container at a temperature not exceeding 25C. If the substance is sterile, store
in a sterile, airtight, tamper-proof container.
LABELLING
The label states:
__________________________________________________________________________________________________________ Ph Eur
40-40
Alpha Tocopherol
Me
Me
Me
Me
H Me
H Me
H Me
HO
Me
and enantiomer
C29H50O2
430.7
59-02-9
Alpha Tocopherol complies with the requirements of the 3rd edition of the European Pharmacopoeia for
-Tocopherol [0692]. These requirements are reproduced after the heading Definition below.
Action and use Used in prevention and treatment of vitamin E deficiencies.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
-Tocopherol contains not less than 96.0 per cent and not more than the equivalent of 102.0 per
cent of (2RS)-2,5,7,8-tetramethyl-2-[(4RS,8RS)-4,8,12-trimethyltridecyl]chroman-6-ol.
CHARACTERS
A clear, colourless or yellowish-brown, viscous, oily liquid, practically insoluble in water, freely
soluble in acetone, in ethanol, in ether, in methylene chloride and in fatty oils.
IDENTIFICATION
obtained with -tocopherol CRS.
C. Examine by thin-layer chromatography (2.2.27), using silica gel HF254 R as the coating substance.
Test solution. Dissolve 10 mg of the substance to be examined in 2 ml of cyclohexane R.
Reference solution. Dissolve 10 mg of -tocopherol CRS in 2 ml of cyclohexane R.
20 volumes of ether R and 80 volumes of cyclohexane R. Dry the plate in a current of air and examine
is similar in position and size to the principal spot in the chromatogram obtained with the reference
solution. Spray the plate with a mixture of 10 volumes of hydrochloric acid R, 40 volumes of a 2.5 g/l
solution of ferric chloride R in alcohol R and 40 volumes of a 10 g/l solution of phenanthroline hydrochloride R in alcohol R. After 1 h to 2 h the principal spots are orange.
D. It complies with the test for optical rotation (see Tests).
TESTS
Optical rotation (2.2.7). Dissolve 2.50 g in ethanol R and dilute to 25.0 ml with the same solvent.
Absorbance (2.2.25). Dissolve 0.100 g in ethanol R and dilute to 100.0 ml with the same solvent
(solution a). Dilute 10.0 ml of solution (a) to 100.0 ml with ethanol R (solution b). Measure the
absorbance of solution (b) at the maximum at 292 nm and of solution (a) at the minimum at
255 nm. The specific absorbance at the maximum is 72.0 to 76.0 and that at the minimum is 6.0 to
8.0.
Acid value (2.5.1). Not more than 2, determined on 2.00 g.
Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. Use sulphuric acid R
instead of dilute sulphuric acid R.
ASSAY
Examine by gas chromatography (2.2.28), using dotriacontane R as the internal standard.
Internal standard solution. Dissolve 0.20 g of dotriacontane R in hexane R and dilute to 100.0 ml with
the same solvent.
40-41
Test solution. Dissolve 0.100 g of the substance to be examined in the internal standard solution and
dilute to 50.0 ml with the internal standard solution.
Reference solution. Dissolve 0.100 g of -tocopherol CRS in the internal standard solution and dilute to
50.0 ml with the same solution.
a silanised glass column 2.0 m to 3.0 m long and 2.2 mm to 4.0 mm in internal diameter packed
with silanised diatomaceous earth for gas chromatography R (125 m to 150 m or 150 m to
180 m), impregnated with 1 per cent m/m to 5 per cent m/m of polydimethylsiloxane R; a plug of
silanised glass wool is placed at each end of the column,
nitrogen for chromatography R as the carrier gas at a flow rate of 25 ml/min to 90 ml/min,
maintaining the column at a constant temperature between 245C and 280C and the injection port
and the detector each at a constant temperature between 270C and 320C. Set the temperature of
the column and the flow rate of the carrier gas in such a manner that the required resolution is
achieved.
Make the injections directly onto the column or via an injection port (preferably glass-lined) using
an automatic injection device or some other reproducible injection method. Measure the peak areas
by electronic integration.
Resolution. Inject 1 l of the reference solution. Repeat this operation until the response factor (RF)
determined as described below is constant to within 2 per cent. The resolution (Rs) between the
dotriacontane peak and the -tocopherol peak is at least 2.6.
Interference test. Dissolve 0.100 g of the substance to be examined in hexane R and dilute to 50.0 ml
with the same solvent. Inject 1 l and record the chromatogram choosing an attenuation such that
the height of the peak corresponding to -tocopherol is greater than 50 per cent of the maximum
recorder response; during the recording, change the attenuation so that any peak appearing with the
same tR value as for dotriacontane is recorded with a sensitivity at least eight times greater than for
the -tocopherol peak. If a peak with a height of at least 5 mm for a recorder paper width of 250 mm
is detected with the same tR value as for dotriacontane, use the corrected peak area SD(corr) for the
final calculation.
SD(corr)
= SD
SI ST
f STI
SD = area of the peak corresponding to the internal standard in the chromatogram obtained
with the test solution,
SI = area of the interfering peak (same tR value as that of the internal standard) in the chromatogram obtained in the interference test,
ST = area of the peak corresponding to -tocopherol in the chromatogram obtained with the
test solution,
STI = area of the peak corresponding to -tocopherol in the chromatogram obtained in the
interference test,
f = factor by which the attenuation was changed.
Inject 1 l of the reference solution and record the chromatogram, choosing an attenuation such
that the peak corresponding to -tocopherol is greater than 50 per cent of the maximum recorder
response. Measure the areas of the peaks corresponding to -tocopherol (ST) and to dotriacontane
(SD) and determine the response factor (RF) as described below. Inject 1 l of the test solution in the
same manner. Measure the areas of the peaks corresponding to -tocopherol (ST) and to
dotriacontane (SD).
Determine the response factor (RF) for -tocopherol in the chromatogram obtained with the
reference solution from the areas of the peak corresponding to =-tocopherol and the peak corresponding to dotriacontane using the expression:
S D mT
S T mD
Calculate the percentage content of -tocopherol using the expression:
100 S T
mD RF
S D(corr.) m
with the reference solution,
SD (corr.) = corrected area of the peak corresponding to the internal standard in the chromatogram
ST = area of the peak corresponding to -tocopherol CRS in the chromatogram obtained with
the reference solution,
test solution,
40-42
mD = mass of the internal standard in the test solution and in the reference solution in
milligrams,
mT = mass of -tocopherol CRS in the reference solution in milligrams,
m = mass of the substance to be examined in the test solution in milligrams.
STORAGE
Store in an airtight container, under an inert gas, protected from light.
__________________________________________________________________________________________________________ Ph Eur
40-43
RRR-Alpha-Tocopherol
Me
Me
Me
Me
H
Me
H Me
H Me
HO
Me
C29H50O2
430.7
59-02-9
RRR-Alpha-Tocopherol complies with the requirements of the 3rd edition of the European Pharmacopoeia for
RRR--Tocopherol [1256]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
RRR--Tocopherol contains not less than 96.0 per cent and not more than the equivalent of
102.0 per cent of (2R)-2,5,7,8-tetramethyl-2-[(4R,8R)-4,8,12-trimethyltridecyl]chroman-6-ol.
CHARACTERS
A clear, colourless or yellowish-brown, viscous, oily liquid, practically insoluble in water, freely
soluble in acetone, in ethanol, in ether, in methylene chloride and in fatty oils.
IDENTIFICATION
obtained with -tocopherol CRS.
Test solution. Dissolve 10 mg of the substance to be examined in 2 ml of cyclohexane R.
Reference solution. Dissolve 10 mg of -tocopherol CRS in 2 ml of cyclohexane R.
solution. Spray the plate with a mixture of 10 volumes of hydrochloric acid R, 40 volumes of a 2.5 g/l
solution of ferric chloride R in alcohol R and 40 volumes of a 10 g/l solution of phenanthroline hydrochloride R in alcohol R. After 1 h to 2 h, the principal spots are orange.
D. RRR--tocopherol is dextrorotatory (2.2.7). The specific optical rotation after oxidation to the
quinone form is not less than +24.
Dissolve 1.0 g of the substance to be examined in 50 ml of ether R. To the solution add 20 ml of a
100 g/l solution of potassium ferricyanide R in an 8 g/l solution of sodium hydroxide R and shake for
3 min. Wash the ether solution with four quantities, each of 50 ml, of water R, discard the washings
and dry the ether layer over anhydrous sodium sulphate R. Evaporate the ether on a water-bath under
reduced pressure or in an atmosphere of nitrogen until a few millilitres remain, then complete the
evaporation removing the last traces of ether without the application of heat. Immediately dissolve
the residue in 5.0 ml of trimethylpentane R and determine the optical rotation.
Calculate the specific optical rotation of the substance in the test solution using as c the number of
grams of RRR--tocopherol in 1000 ml.
TESTS
Absorbance (2.2.25). Dissolve 0.100 g in ethanol R and dilute to 100 ml with the same solvent
(solution a). Dilute 10.0 ml of solution (a) to 100.0 ml with ethanol R (solution b). Measure the
absorbance of solution (b) at the maximum at 292 nm and of solution (a) at the minimum at
255 nm. The specific absorbance at the maximum is 72.0 to 76.0 and that at the minimum is 5.5 to
8.0.
40-44
ASSAY
the same solvent.
Test solution. Dissolve 0.100 g of the substance to be examined in 10.0 ml of the internal standard
solution, dilute to 50.0 ml with hexane R and mix.
Reference solution. Dissolve 0.100 g of -tocopherol CRS in 10.0 ml of the internal standard solution,
dilute to 50.0 ml with hexane R and mix.
a fused-silica column, 15 m long and 0.32 mm in internal diameter coated with a 0.25 m layer
of polydimethylsiloxane R,
helium for chromatography R as the carrier gas at a flow rate of 3 ml to 6 ml per minute,
maintaining the temperature of the injection port at 300C and that of the detector at 330C. Adjust
the split ratio between 1 to 10 and 1 to 20. Set the temperature of the column at 200C, then raise
the temperature at a rate of 5C per minute to 250C and maintain at 250C for 10 min.
Inject directly onto the column or via a glass-lined injection port using an automatic injection
device or some other reproducible injection method. Measure the peak areas by electronic integration. The test is not valid unless in the chromatogram obtained with the reference solution, the
resolution between the peaks corresponding to dotriacontane and -tocopherol is at least 9.0.
with the same solvent. Inject 1 l of the solution and record the chromatogram. If a peak is detected
with the same tR value as for dotriacontane, calculate the area of this peak relative to the peak area
of -tocopherol. If the relative peak area is greater than 0.5 per cent, use the corrected peak area
SD(corr.) for the final calculation.
SD(corr)
= SD
SI ST
STI
test solution,
STl = area of the peak corresponding to -tocopherol in the chromatogram obtained in the
interference test.
Once the system suitability has been established, inject 1 l of the reference solution and record the
chromatogram. Measure the areas of the peaks corresponding to -tocopherol (ST) and
dotriacontane (SD) and determine the response factor (RF) as described below.
Determine the response factor (RF) for -tocopherol in the chromatogram obtained with the
reference solution from the area of the peak corresponding to -tocopherol and the peak corresponding to dotriacontane using the expression:
RF =
SD mT
ST mD
Inject 1 l of the test solution in the same manner. Measure the areas of the peaks corresponding to
-tocopherol (ST) and dotriacontane (SD).
Calculate the percentage content of -tocopherol using the expression.
100( ST
mD RF
SD(corr)
m
SD(CORR) = corrected area of the peak corresponding to the internal standard in the chromatogram
ST = area of the peak corresponding to -tocopherol CRS in the chromatogram obtained with
the reference solution,
test solution,
mD = mass of the internal standard in the test solution and in the reference solution in milligrams,
40-45
mT = mass of -tocopherol CRS in the reference solution, in milligrams,
STORAGE
Store in an airtight container, under inert gas, protected from light.
__________________________________________________________________________________________________________ Ph Eur
40-46
Alpha Tocopheryl Acetate

Me
Me
Me
Me
H Me
H Me
H Me
AcO
Me
and enantiomer
C31H52O3
472.7
7695-91-2
Alpha Tocopheryl Acetate complies with the requirements of the 3rd edition of the European Pharmacopoeia
for -Tocopheryl Acetate [0439]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
-Tocopheryl acetate contains not less than 96.0 per cent and not more than the equivalent of
102.0 per cent of (2RS)-2,5,7,8-tetramethyl-2-[(4RS,8RS)-4,8,12-trimethyltridecyl]chroman-6-yl
acetate.
CHARACTERS
A clear, slightly greenish-yellow, viscous, oily liquid, practically insoluble in water, freely soluble in
acetone, in ethanol, in ether and in fatty oils, soluble in alcohol.
IDENTIFICATION
A. Dissolve 10 mg in ethanol R and dilute to 100 ml with the same solvent. Examined between
230 nm and 350 nm (2.2.25), the solution shows a maximum at 284 nm, a shoulder at 278 nm and a
minimum at 254 nm.
obtained with -tocopheryl acetate CRS.
Test solution (a). Dissolve about 10 mg of the substance to be examined in 2 ml of cyclohexane R.
Test solution (b). In a glass-stoppered test-tube, dissolve about 10 mg of the substance to be examined
in 2 ml of 2.5M alcoholic sulphuric acid R. Heat on a water-bath for 5 min. Cool and add 2 ml of
water R and 2 ml of cyclohexane R. Shake for 1 min. Use the upper layer.
Reference solution (a). Dissolve about 10 mg of -tocopheryl acetate CRS in 2 ml of cyclohexane R.
Reference solution (b). Prepare as prescribed for test solution (b), using -tocopheryl acetate CRS instead
in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with test solution (a)
solution (a). In the chromatograms obtained with test solution (b) and reference solution (b), there
are two spots: the spot with the higher Rf value is due to -tocopheryl acetate and corresponds to the
spot in the chromatogram obtained with reference solution (a); the spot with the lower Rf value is due
to -tocopherol. Spray the plate with a mixture of 10 volumes of hydrochloric acid R, 40 volumes of a
2.5 g/l solution of ferric chloride R in alcohol R and 40 volumes of a 10 g/l solution of phenanthroline
hydrochloride R in alcohol R. In the chromatograms obtained with test solution (b) and reference
solution (b), the spot due to -tocopherol is orange.
TESTS
Absorbance (2.2.25). Dissolve 0.150 g in ethanol R and dilute to 100.0 ml with the same solvent.
Dilute 10.0 ml of the solution to 100.0 ml with ethanol R (solution a). Dilute 20.0 ml of the initial
solution to 50.0 ml with ethanol R (solution b). Measure the absorbance of solution (a) at the maximum at 284 nm and the absorbance of solution (b) at the minimum at 254 nm. The specific absorbance at the maximum is 42.0 to 45.0 and that at the minimum is 7.0 to 9.0.
40-47
Free tocopherol Not more than 1.0 per cent. Dissolve 0.500 g in 100 ml of 0.25M alcoholic sulphuric
acid R. Add 20 ml of water R and 0.1 ml of a 2.5 g/l solution of diphenylamine R in sulphuric acid R.
Titrate with 0.01M ammonium and cerium sulphate until a blue colour is obtained which persists for at
least 5 s. Carry out a blank titration.
1 ml of 0.01M ammonium and cerium sulphate is equivalent to 2.154 mg of free tocopherol.
ASSAY
Internal standard solution. Dissolve 1.0 g of dotriacontane R in hexane R and dilute to 100.0 ml with the
same solvent.
Reference solution. Dissolve 0.100 g of -tocopheryl acetate CRS in 10.0 ml of the internal standard
with silanised diatomaceous earth for gas chromatography R (125 m to 150 m or 150 m to
180 m), impregnated with 1 per cent m/m to 5 per cent m/m of polydimethylsiloxane R; a plug of
silanised glass wool is placed at each end of the column,
nitrogen for chromatography R as the carrier gas at a flow rate of 25 ml/min to 90 ml/min,
Maintain the column at a constant temperature between 245C and 280C and the injection port
achieved.
Make the injections either directly onto the column or via a glass-lined injection port using an
automatic injection device or some other reproducible injection method. Measure the peak areas by
electronic integration.
determined as described below is constant to within 2 per cent. The resolution (Rs) must be
greater than 1.4.
with the same solvent. Inject 1 l and record the chromatogram choosing an attenuation such that
the height of the peak corresponding to -tocopheryl acetate is greater than 50 per cent of the
maximum recorder response; during the recording, change the attenuation so that any peak appearing with the same tR value as for dotriacontane is recorded with a sensitivity at least eight times
greater than for the -tocopheryl acetate peak. If a peak with a height of at least 5 mm for a recorder
paper width of 250 mm is detected with the same tR value as for dotriacontane, use the corrected
peak area SD(corr) if necessary, for the final calculation.
SD(corr)
= SD
SI ST
f STI
ST = area of the peak corresponding to -tocopherol acetate in the chromatogram obtained
STI = area of the peak corresponding to -tocopherol acetate in the chromatogram obtained in
the interference test,
After having verified the resolution of the column, inject 1 l of the reference solution and record the
chromatogram, choosing an attenuation such that the peak corresponding to -tocopherol acetate is
greater than 50 per cent of the maximum recorder response. Measure the areas of the peaks corresponding to -tocopheryl acetate (ST) and to dotriacontane (SD) and determine the response factor
(RF). Inject 1 l of the test solution in the same manner. Measure the areas of the peaks corresponding to -tocopheryl acetate (ST) and to dotriacontane (SD).
Determine the response factor (RF) for -tocopheryl acetate in the chromatogram obtained with the
reference solution from the areas of the peak corresponding to -tocopheryl acetate and the peak
40-48
corresponding to dotriacontane using the expression:
S D mT
S T mD
Calculate the percentage content of -tocopheryl acetate using the expression:
100 S T
mD RF
S D(corr.) m
SD(corr.) = corrected area of the peak corresponding to the internal standard in the chromatogram
ST = area of the peak corresponding to -tocopheryl acetate CRS in the chromatogram obtained
ST = area of the peak corresponding to -tocopheryl acetate in the chromatogram obtained
milligrams,
mT = mass of -tocopheryl acetate CRS in the reference solution in milligrams,
STORAGE
__________________________________________________________________________________________________________ Ph Eur
40-49
Alpha Tocopheryl Acetate Concentrate (Powder Form)

Alpha Tocopheryl Acetate Concentrate (Powder Form) complies with the requirements of the 3rd edition of
the European Pharmacopoeia for -Tocopherol Acetate Concentrate (Powder Form) [0691]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
-Tocopherol acetate concentrate (powder form) is prepared either by finely dispersing -Tocopherol
acetate (439) in a suitable carrier of suitable quality (for example gelatin, acacia, carbohydrates,
lactoproteins or a mixture thereof) or by adsorbing -Tocopherol acetate on silicic acid of suitable
quality.
The declared content of -tocopherol acetate is not less than 25 g per 100 g of concentrate. The
concentrate contains not less than 90.0 per cent and not more than 115.0 per cent of the content
stated on the label.
CHARACTERS
Almost white, yellowish or light-brown small particles which, depending on their formulation, may be
practically insoluble in water or may swell or form a dispersion.
IDENTIFICATION
Examine by thin-layer chromatography (2.2.27), using silica gel HF254 R as the coating substance.
Test solution. To a quantity of concentrate corresponding to 50 mg of -tocopherol acetate add 5 ml
of 0.01M hydrochloric acid and treat with ultrasound at 60C. Add 5 ml of ethanol R and 10 ml of
cyclohexane R, shake for 1 min and centrifuge for 5 min. Use the upper layer.
Reference solution. Dissolve 50 mg of -tocopherol acetate CRS in cyclohexane R and dilute to 10 ml with
the same solvent.
solution.
ASSAY
the same solvent.
Test solution. Weigh accurately a quantity of the preparation to be examined corresponding to about
0.100 g of -tocopherol acetate in a 250 ml conical flask. Add 20 ml of 1M hydrochloric acid and treat
with ultrasound at 70C for 20 min. Add 50 ml of ethanol R and 50.0 ml of the internal standard
solution and thoroughly mix the two phases for 30 min. Allow to separate, and use the upper layer.
Reference solution. Dissolve 0.100 g of -tocopherol acetate CRS in the internal standard solution and
dilute to 50.0 ml with the internal standard solution.
with diatomaceous earth for gas chromatography R (125 m to 150 m or 150 m to 180 m),
silanised with dimethyldichlorosilane and impregnated with 1 per cent m/m to 5 per cent m/m of
polydimethylsiloxane R; a plug of silanised glass wool is placed at each end of the column,
nitrogen for chromatography R as the carrier gas at a flow rate of 25 ml to 90 ml per minute,
maintaining the column at a constant temperature between 245C and 280C and the injection port
achieved.
Make the injections directly onto the column or via an injection port (preferably glass-lined) using
an automatic injection device or some other reproducible injection method. Measure the peak areas
by electronic integration.
determined as described below is constant to within 2 per cent. The resolution (RS) between the
dotriacontane peak and the -tocopherol acetate peak is at least 1.4.
40-50
Interference test. Weigh accurately a quantity of the substance to be examined corresponding to about
0.100 g of -tocopherol acetate in a 250 ml conical flask. Add 20 ml of 1M hydrochloric acid and treat
with ultrasound at 70C for 20 min. Add 50 ml of ethanol R and 50 ml of hexane R and thoroughly
mix the two phases for 30 min. Allow to separate. Inject 1 l of the upper layer and record the
chromatogram, choosing an attenuation such that the height of the peak corresponding to tocopherol acetate is greater than 50 per cent of the maximum recorder response; during the recording, change the attenuation so that any peak appearing with the same tR value as for dotriacontane is
recorded with a sensitivity at least eight times greater than for the -tocopherol acetate peak. If a peak
with a height of at least 5 mm for a recorder paper width of 250 mm is detected with the same tR
value as for dotriacontane, use the corrected peak area SD(corr.) for the final calculation.
SD(corr)
= SD
SI ST
f STI
STI = area of the peak corresponding to -tocopherol acetate in the chromatogram obtained in
the interference test,
Inject 1 l of the reference solution and record the chromatogram, choosing an attenuation such
that the peak corresponding to -tocopherol acetate is greater than 50 per cent of the maximum
recorder response. Measure the areas of the peaks corresponding to -tocopherol acetate (ST) and to
dotriacontane (SD) and determine the response factor (RF) as described below. Inject 1 l of the test
solution in the same manner. Measure the areas of the peaks corresponding to -tocopherol acetate
(ST) and to dotriacontane (SD).
Determine the response factor (RF) for -tocopherol acetate in the chromatogram obtained with
the reference solution from the areas of the peak corresponding to -tocopherol acetate and the peak
S D mT
S T mD
Calculate the percentage content of -tocopherol acetate using the expression:
100 S T
mD RF
S D(corr.) m
SD(corr.) = corrected area of the peak corresponding to the internal standard in the chromatogram
ST = area of the peak corresponding to -tocopherol acetate CRS in the chromatogram obtained
milligrams,
mT = mass of -tocopherol acetate CRS in the reference solution in milligrams,
STORAGE
LABELLING
The label states the content of -tocopherol acetate, expressed in grams per 100 g of concentrate.
__________________________________________________________________________________________________________ Ph Eur
40-51
RRR-Alpha-Tocopheryl Acetate
Me
Me
Me
Me
H Me
H Me
H Me
AcO
Me
C31H52O3
472.7
RRR-Alpha-Tocopheryl Acetate complies with the requirements of the 3rd edition of the European Pharmacopoeia for RRR--Tocopheryl Acetate [1257]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
RRR--Tocopheryl acetate contains not less than 96.0 per cent and not more than the equivalent of
102.0 per cent of (2R)-2,5,7,8-tetramethyl-2-[(4R,8R)-4,8,12-trimethyltridecyl]-chroman-6-yl
acetate.
CHARACTERS
A clear, pale greenish-yellow, viscous, oily liquid, practically insoluble in water, soluble in alcohol,
freely soluble in acetone, in ethanol, in ether and in fatty oils.
IDENTIFICATION
obtained with -tocopheryl acetate CRS.
Test solution (a). Dissolve 10 mg of the substance to be examined in 2 ml of cyclohexane R.
Test solution (b). In a ground glass-stoppered tube, dissolve 10 mg of the substance to be examined in
2 ml of 2.5M alcoholic sulphuric acid. Heat on a water-bath for 5 min. Cool and add 2 ml of water R
and 2 ml of cyclohexane R. Shake for 1 min. Use the upper layer.
Reference solution (a). Dissolve 10 mg of -tocopheryl acetate CRS in 2 ml of cyclohexane R.
Reference solution (b). Prepare as described for test solution (b), using -tocopheryl acetate CRS instead
in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with test solution (a)
solution (a). In the chromatograms obtained with test solution (b) and reference solution (b), there
are two spots: the spot with the higher Rf value is due to -tocopheryl acetate and corresponds to the
spot in the chromatogram obtained with reference solution (a); the spot with the lower Rf value is due
to -tocopherol. Spray the plate with a mixture of 10 volumes of hydrochloric acid R, 40 volumes of a
2.5 g/l solution of ferric chloride R in alcohol R and 40 volumes of a 10 g/l solution of phenanthroline
hydrochloride R in alcohol R. In the chromatograms obtained with test solution (b) and reference
solution (b), the spot due to -tocopherol is orange.
D. After saponification, the resulting RRR--tocopherol is dextrorotatory (2.2.7). The specific optical
rotation after oxidation to the quinone form is at least +24.
Carry out the test avoiding exposure to actinic light. Transfer 1.0 g of the substance to be examined to a
round-bottomed, ground glass-stoppered, 250 ml flask; dissolve in 30 ml of ethanol R and heat under
reflux for 3 min. While the solution is boiling, add, through the condenser, 20 ml of 2M alcoholic
potassium hydroxide solution R. Continue heating under reflux for 20 min and, without cooling, add
4.0 ml of hydrochloric acid R dropwise through the condenser. Cool, rinse the condenser with 10 ml of
ethanol R, transfer the contents of the flask to a 500 ml separating funnel, rinse the flask with four
quantities, each of 25 ml, of water R and four quantities, each of 25 ml, of ether R. Add the rinsings to
the separating funnel. Shake vigorously for 2 min, allow the layers to separate and collect each of the
two layers in individual separating funnels. Shake the aqueous layer with two quantities, each of
40-52
50 ml, of ether R and add these extracts to the main ether extract. Wash the combined ether extracts
with four quantities, each of 100 ml, of water R and discard the washings.
To the ether solution add 40 ml of a 100 g/l solution of potassium ferricyanide R in an 8 g/l solution
of sodium hydroxide R and shake for 3 min. Wash the ether solution with four quantities, each of
50 ml, of water R, discard the washings and dry the ether layer over anhydrous sodium sulphate R.
Evaporate the ether on a water-bath under reduced pressure or in an atmosphere of nitrogen until a
few millilitres remain, then complete the evaporation removing the last traces of ether without the
application of heat. Immediately dissolve the residue in 25.0 ml of trimethylpentane R and determine
the optical rotation.
grams equivalent to RRR--tocopherol (factor 0.911) in 1000 ml.
TESTS
Absorbance (2.2.25). Dissolve 0.150 g in ethanol R and dilute to 100 ml with the same solvent.
Dilute 10.0 ml of the solution to 100.0 ml with ethanol R (solution A). Dilute 20.0 ml of the initial
solution to 50.0 ml with ethanol R (solution B).
Measure the absorbance of solution (A) at the maximum at 284 nm and the absorbance of solution
(B) at the minimum at 254 nm. The specific absorbance at the maximum is 42.0 to 45.0 and that at
the minimum is 7.0 to 9.0.
acid R. Add 20 ml of water R and 0.1 ml of a 2.5 g/l solution of diphenylamine R in sulphuric acid R.
ASSAY
the same solvent.
Reference solution. Dissolve 0.100 g of -tocopheryl acetate CRS in 10.0 ml of the internal standard
a fused-silica column 15 m long and 0.32 mm in internal diameter coated with a 0.25 m layer
of polydimethylsiloxane R,
device or some other reproducible injection method. Measure the peak areas by electronic integration. The test is not valid unless: in the chromatogram obtained with the reference solution, the
resolution between the peaks corresponding to dotriacontane and -tocopheryl acetate is at least 4.0.
of -tocopheryl acetate. If the relative peak area is greater than 0.5 per cent, use the corrected peak
area SD(corr.) for the final calculation.
SD(corr)
= SD
SI ST
STI
40-53
STl = area of the peak corresponding to -tocopheryl acetate in the chromatogram obtained in
the interference test.
After the system suitability has been established, inject 1 l of the reference solution and record the
chromatogram. Measure the areas of the peaks corresponding to -tocopheryl acetate (ST) and
dotriacontane (SD) and determine the response factor (RF) as described below.
Determine the response factor (RF) for -tocopheryl acetate in the chromatogram obtained with
the reference solution from the areas of the peak corresponding to -tocopheryl acetate and the peak
RF =
SD mT
ST mD
Inject 1 l of the test solution in the same manner. Measure the areas of the peaks corresponding
to -tocopheryl acetate (ST) and dotriacontane (SD).
Calculate the percentage content of RRR--tocopheryl acetate using the expression:
100( ST
mD RF
SD(corr)
m
ST = area of the peak corresponding to -tocopheryl acetate CRS in the chromatogram obtained
mT = mass of -tocopheryl acetate CRS in the reference solution, in milligrams,
STORAGE
__________________________________________________________________________________________________________ Ph Eur
40-54
Alpha Tocopheryl Hydrogen Succinate

Me
Me
Me
Me
HOOC
H
O
Me
H Me
Me
Me
and enantiomer
C33H54O5
530.8
Alpha Tocopheryl Hydrogen Succinate complies with the requirements of the 3rd edition of the European
Pharmacopoeia for DL--Tocopheryl Hydrogen Succinate [1258]. These requirements are reproduced after the

Ph Eur ___________________________________________________________________________________________________________
DEFINITION
DL--Tocopheryl hydrogen succinate contains not less than 96.0 per cent and not more than the
equivalent of 102.0 per cent of (2RS)-2,5,7,8-tetramethyl-2-[(4RS,8RS)(4,8,12-trimethyltridecyl)chroman-6-yl hydrogen succinate.
CHARACTERS
ethanol and in ether, very soluble in methylene chloride.
IDENTIFICATION
obtained with RRR--tocopheryl hydrogen succinate CRS.
2 ml of 2.5M alcoholic sulphuric acid. Heat on a water-bath for 5 min. Cool and add 2 ml of water R
Reference solution (a). Dissolve 10 mg of RRR--tocopheryl hydrogen succinate CRS in 2 ml of cyclohexane R.
Reference solution (b). Prepare as described for test solution (b), using RRR--tocopheryl hydrogen
succinate CRS instead of the substance to be examined.
0.2 ml of glacial acetic acid R, 20 volumes of ether R and 80 volumes of cyclohexane R. Dry the plate in
a current of air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram
obtained with test solution (a) is similar in position and size to the principal spot in the chromatogram obtained with the reference solution (a). In the chromatograms obtained with test solution (b)
and reference solution (b), there are two spots: the spot with the higher Rf value is due to
-tocopherol, the spot with the lower Rf value is due to -tocopheryl acid succinate and corresponds
to the spot obtained with reference solution (a). Depending on the degree of hydrolysis, the lower
spot may be weak or even absent. Spray the plate with a mixture of 10 volumes of hydrochloric acid R,
40 volumes of a 2.5 g/l solution of ferric chloride R in alcohol R and 40 volumes of a 10 g/l solution of
phenanthroline hydrochloride R in alcohol R. In the chromatograms obtained with test solution (b) and
reference solution (b), the spot due to -tocopherol is orange.
TESTS
solution to 50.0 ml with ethanol R (solution b). Measure the absorbance of solution (a) at the maximum at 284 nm and the absorbance of solution (b) at the minimum at 254 nm. The specific absorbance at the maximum is 35 to 38 and that at the minimum is 6.0 to 8.0.
40-55
Acid value (2.5.1). Between 101 and 108, determined on 1.00 g.
acid. Add 20 ml of water R and 0.1 ml of a 2.5 g/l solution of diphenylamine R in sulphuric acid R.
ASSAY
the same solvent.
Test solution. Weigh 30.0 mg of the substance to be examined into a 20 ml vial. Pipette 2.0 ml of
methanol R, 1.0 ml of dimethoxypropane R and 0.1 ml of hydrochloric acid R into the vial. Cap tightly
and sonicate the sample. Allow to stand in the dark for 1 h ( 5 min). Remove the sample from the
dark and place on a steam bath under a nitrogen blanket for 10 min. Pipette 10.0 ml of the internal
standard solution into the sample vial. Vortex into solution.
Reference solution. Weigh 30.0 mg of RRR--tocopheryl hydrogen succinate CRS into a 20 ml vial, weigh
to the nearest 0.01 mg. Pipette 2.0 ml of methanol R, 1.0 ml of dimethoxypropane R and 0.1 ml of
hydrochloric acid R into the vial. Cap tightly and sonicate the reference solution. Allow to stand in the
dark for 1 h ( 5 min). Remove the reference solution from the dark and place on a steam bath under
a nitrogen blanket for 10 min. Pipette 10.0 ml of the internal standard solution into the reference
solution vial. Vortex into solution.
a fused-silica open tubular capillary column 15 m long and 0.32 mm in internal diameter coated
with a 0.25 m layer of polydimethylsiloxane R,
resolution between the peaks corresponding to dotriacontane and -tocopherol acid succinate is at
least 12.0.
of -tocopheryl succinate. If the relative peak area is greater than 0.5 per cent, use the corrected peak
SD(corr)
= SD
SI ST
STI
ST = area of the peak corresponding to -tocopheryl hydrogen succinate in the chromatogram
STl = area of the peak corresponding to -tocopheryl hydrogen succinate in the chromatogram
obtained in the interference test.
chromatogram. Measure the areas of the peaks corresponding the -tocopheryl hydrogen succinate
(ST) and dotriacontane (SD) and determine the response factor (RF) as described below.
Determine the response factor (RF) for -tocopheryl hydrogen succinate in the chromatogram
obtained with the reference solution from the area of the peak corresponding to -tocopheryl
hydrogen succinate and the peak corresponding to dotriacontane using the expression:
40-56
RF =
SD mT
ST mD
to -tocopheryl hydrogen succinate (ST) and dotriacontane (SD).
Calculate the percentage content of -tocopheryl acid succinate using the expression:
100( ST
mD RF
SD(corr)
m
ST = area of the peak corresponding to RRR-tocopheryl hydrogen succinate CRS in the chromatogram obtained with the reference solution,
ST = area of the peak corresponding to DL-tocopheryl hydrogen succinate in the chromatogram obtained with the test solution,
mT = mass of RRR--tocopheryl hydrogen succinate CRS in the reference solution, in milligrams,
STORAGE
__________________________________________________________________________________________________________ Ph Eur
40-57
RRR-Alpha Tocopheryl Hydrogen Succinate

Me
Me
Me
Me
HOOC
H Me
O
H Me
Me
Me
C33H54O5
530.8
4345-03-3
RRR-Alpha Tocopheryl Hydrogen Succinate complies with the requirements of the 3rd edition of the European
Pharmacopoeia for RRR--Tocopheryl Hydrogen Succinate [1259]. These requirements are reproduced after
the heading Definition below.
Preparation
Alpha Tocopheryl Succinate Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
RRR--Tocopheryl hydrogen succinate contains not less than 96.0 per cent and not more than the
equivalent of 102.0 per cent of (2R)-2,5,7,8-tetramethyl-2-[(4R,8R)-4,8,12-trimethyltridecyl]chroman-6-yl hydrogen succinate.
CHARACTERS
ethanol and in ether, very soluble in methylene chloride.
IDENTIFICATION
obtained with RRR--tocopheryl hydrogen succinate CRS.
2 ml of 2.5M alcoholic sulphuric acid. Heat on a water bath for 5 min. Cool and add 2 ml of water R
Reference solution (a). Dissolve 10 mg of RRR--tocopheryl hydrogen succinate CRS in 2 ml of cyclohexane R.
Reference solution (b). Prepare as described for test solution (b), using RRR--tocopheryl hydrogen
succinate CRS instead of the substance to be examined.
0.2 ml of glacial acetic acid R, 20 volumes of ether R and 80 volumes of cyclohexane R. Dry the plate in
a current of air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram
obtained with test solution (a) is similar in position and size to the principal spot in the chromatogram obtained with the reference solution (a). In the chromatograms obtained with test solution (b)
and reference solution (b), there are two spots: the spot with the higher Rf value is due to
-tocopherol, the spot with the lower Rf value is due to -tocopheryl hydrogen succinate and corresponds to the spot obtained with reference solution (a). Depending on the degree of hydrolysis, the
lower spot may be weak or even absent. Spray the plate with a mixture of 10 volumes of hydrochloric
acid R, 40 volumes of a 2.5 g/l solution of ferric chloride R in alcohol R and 40 volumes of a 10 g/l
solution of phenanthroline hydrochloride R in alcohol R. In the chromatograms obtained with test
solution (b) and reference solution (b), the spot due to -tocopherol is orange.
D. After saponification, the resulting RRR--tocopherol is dextrorotatory (2.2.7). The specific optical
rotation after oxidation to the quinone form is at least +24.
Carry out the test avoiding exposure to actinic light. Transfer 1.0 g of the substance to be examined to a
round-bottomed, ground-glass-stoppered, 250 ml flask and dissolve in 30 ml of ethanol R and heat
under reflux for 3 min. While the solution is boiling, add, through the condenser, 20 ml of 2M
alcoholic potassium hydroxide solution R. Continue heating under reflux for 20 min and, without cooling, add 4.0 ml of hydrochloric acid R dropwise through the condenser. Cool, rinse the condenser with
10 ml of ethanol R, transfer the contents of the flask to a 500 ml separating funnel, rinse the flask with
40-58
four quantities, each of 25 ml, of water R and four quantities, each of 25 ml, of ether R. Add the
rinsings to the separating funnel. Shake vigorously for 2 min, allow the layers to separate and collect
each of the two layers in individual separating funnels. Shake the aqueous layer with two quantities,
each of 50 ml, of ether R and add these extracts to the main ether extract. Wash the combined ether
extracts with four quantities, each of 100 ml, of water R and discard the washings.
To the ether solution add 40 ml of a 100 g/l solution of potassium ferricyanide R in an 8 g/l solution
of sodium hydroxide R and shake for 3 min. Wash the ether solution with four quantities, each of
50 ml, of water R, discard the washings and dry the ether layer over anhydrous sodium sulphate R.
Evaporate the ether on a water-bath under reduced pressure or in an atmosphere of nitrogen until a
few millilitres remain, then complete the evaporation removing the last traces of ether without the
application of heat. Immediately dissolve the residue in 25.0 ml of trimethylpentane R and determine
the optical rotation.
grams equivalent to -tocopherol (factor 0.811) in 1000 ml.
TESTS
solution to 50.0 ml with ethanol R (solution b). Measure the absorbance of solution (a) at the maximum at 284 nm and the absorbance of solution (b) at the minimum at 254 nm. The specific absorbance at the maximum is 35 to 38 and that at the minimum is 6.0 to 8.0.
Acid value (2.5.1). Between 101 and 108, determined on 1.00 g.
acid. Add 20 ml of water R and 0.1 ml of a 2.5 g/l solution of diphenylamine R in sulphuric acid. Titrate
with 0.01M ammonium and cerium sulphate until a blue colour is obtained which persists for at least
5 s. Carry out a blank titration.
ASSAY
the same solvent.
Test solution. Weigh 30.0 mg of the substance to be examined into a 20 ml vial. Pipette 2.0 ml of
methanol R, 1.0 ml of dimethoxypropane R and 0.1 ml of hydrochloric acid R into the vial. Cap tightly
and sonicate the sample. Allow to stand in the dark for 1 h ( 5 min). Remove the sample from the
dark and place on a steam bath under a nitrogen blanket for 10 min. Pipette 10.0 ml of the internal
standard solution into the sample vial. Vortex into solution.
Reference solution. Weigh 30.0 mg of RRR--tocopheryl hydrogen succinate CRS into a 20 ml vial.
Pipette 2.0 ml of methanol R, 1.0 ml of dimethoxypropane R and 0.1 ml of hydrochloric acid R into the
vial. Cap tightly and sonicate the reference solution. Allow to stand in the dark for 1 h ( 5
min). Remove the reference solution from the dark and place on a steam bath under a nitrogen
blanket for 10 min. Pipette 10.0 ml of the internal standard solution into the reference solution vial.
Vortex into solution.
a fused-silica open tubular capillary column 15 m long and 0.32 mm in internal diameter coated
with a 0.25 m layer of polydimethylsiloxane R,
resolution between the peaks corresponding to dotriacontane and -tocopherol hydrogen succinate is
at least 12.0.
Interference test. Dissolve 0.100 g of the solution to be examined in hexane R and dilute to 50.0 ml
40-59
of -tocopheryl succinate. If the relative peak area is greater than 0.5 per cent, use the corrected peak
SD(corr)
= SD
SI ST
STI
STI = area of the peak corresponding to -tocopheryl hydrogen succinate in the chromatogram
obtained in the interference test.
chromatogram. Measure the areas of the peaks corresponding the -tocopheryl hydrogen succinate
(ST) and dotriacontane (SD) and determine the response factor (RF) as described below.
Determine the response factor (RF) for -tocopheryl hydrogen succinate in the chromatogram
obtained with the reference solution from the area of the peak corresponding to -tocopheryl
hydrogen succinate and the peak corresponding to dotriacontane using the expression:
RF =
SD mT
ST mD
to -tocopheryl hydrogen succinate (ST) and dotriacontane (SD).
Calculate the percentage content of -tocopheryl hydrogen succinate using the expression:
100( ST
mD RF
SD(corr)
m
ST = area of the peak corresponding to RRR--tocopheryl hydrogen succinate CRS in the chromatogram obtained with the reference solution,
mT = mass of RRR--tocopheryl hydrogen succinate CRS in the reference solution, in milligrams,
STORAGE
__________________________________________________________________________________________________________ Ph Eur
41-1
Tolazamide
O
O
S
N
H
N
H
Me
C14H21N3O3S
311.4
1156-19-0
Definition Tolazamide is 1-perhydroazepin-1-yl-3-tolyl-p-sulphonylurea. It contains not less than

98.0% and not more than 101.0% of C14H21N3O3S, calculated with reference to the dried substance.
Very slightly soluble in water; freely soluble in chloroform; soluble in acetone; slightly soluble in
ethanol (96%).
Identification
tolazamide (RS 342).
B. The light absorption, Appendix II B, in the range 230 to 350 nm of a 0.04% w/v solution in ethanol
(96%) exhibits maxima at 256, 263 and 275 nm and a shoulder at 268 nm. The absorbances at the
maxima are about 0.78, about 0.83 and about 0.62, respectively.
Heavy metals Moisten the residue obtained in the test for Sulphated ash with 1 ml of hydrochloric
acid, evaporate to dryness and dissolve the residue in 20 ml of water. 12 ml of the resulting solution
complies with limit test A for heavy metals, Appendix VII. Use lead standard solution (1 ppm Pb) to
prepare the standard (20 ppm).
gel G as the coating substance and a mixture of 200 volumes of chloroform, 100 volumes of methanol,
60 volumes of cyclohexane and 23 volumes of 13.5M ammonia as the mobile phase. Apply separately
to the plate 10 l of each of two solutions in acetone containing (1) 2.0% w/v of the substance being
examined and (2) 0.010% w/v of toluene-p-sulphonamide. After removal of the plate, dry it in a current
of cold air, heat at 110 for 10 minutes, place the hot plate in a tank of chlorine gas prepared by the
addition of hydrochloric acid to a 5% w/v solution of potassium permanganate contained in a beaker
placed in the tank and allow to stand for 2 minutes. Dry it in a current of cold air until an area of the
plate below the line of application gives at most a very faint blue colour with a 0.5% w/v solution of
potassium iodide in starch mucilage; avoid prolonged exposure to cold air. Spray the plate with a 0.5%
w/v solution of potassium iodide in starch mucilage. Any secondary spot in the chromatogram obtained
with solution (1) is not more intense than the spot in the chromatogram obtained with solution (2).
Assay Dissolve 0.5 g in 20 ml of butan-2-one with the aid of gentle heat. Allow to cool, add 30 ml of
ethanol (96%) and titrate with 0.1M sodium hydroxide VS using phenolphthalein solution R1 as indicator.
Each ml of 0.1M sodium hydroxide VS is equivalent to 31.14 mg of C14H21N3O3S.
Storage Tolazamide should be kept in a well-closed container.
Preparation
Tolazamide Tablets
41-2
Tolbutamide
O
O
S
N
H
NHBun
Me
C12H18N2O3S
270.3
64-77-7
Tolbutamide complies with the requirements of the 3rd edition of the European Pharmacopoeia [0304]. These
Preparation
Tolbutamide Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Tolbutamide contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of 3-butyl-1-[(4-methylphenyl)sulphonyl]urea, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder, practically insoluble in water, soluble in acetone, in alcohol, slightly
soluble in ether. It dissolves in dilute solutions of alkali hydroxides.
IDENTIFICATION
B. Dissolve 25.0 mg in methanol R and dilute to 100.0 ml with the same solvent. Examined between
245 nm and 300 nm (2.2.25), the solution shows three absorption maxima, at 258 nm, 263 nm and
275 nm, and a shoulder at 268 nm. Dilute 10.0 ml of the solution to 250.0 ml with methanol R.
Examined between 220 nm and 235 nm, the solution shows a single absorption maximum, at
228 nm. The specific absorbance at this maximum is 480 to 520.
obtained with tolbutamide CRS.
D. To 0.2 g add 8 ml of a 500 g/l solution of sulphuric acid R and heat under a reflux condenser for
30 min. Allow to cool. Crystals are formed which, after recrystallisation from hot water R and drying
at 100C to 105C, melt (2.2.14) at 135C to 140C.
TESTS
Appearance of solution Dissolve 0.2 g in 5 ml of dilute sodium hydroxide solution R and add 5 ml of
water R. The solution is clear (2.2.1) and colourless (Method II, 2.2.2).
pH (2.2.3). To 2.0 g add 50 ml of carbon dioxide-free water R and heat at 70C for 5 min. Cool
rapidly and filter. The pH of the filtrate is 4.5 to 5.5.
coating substance.
Test solution. Dissolve 0.50 g of the substance to be examined in acetone R and dilute to 10 ml with
the same solvent.
Reference solution (a). Dissolve 15 mg of toluenesulphonamide R in acetone R and dilute to 100 ml with
the same solvent.
Reference solution (b). To 5 ml of the test solution add 5 ml of reference solution (a).
Apply separately to the plate 5 l of the test solution and of reference solution (a) and 10 l of reference solution (b). Develop over a path of 15 cm using a mixture of 2 volumes of anhydrous formic
acid R, 8 volumes of methanol R and 90 volumes of chloroform R. Dry the plate in a current of warm
air and heat at 110C for 10 min. At the bottom of a chromatography tank, place an evaporating dish
containing a 50 g/l solution of potassium permanganate R and add an equal volume of hydrochloric
acid R. Place the plate whilst still hot in the tank and close the tank. Leave the plate in contact with
the chlorine vapour for 2 min. Withdraw the plate and place it in a current of cold air until the excess
of chlorine is removed and an area of coating below the points of application gives at most a very
41-3
faint blue colour with a drop of potassium iodide and starch solution R; avoid prolonged exposure to
cold air. Spray with potassium iodide and starch solution R and allow to stand for 5 min. Any spot in the
the spot in the chromatogram obtained with reference solution (a) (0.3 per cent). The test is not valid
obtained by diluting lead standard solution (100 ppm Pb) R with a mixture of 15 volumes of water R
and 85 volumes of acetone R.
100C to 105C.
ASSAY
Dissolve 0.2500 g in a mixture of 20 ml of water R and 40 ml of alcohol R. Titrate with 0.1M sodium
hydroxide, using 1 ml of phenolphthalein solution R as indicator.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
41-4
Tolnaftate
Me
Me
O
S
C19H17NOS
307.4
2398-96-1
Tolnaftate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1158]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Tolnaftate contains not less than 97.0 per cent and not more than the equivalent of 103.0 per cent of
O-(2-naphthyl) N-methyl-N-(3-methylphenyl)thiocarbamate, calculated with reference to the dried
substance.
CHARACTERS
A white or yellowish-white powder, practically insoluble in water, freely soluble in acetone and in
methylene chloride, sparingly soluble in ether, very slightly soluble in alcohol.
IDENTIFICATION
obtained with tolnaftate CRS. Examine the substances prepared as discs.
D. Mix about 1 mg with 0.5 ml of sulphuric acid R. Add 0.05 ml of formaldehyde solution R. A
greenish-blue colour develops.
TESTS
Appearance of solution Dissolve 0.5 g in 10 ml of acetone R. The solution is clear (2.2.1) and not
coating substance.
the same solvent.
Test solution (b). Dilute 0.5 ml of test solution (a) to 10 ml with acetone R.
Reference solution (a). Dissolve 25 mg of tolnaftate CRS in acetone R and dilute to 10 ml with the same
solvent.
Reference solution (c). Dissolve 50 mg of -naphthol R in 1 ml of test solution (a) and dilute to 10 ml
with acetone R.
Apply separately to the plate 5 l of each solution. Develop over a path of 12 cm using toluene R.
Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with test solution (a), apart from the principal spot, is not more intense than the
spot in the chromatogram obtained with reference solution (b) (0.5 per cent). The test is not valid
unless the chromatogram obtained with reference solution (c) shows two clearly separated spots.
41-5
ASSAY
Dissolve 50.0 mg in methanol R and dilute to 250.0 ml with the same solvent. Dilute 2.0 ml to
50.0 ml with methanol R. Measure the absorbance (2.2.25) at the maximum at 257 nm.
Calculate the content of C19H17NOS taking the specific absorbance to be 720.
STORAGE
IMPURITIES
OH
A. -naphthol (2-naphthol),
O
O
S
B. O,O-di(2-naphthyl)thiocarbonate,
O
Cl
S
C. O-(2-naphthyl)chlorothioformate,
MeHN
Me
D. methyl(3-methylphenyl)amine.
__________________________________________________________________________________________________________ Ph Eur
41-6
Tormentil
Tormentil complies with the requirements of the 3rd edition of the European Pharmacopoeia [1478]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Tormentil consists of the whole or cut, dried rhizome, freed from the roots, of Potentilla erecta
(L.) Raeusch. (P. tormentilla Stokes). It contains not less than 7 per cent of tannins expressed as
pyrogallol (C6H6O3; Mr 126.1) calculated with reference to the dried drug.
CHARACTERS
IDENTIFICATION
A. The rhizome is cylindrically spindle-shaped, with a very irregular appearance, often forming,
twisted, knotty tubers, up to 10 cm long and 1 cm to 2 cm thick, very hard and scarcely branched.
The surface is brown to reddish-brown, rugose and has remains of roots and transversely elongated
depressed whitish scars from the stems. At the top of the rhizome the remains of numerous aerial
stems may be present. The fracture is short and granular, dark red to brownish-yellow; the smoothed
transverse surface shows a cambium line separating the narrow outer region from an indistinctly
radiate xylem, with widely-separated small groups of lignified tissue arranged in concentric rings,
surrounding a large central pith.
B. Reduce to a powder (355). The powder is reddish-brown. Examine under a microscope, using
chloral hydrate solution R. The powder shows coarsely serrate cluster crystals of calcium oxalate, up to
60 m in diameter; fragments of thin-walled parenchyma containing reddish-brown tannin; groups of
narrow, bordered-pitted vessels with lateral pores; thick-walled and pitted, polygonal parenchyma;
groups and fragments of sclerenchymatous thick-walled fibres; occasional fragments of cork with
thin-walled, brown, tabular cells. Examine under a microscope using a 50 per cent V/V solution of
glycerol R. The powder shows spherical or elliptical starch granules, up to about 20 m in length.
Test solution. Shake 0.5 g of the powdered drug (355) with 10 ml of water R for 10 min and filter.
Shake the filtrate with two quantities, each of 10 ml, of ethyl acetate R and filter the combined upper
phases over 6 g of anhydrous sodium sulphate R. Evaporate the filtrate to dryness at reduced pressure
and dissolve the residue in 1.0 ml of ethyl acetate R.
Reference solution. Dissolve 1.0 mg of catechin R in 1.0 ml of methanol R.
20 volumes of glacial acetic acid R, 20 volumes of ether R, 20 volumes of hexane R and 40 volumes of
ethyl acetate R. Allow the plate to dry in air for 10 min to 15 min. Spray the plate with a freshly
prepared 5 g/l solution of fast blue B salt R. Reddish zones appear. Expose the plate to ammonia
vapour, the zones become more intense turning reddish-brown. Examine in daylight. The chromatogram obtained with the reference solution shows in the upper third an intense zone (catechin). The
chromatogram obtained with the test solution also shows a zone due to catechin which is usually
stronger than the zone of catechin in the chromatogram obtained with the reference solution; below it
an intense zone and other fainter zones are present.
TESTS
Foreign matter (2.8.2). Not more than 3 per cent of root and stems as well as rhizomes with black
fracture and not more than 2 per cent of other foreign matter.
ASSAY
Carry out the determination of tannins in herbal drugs (2.8.14). Use 0.500 g of the powdered drug
(180).
STORAGE
__________________________________________________________________________________________________________ Ph Eur
41-7
Tosylchloramide Sodium
O
ONa
S
N
Cl
Me
C7H7ClNNaO2S,3H2O 281.7
127-65-1
Tosylchloramide Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia
for Chloramine [0381]. These requirements are reproduced after the heading Definition below.
Action and use Antiseptic; disinfectant.
When chloramine is prescribed or demanded, Tosylchloramide Sodium shall be dispensed or
supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Chloramine contains not less than 98.0 per cent and not more than the equivalent of 103.0 per cent
of sodium N-chloro-4-methylbenzene-sulphonimidate trihydrate.
CHARACTERS
A white or slightly yellow, crystalline powder, freely soluble in water, soluble in alcohol, practically
insoluble in ether.
IDENTIFICATION
A. Solution S (see Tests) turns red litmus paper R blue and then bleaches it.
B. To 10 ml of solution S add 10 ml of dilute hydrogen peroxide solution R. A white precipitate is
formed which dissolves on heating. Filter the hot solution and allow to cool. White crystals are
formed which, when washed and dried at 100C to 105C, melt (2.2.14) at 137C to 140C.
C. Ignite 1 g (cautiously, because of the risk of deflagration). Dissolve the residue in 10 ml of
water R. The solution gives reaction (a) of chlorides (2.3.1).
D. The solution prepared for identification test C gives reaction (a) of sulphates (2.3.1).
E. The solution prepared for identification test C gives reaction (b) of sodium (2.3.1).
TESTS
Ortho compound To 2 g add 10 ml of water R, mix, add 1 g of sodium metabisulphite R and heat to
boiling. Cool to OC, filter rapidly and wash with three quantities, each of 5 ml, of iced water R. The
precipitate, dried over diphosphorus pentoxide R at a pressure not exceeding 600 Pa melts (2.2.14) at a
temperature not lower than 134C.
Residue insoluble in ethanol Shake 1.00 g for 30 min with 20 ml of ethanol R, filter on a tared
filter, wash any residue with 5 ml of ethanol R and dry at 100C to 105C. The residue weighs not
more than 20 mg (2 per cent).
ASSAY
Dissolve 0.125 g in 100 ml of water R in a ground-glass-stoppered flask. Add 1 g of potassium iodide R
and 5 ml of dilute sulphuric acid R. Allow to stand for 3 min. Titrate with 0.1M sodium thiosulphate,
using 1 ml of starch solution R as indicator.
1 ml of 0.1M sodium thiosulphate is equivalent to 14.08 mg of C7H7ClNNaO2S,3H2O.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
41-8
Tragacanth
Tragacanth complies with the requirements of the 3rd edition of the European Pharmacopoeia [0532]. These
When Powdered Tragacanth is prescribed or demanded, material complying with the requirements
below with the exception of Identification test A shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Tragacanth is the air-hardened, gummy exudate, flowing naturally or obtained by incision from the
trunk and branches of Astragalus gummifer Labill. and certain other species of Astragalus from western
Asia.
CHARACTERS
IDENTIFICATION
A. Tragacanth occurs in thin, flattened, ribbon-like, white or pale yellow, translucent strips, about
30 mm long and 10 mm wide and up to 1 mm thick, more or less curved; horny, fracture short; the
surface is marked by fine longitudinal striae and concentric transverse ridges. It may also contain
pieces similar in shape but somewhat thicker, more opaque and more difficult to fracture.
B. Reduce to a powder (355). The powder is white or almost white and it forms a mucilaginous gel
with about ten times its mass of water R. Examine under a microscope using a 50 per cent V/V
solution of glycerol R. The powder shows in the gummy mass numerous stratified cellular membranes
turning slowly violet when treated with iodinated zinc chloride solution R. The gummy mass includes
starch grains, isolated or in small groups, usually rounded in shape and sometimes deformed, with
diameters varying between 4 m and 10 m, occasionally up to 20 m, and a central hilum visible
between crossed nicol prisms.
C. Examine the chromatograms obtained in the test for acacia. The chromatogram obtained with the
test solution shows three zones, corresponding to galactose, arabinose and xylose. A faint yellowish
zone at the solvent front and a greyish-green zone between the zones due to galactose and arabinose
may be present.
D. Moisten 0.5 g of the powdered drug (355) with 1 ml of alcohol R and add gradually, while
shaking, 50 ml of water R until a homogeneous mucilage is obtained. To 5 ml of the mucilage add
5 ml of water R and 2 ml of barium hydroxide solution R. A slight flocculent precipitate is formed. Heat
on a water-bath for 10 min. An intense yellow colour develops.
TESTS
Acacia Examine by thin-layer chromatography (2.2.27), using a TLC silica gel plate R.
Test solution. To 100 mg of the powdered drug (355) in a thick-walled, centrifuge test tube, add 2 ml
of a 100 g/l solution of trifluoroacetic acid R, shake vigorously to dissolve the forming gel, stopper the
test tube and heat the mixture at 120C for 1 h. Centrifuge the hydrolysate, transfer the clear
supernatant carefully into a 50 ml flask, add 10 ml of water R and evaporate the solution to dryness
under reduced pressure. To the resulting clear film add 0.1 ml of water R and 0.9 ml of methanol R.
Centrifuge to separate the amorphous precipitate and dilute the supernatant if necessary to 1 ml with
methanol R.
Reference solution. Dissolve 10 mg of arabinose R, 10 mg of galactose R, 10 mg of rhamnose R and
10 mg of xylose R in 1 ml of water R and dilute to 10 ml with methanol R.
10 volumes of a 16 g/l solution of sodium dihydrogen phosphate R, 40 volumes of butanol R and 50
volumes of acetone R. Dry the plate in a current of warm air for a few minutes and again develop over
a path of 15 cm using the same mobile phase. Dry the plate at 110C for 10 min, spray with
anisaldehyde solution R and dry again at 110C for 10 min. The chromatogram obtained with the
reference solution shows four clearly separated coloured zones due to galactose (greyish-green to
green), arabinose (yellowish-green), xylose (greenish-grey or yellowish-grey) and rhamnose
(yellowish-green), in order of increasing Rf value. The chromatogram obtained with the test solution
does not show a yellowish-green zone corresponding to the zone of rhamnose in the chromatogram
Methylcellulose Examine the chromatograms obtained in the test for acacia. The chromatogram
obtained with the test solution does not show a red zone near the solvent front.
Sterculia gum
41-9
A. Place 0.2 g of the powdered drug (355) in a 10 ml ground glass-stoppered cylinder graduated in
0.1 ml. Add 10 ml of alcohol (60 per cent V/V) R and shake. Any gel formed occupies not more than
1.5 ml.
B. To 1.0 g of the powdered drug (355) add 100 ml of water R and shake. Add 0.1 ml of methyl red
indicator.
Foreign matter Place 2.0 g of the powdered drug (355) in a 250 ml round-bottomed flask and add
95 ml of methanol R. Swirl to moisten the powder and add 60 ml of hydrochloric acid R1. Add a few
glass beads about 4 mm in diameter and heat on a water-bath under a reflux condenser for 3 h,
shaking occasionally. Remove the glass beads and filter the hot suspension in vacuo through a
sintered-glass filter (160). Rinse the flask with a small quantity of water R and pass the rinsings
through the filter. Wash the residue on the filter with about 40 ml of methanol R and dry to constant
mass at 110C (about 1 h). Allow to cool in a desiccator and weigh. The residue weighs not more
than 20 mg (1.0 per cent).
Flow time Not less than 10 s or, if the substance to be examined is to be used for the preparation of
emulsions, not less than 50 s. Place 1.0 g of the powdered drug (125 to 250) in a 1000 ml round
bottomed flask with a ground-glass stopper and add 8.0 ml of alcohol R and close the flask. Disperse
the suspension over the inner surface of the flask by shaking, taking care not to wet the stopper. Open
the flask and add in one portion 72.0 ml of water R. Stopper the flask and shake vigorously for 3 min.
Allow to stand for 24 h and shake vigorously again for 3 min. Eliminate air bubbles by applying
vacuum above the mucilage for 5 min. Transfer the mucilage to a 50 ml cylinder. Dip in the mucilage
a piece of glass tubing 200 mm long and 6.0 mm in internal diameter and graduated at 20 mm and
120 mm from the lower end; the tubing must not be rinsed with surface-active substances. When the
mucilage has reached the upper mark, close the tube with a finger. Withdraw the closed tube, remove
the finger and measure with a stop-watch the time needed for the meniscus to reach the lower
graduation. Carry out this operation four times and determine the average value of the last three
determinations.
per gram, determined by plate count. It complies with the tests for Escherichia coli and Salmonella
(2.6.13).
STORAGE
Store in a well-closed container protected from light.
LABELLING
The label states whether or not the contents are suitable for preparing emulsions.
__________________________________________________________________________________________________________ Ph Eur
41-10
Tranexamic Acid
H
COOH
2N
H
C8H15NO2
157.2
1197-18-8
Tranexamic Acid complies with the requirements of the 3rd edition of the European Pharmacopoeia [0875].
Action and use Antifibrinolytic; haemostatic.
Preparations
Tranexamic Acid Injection
Tranexamic Acid Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Tranexamic acid is trans-4-(aminomethyl)cyclohexane carboxylic acid. It contains not less than
99.0 per cent and not more than the equivalent of 101.0 per cent of C8H15NO2, calculated with
CHARACTERS
A white, crystalline powder, freely soluble in water and in glacial acetic acid, practically insoluble in
alcohol and in acetone.
IDENTIFICATION
with tranexamic acid CRS. Examine the substances prepared as discs.
TESTS
the same solvent.
Reference solution (a). Dissolve 5.0 ml of the test solution to 100.0 ml with water R. Dilute 1.0 ml of
this solution to 10.0 ml with water R.
Reference solution (b). Dissolve 10 mg of tranexamic acid impurity C CRS in water R and dilute to
100.0 ml with the same solvent. To 1.0 ml of this solution add 1.0 ml of the test solution and dilute
to 50.0 ml with water R.
Column:
material: stainless steel,
stationary phase: octadecylsilyl silica gel for chromatography R (5 m),
size: L = 0.25 m, = 4.6 mm or L = 0.25 m, = 6.0 mm.
Mobile phase: dissolve 11.0 g of anhydrous sodium dihydrogen phosphate R in 500 ml of water R, add
5 ml of triethylamine R and 1.4 g of sodium laurilsulfate R. Adjust to pH 2.5 with dilute phosphoric
acid R and dilute to 600 ml with water R. Add 400 ml of methanol R and mix.
Injection: 20 l.
Sensitivity: the height of the principal peak is at least 50 per cent of the full scale of the recorder for
Run time: 3 times the retention time of tranexamic acid (about 13 min).
System suitability:
relative retention times:
impurity C = about 1.1
impurity D = about 1.3
41-11
impurity B = about 1.5
impurity A = about 2.1
resolution: minimum of 2.0 between the peaks due to tranexamic acid and to impurity C in the
Limits:
impurity A: not more than 0.2 times the area of the principal peak in the chromatogram obtained
with reference solution (a) (0.1 per cent),
impurity B: not more than 0.4 times the area of the principal peak in the chromatogram obtained
with reference solution (a) (0.2 per cent), taking into account a correction factor of 1.2,
any other impurity: not more than 0.2 times the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.1 per cent), taking into account a correction factor of 0.005
for impurity C and of 0.006 for impurity D,
total of all other impurities: not more than 0.4 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.2 per cent), taking into account a correction factor of
0.005 for impurity C and of 0.006 for impurity D,
Halides expressed as chlorides (2.4.4). Dissolve 1.2 g in water R and dilute to 50 ml with the
same solvent. 15 ml of this solution complies with the limit test for chlorides (140 ppm).
this solution complies with limit test A for heavy metals (10 ppm). Prepare the standard using lead
100C to 105C for 2 h.
ASSAY
IMPURITIES
Qualified impurities
OOC
COOH
H
N
H
A. trans,trans-4,4-(iminodimethylene)di(cyclohexanecarboxylic) acid,
H
COOH
H2N
H
B. cis-4-(aminomethyl)cyclohexanecarboxylic acid,
Other detectable impurities

COOH
H2N
H
C. (RS)-4-(aminomethyl)cyclohex-1-enecarboxylic acid,
COOH
H2N
D. 4-aminomethylbenzoic acid.
__________________________________________________________________________________________________________ Ph Eur
41-12
Products with Risk of Transmitting Agents of Animal Spongiform

Encephalopathies
Products with Risk of Transmitting Agents of Animal Spongiform Encephalopathies comply with the requirements of the 3rd edition of the European Pharmacopoeia [1483].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Products with risk of transmitting agents of animal spongiform encephalopathies are those derived
from tissues or secretions of animals susceptible to transmissible spongiform encephalopathies other
than by experimental challenge. This monograph applies to all substances or preparations obtained
from such animals and applies to all substances or preparations where products obtained from such
animals are included as active substances or excipients or have been used during production, for
example, as raw or source materials, starting materials or reagents.
PRODUCTION
Production complies with chapter 5.2.8.
__________________________________________________________________________________________________________ Ph Eur
41-13
Tranylcypromine Sulphate
,H2SO4
NH2
and enantiomer
(C9H11N)2,H2SO4
364.5
13492-01-8
Definition Tranylcypromine Sulphate is (1RS,2SR)-2-phenylcyclopropylamine sulphate. It contains

not less than 98.0% and not more than 101.0% of (C9H11N)2,H2SO4, calculated with reference to
Characteristics A white or almost white, crystalline powder; odourless or with a faint odour similar
to that of cinnamaldehyde.
Soluble in water; very slightly soluble in ethanol (96%) and in ether; insoluble in chloroform.
Identification
tranylcypromine sulphate (RS 345).
B. Yields the reactions characteristic of sulphates, Appendix VI.
Related substances Dissolve 10 mg of 4-chloroaniline (internal standard) in sufficient 0.1M hydrochloric acid to produce 20 ml (solution A). Carry out the method for gas chromatography, Appendix III
B, using the following solutions. For solution (1) add 5 ml of 1M sodium hydroxide to 1 ml of solution
A, extract with 10 ml of dichloromethane, add 1 ml of trifluoroacetic anhydride to the dichloromethane
extract and allow to stand for 10 minutes. Evaporate the solution at a pressure of 2 kPa using a rotary
evaporator and a water bath at 20 and dissolve the residue in 2 ml of dichloromethane. For solution
(2) dissolve 0.1 g of the substance being examined in 5 ml of water, add 1 ml of 5M sodium hydroxide
and continue as for solution (1), beginning at the words extract with .... For solution (3) dissolve
0.1 g of the substance being examined in 5 ml of water, add 1 ml of solution A and 1 ml of 5M sodium
hydroxide and continue as for solution (1), beginning at the words extract with ....
with acid-washed, silanised diatomaceous support (100 to 120 mesh) coated with 3% w/w of
cyanopropylmethylphenyl methyl silicone fluid (OV-225 is suitable) and maintained at 170.
that of the peak due to the trifluoracetyl derivative of 4-chloroaniline (0.5%).
1 g.
(C9H11N)2,H2SO4.
Storage Tranylcypromine Sulphate should be kept in a well-closed container.
Preparation
Tranylcypromine Tablets
41-14
Trapidil
1/01
NEt2
N
e
C10H15N5
N
N
205.3
15421-84-8
Trapidil complies with the requirements of the 3rd edition of the European Pharmacopoeia [1576]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
N,N-Diethyl-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine.
CHARACTERS
Appearance: white or almost white, crystalline powder.
Solubility: freely soluble in water, soluble in ethanol and in methylene chloride.
mp: about 102C.
IDENTIFICATION
Comparison: trapidil CRS.
TESTS
Appearance of solution Solution S is clear (2.2.1) and colourless (2.2.2, Method II).
Acidity or alkalinity. To 10 ml of solution S add 0.2 ml of methyl red solution R and 0.2 ml of 0.01M
hydrochloric acid. The solution is red. Add 0.4 ml of 0.01M sodium hydroxide. The solution is yellow.
Reference solution (a). Dissolve 5.0 mg of trapidil impurity A CRS in the mobile phase and dilute to
Reference solution (b). Dissolve 5.0 mg of trapidil impurity B CRS in the mobile phase and dilute to
Reference solution (c). Mix equal volumes of reference solution (a) and reference solution (b).
Column:
size: l = 0.125 m, = 4.0 mm,
stationary phase: base-deactivated octadecylsilyl silica gel for chromatography R (5 m),
Mobile phase: 50 ml of methanol R, 75 ml of acetonitrile R and 800 ml of a 1.7 g/l solution of potassium
dihydrogen phosphate R adjusted to pH 2.45 with phosphoric acid R; dilute to 1000 ml with water R.
Injection: 10 l.
Run time: 3 times the retention time of trapidil.
System suitability:
resolution: minimum of 4.0 between the peaks due to impurity A and impurity B in the chromatogram obtained with reference solution (c).
Limits:
impurity A: not more than the area of the principal peak in the chromatogram obtained with
41-15
any other impurity: not more than the area of the principal peak in the chromatogram obtained with
disregard limit: 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.01 per cent).
Chlorides (2.4.4): maximum 100 ppm.
Dissolve 0.25 g in 10 ml of water R and dilute to 15 ml with water R. The solution complies with the
limit test for chlorides. Prepare the standard using 5 ml of chloride standard solution (5 ppm Cl) R.
Ammonium (2.4.1): maximum 20 ppm.
0.50 g complies with limit test A. Prepare the standard using 0.1 ml of ammonium standard solution
(100 ppm NH4) R.
Dissolve 2.0 g in 20 ml of water R. 12 ml of the solution complies with limit test A. Prepare the
Loss on drying (2.2.32): maximum 0.5 per cent, determined on 1.000 g by drying in vacuo at 60C
for 3 h.
ASSAY
STORAGE
IMPURITIES
OH
N N
Me
A. 5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol,
H
N N
H2N
B. 1,2,4-triazol-3-amine.
__________________________________________________________________________________________________________ Ph Eur
41-16
Trazodone Hydrochloride
N
N
N
N
O
C19H22ClN5O,HCl
Cl
,HCl
408.3
25332-39-2
Definition Trazodone Hydrochloride is 2-{3-[4-(3-chloro)phenylpiperazin-1-yl]propyl}-1,2,4triazolo[4,3-a]pyridin-3(2H)-one hydrochloride. It contains not less than 99.0% and not more than
101.0% of C19H22ClN5O, HCl, calculated with reference to the dried substance.
Soluble in water; sparingly soluble in ethanol (96%); practically insoluble in ether.
Identification
trazodone hydrochloride (RS 346). In the preparation of the disc, avoid excessive grinding when
triturating the substance being examined with the potassium chloride.
B. Yields the reactions characteristic of chlorides, Appendix VI.
3-Chloroaniline To 10 ml of a 1% w/v solution of the substance being examined in a mixture of
equal volumes of water and ethanol add 2 ml of a freshly prepared 5% w/v solution of 4-dimethylaminobenzaldehyde in ethanol and 0.1 ml of hydrochloric acid. Shake well and allow to stand for 5
minutes. Any yellow colour produced within 5 minutes of preparation of the solution is not more
intense than that produced by treating at the same time and in the same manner 10 ml of a 1 g
per ml solution of 3-chloro-aniline in a mixture of equal volumes of water and ethanol beginning at the
words add 2 ml... (100 ppm).
Related substances The combined nominal total content of impurities determined in tests A and B
below is not more than 1.0% and no single impurity is more than 0.3%.
A. Carry out the method for liquid chromatography, Appendix III D, using three solutions in the
mobile phase containing (1) 0.10% w/v of the substance being examined, (2) 0.00050% w/v of the
substance being examined and (3) 0.10% w/v of trazodone hydrochloride impurity standard BPCRS.
(25 cm 4.6 mm) packed with stationary phase C (5 m) (Supelco Suplex pKb 100 is suitable) and a
pre-column (1 cm 4.6 mm) packed with the same material placed between the pump and the
injector, (b) a mixture of 0.4 volume of diethylamine, 350 volumes of acetonitrile and 650 volumes of
water as the mobile phase with a flow rate of 2.0 ml per minute and (c) a detection wavelength of
254 nm. If necessary adjust the proportions of acetonitrile and water in the mobile phase to obtain a
retention time of about 10 minutes for the principal peak. For solution (1) allow the chromatography
to proceed for three times the retention time of the principal peak.
The test is not valid unless the chromatogram obtained with solution (3) closely resembles reference chromatogram A supplied with trazodone hydrochloride impurity standard BPCRS.
From any secondary peaks in the chromatogram obtained with solution (1) calculate the nominal
content of each impurity with a retention time of up to and including that of impurity E, identified
from reference chromatogram A. Use, as reference, the principal peak in the chromatogram obtained
with solution (2). Disregard any peak with an area less than 0.1 times that of the principal peak in the
chromatogram obtained with solution (2) (0.05%).
B. Carry out the method for liquid chromatography, Appendix III D, using three solutions in the
mobile phase containing (1) 0.10% w/v of the substance being examined, (2) 0.00050% w/v of the
substance being examined and (3) 0.10% w/v of trazodone hydrochloride impurity BPCRS.
(25 cm 4.6 mm) packed with stationary phase C (5 m) (Supelco Suplex pKb 100 is suitable) and a
pre-column (1 cm 4.6 mm) packed with the same material placed between the pump and the
injector, (b) a mixture of 0.4 volume of diethylamine, 320 volumes of water and 680 volumes of
acetonitrile as the mobile phase with a flow rate of 1.7 ml per minute and (c) a detection wavelength
of 254 nm. If necessary adjust the proportions of acetonitrile and water in the mobile phase to obtain a
retention time of about 2.5 minutes for the principal peak. For solution (1) allow the chromatography to proceed for at least five times the retention time of the principal peak.
41-17
From any secondary peaks in the chromatogram obtained with solution (1) calculate the nominal
content of each impurity with a retention time longer than that of impurity E identified from reference chromatogram B, supplied with trazodone hydrochloride impurity standard BPCRS. Use, as reference, the principal peak in the chromatogram obtained with solution (2). Disregard any peak with an
area less than 0.1 times that of the principal peak in the chromatogram obtained with solution (2)
(0.05%).
Loss on drying When dried to constant weight at 105 at a pressure of 3.5 to 6.5 kPa, loses not
Assay Dissolve 0.3 g in 60 ml of glacial acetic acid, add 5 ml of mercury(II) acetate solution and carry
out Method I for non-aqueous titration, Appendix VIII A, determining the end point potentiometrically. Each ml of 0.1M perchloric acid VS is equivalent to 40.83 mg of C19H22ClN5O,HCl.
Storage Trazodone Hydrochloride should be kept in an airtight container and protected from light.
IMPURITIES
Cl
N
O
A. 4-(3-chlorophenyl)-1-[3-(3-oxo-2,3-dihydro-1,2,4-triazolo[4,3-a]pyridin-2yl)propyl]piperazine N1-oxide
B. 2-[3-(4-phenylpiperazin-1-yl)propyl]-1,2,4-triazolo[4,3-a]pyridin-3(2H)-one
Cl
N
C. 2-{3-[4-(4-chlorophenyl)piperazin-1-yl]propyl}-1,2,4-triazolo[4,3-a]pyridin-3(2H)-one
Br
D. 2-{3-[4-(3-bromophenyl)piperazin-1-yl]propyl}-1,2,4-triazolo[4,3-a]pyridin-3(2H)-one
Et
N
O
Cl
E. 2-{3-[4-(3-chloro-4-ethylphenyl)piperazin-1-yl]propyl}-1,2,4-triazolo[4,3-a]pyridin-3(2H)one
N
Cl
Cl
F. 1-(3-chloropropyl)-3-chlorophenylpiperazine
41-18
Me
Me
N
O
Cl
G. 3-[4-(3-chlorophenyl)piperazin-1-yl]propyl isobutyl ether
Cl
N
N
Cl
H. 1,3- bis-[4-(3-chlorophenyl)piperazin-1-yl]propane
41-19
Tretinoin
e
Me
Me
Me
COOH
Me
C20H28O2
300.4
302-79-4
Tretinoin complies with the requirements of the 3rd edition of the European Pharmacopoeia [0693]. These
Action and use Used in treatment of acne.
Preparations
Tretinoin Gel
Tretinoin Solution
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Tretinoin contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent of
(2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-enyl)nona-2,4,6,8-tetraenoic acid,
CHARACTERS
A yellow or light orange, crystalline powder, practically insoluble in water, soluble in methylene
chloride, slightly soluble in alcohol. It is sensitive to air, heat and light, especially in solution.
Carry out all operations as rapidly as possible and avoid exposure to actinic light; use freshly prepared
solutions.
IDENTIFICATION
A. Dissolve 75.0 mg in 5 ml of methylene chloride R and dilute immediately to 100.0 ml with acidified
2-propanol (prepared by diluting 1 ml of 0.01M hydrochloric acid to 1000 ml with 2-propanol R).
Dilute 5.0 ml of this solution to 100.0 ml with the acidified 2-propanol. Dilute 5.0 ml of this latter
solution to 50.0 ml with the acidified 2-propanol. Examined between 300 nm and 400 nm (2.2.25),
the solution shows a single maximum, at 353 nm. The specific absorbance at the maximum is 1455
to 1545.
obtained with tretinoin CRS. Examine the substances prepared as discs.
Reference solution. Dissolve 10 mg of tretinoin CRS in methylene chloride R and dilute to 10 ml with the
same solvent.
volumes of glacial acetic acid R, 4 volumes of acetone R, 40 volumes of peroxide-free ether R and 54
volumes of cyclohexane R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm.
to the principal spot in the chromatogram obtained with the reference solution.
D. Dissolve about 5 mg in 2 ml of antimony trichloride solution R. An intense red colour develops and
later becomes violet.
TESTS
Reference solution (a). Dissolve 10.0 mg of isotretinoin CRS in methanol R and dilute to 10.0 ml with
the same solvent.
41-20
Reference solution (b). Dilute 1.0 ml of reference solution (a) to 25.0 ml with methanol R.
Reference solution (c). Mix 1.0 ml of reference solution (a) with 0.5 ml of the test solution and dilute
to 25.0 ml with methanol R.
Reference solution (d). Dilute 0.5 ml of the test solution to 100.0 ml with methanol R.
as mobile phase at a flow rate of 1.0 ml per minute a mixture of 5 volumes of glacial acetic acid R,
225 volumes of water R and 770 volumes of methanol R,
Inject separately 10 l of reference solutions (b), (c) and (d) and of the test solution. Adjust the
sensitivity of the detector so that the height of the principal peak in the chromatogram obtained with
reference solution (b) is about 70 per cent of the full scale of the recorder. The test is not valid unless
the resolution between isotretinoin and tretinoin in the chromatogram obtained with reference
solution (c) is at least 2.0. In the chromatogram obtained with the test solution, the area of the peak
due to isotretinoin is not greater than the area of the principal peak in the chromatogram obtained
with reference solution (b) (2.0 per cent) and the sum of the areas of any peaks, apart from the
principal peak and any peak due to isotretinoin, is not greater than the area of the principal peak in
the chromatogram obtained with reference solution (d) (0.5 per cent).
100C to 105C.
ASSAY
Dissolve 0.200 g in 70 ml of acetone R. Titrate with 0.1M tetrabutylammonium hydroxide determining
1 ml of 0.1M tetrabutylammonium hydroxide is equivalent to 30.04 mg of C20H28O2.
STORAGE
Store in an airtight container, protected from light, at a temperature not exceeding 25C.
It is recommended that the contents of an opened container be used as soon as possible and any
unused part be protected by an atmosphere of an inert gas.
IMPURITIES
A. isotretinoin,
Me
e Me
Me
COOH
Me
B. (2Z,4E,6Z,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-enyl)nona-2,4,6,8-tetraenoic acid
(9,13-di-cis-retinoic acid),
Me
Me Me
Me
Me
COOH
C. (2Z,4Z,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-enyl)nona-2,4,6,8-tetraenoic acid
(11,13-di-cis-retinoic acid),
Me
e Me
Me
Me
COOH
D. (2E,4E,6Z,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-enyl)nona-2,4,6,8-tetraenoic acid
(9-cis-retinoic acid),
E. oxidation products of tretinoin.
__________________________________________________________________________________________________________ Ph Eur
41-21
Triacetin
CH2OAc
CHOAc
CH2OAc
C19H14O6
218.2
102-76-1
Triacetin complies with the requirements of the 3rd edition of the European Pharmacopoeia for Glycerol
Triacetate [1106]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Glycerol triacetate contains not less than 97.0 per cent and not more than the equivalent of 100.5 per
cent of propane-1,2,3-triol triacetate, calculated with reference to the anhydrous substance.
CHARACTERS
A clear, colourless, slightly viscous oily liquid. It is soluble in water, miscible with ethanol and
toluene. It boils at about 260C.
IDENTIFICATION
spectrum of glycerol triacetate. Examine the substance as film on potassium bromide R.
TESTS
Appearance It is clear (2.2.1) and not more intensely coloured than reference solution Y6 (Method
II, 2.2.2).
Acidity Dissolve 5.00 g in 25 ml of ethanol R, previously neutralised to 0.2 ml of phenolphthalein
solution R and add 0.20 ml of 0.1M sodium hydroxide. The pink colour of the mixture persists for 15 s.
of water.
ASSAY
Introduce 0.300 g of the substance to be examined into a 250 ml borosilicate glass flask fitted with a
reflux condenser. Add 25.0 ml of 0.5M alcoholic potassium hydroxide and a few glass beads. Attach the
condenser and heat under reflux for 30 min. Add 1 ml of phenolphthalein solution R1 and titrate
immediately with 0.5M hydrochloric acid. Carry out a blank test under the same conditions. Calculate
the content from the difference in consumption of alkali in the main and the blank procedure.
1 ml of 0.5M alcoholic potassium hydroxide is equivalent to 36.37 mg of C9H14O6.
STORAGE
Store in a well-closed, well-filled container.
__________________________________________________________________________________________________________ Ph Eur
41-22
Triamcinolone
O
H
Me
HO
Me
OH
OH
OH
H
H
F
O
C21H27FO6
394.4
124-94-7
Triamcinolone complies with the requirements of the 3rd edition of the European Pharmacopoeia [1376].
Preparation
Triamcinolone Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Triamcinolone contains not less than 97.0 per cent and not more than the equivalent of 103.0 per
cent of 9-fluoro-11,16,17,21-tetrahydroxypregna-1,4-diene-3,20-dione, calculated with reference
CHARACTERS
A white or almost white, crystalline powder, practically insoluble in water, slightly soluble in
methanol, practically insoluble in methylene chloride.
IDENTIFICATION
obtained with triamcinolone CRS. If the spectra obtained show differences, dissolve the substance to be
examined and the reference substance separately in methanol R, evaporate to dryness, dry the residues
at 60C at a pressure not exceeding 0.7 kPa and record new spectra using the residues.
Prepare the solutions immediately before use and protected from light. Examine the plate under ultraviolet light
immediately after development.
the same solvent.
Reference solution (a). Dissolve 20 mg of triamcinolone CRS in methanol R and dilute to 20 ml with the
same solvent.
Reference solution (b). Dissolve 10 mg of dexamethasone CRS in reference solution (a) and dilute to
(b) shows two clearly separated spots.
TESTS
Specific optical rotation (2.2.7). Dissolve 0.10 g in dimethylformamide R, and dilute to 10.0 ml with
the same solvent. The specific optical rotation is +65 to +72, calculated with reference to the
before use and protect from light.
Reference solution. Dilute 1.0 ml of the test solution to 100.0 ml with methanol R.
41-23
as mobile phase at a flow rate of 2 ml/min a mixture of equal volumes of water R and methanol R,
Inject 20 l of the reference solution. Adjust the sensitivity of the system so that the height of the
full scale of the recorder. When the chromatograms are recorded in the prescribed conditions, the
retention time for triamcinolone is about 5 min. The test is not valid unless in the chromatogram
obtained with the reference solution, the number of theoretical plates calculated for the peak due to
triamcinolone is at least 5000.
Inject 20 l of the test solution. Continue the chromatography for four times the retention time of
the principal peak. In the chromatogram obtained with the test solution: the area of any peak apart
from the principal peak, is not greater than twice the area of the principal peak in the chromatogram
obtained with the reference solution (2 per cent); not more than one such peak has an area greater
than that of the principal peak in the chromatogram obtained with the reference solution (1 per cent);
the sum of the areas of any peak, apart from the principal peak, is not greater than four times the area
of the principal peak in the chromatogram obtained with the reference solution (4 per cent).
Disregard any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with the reference solution.
ASSAY
Prepare the solutions immediately before use and protect from light.
Calculate the content of C21H27FO6 taking the specific absorbance to be 389.
STORAGE
Store in a well closed container, protected from light.
IMPURITIES
O
Me
HO
Me
H
F
OAc
OH
OAc
A. triamcinolone 16,21-diacetate,
O
Me
HO
Me
H
F
OAc
OH
OH
B. triamcinolone 21-acetate.
__________________________________________________________________________________________________________ Ph Eur
41-24
Triamcinolone Acetonide
OH
O
H
Me
HO
Me
Me
O
H
H
F
Me
O
C24H31FO6
434.5
76-25-5
Triamcinolone Acetonide complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparations
Triamcinolone Cream
Triamcinolone Acetonide Injection
Triamcinolone Ointment
Triamcinolone Dental Paste
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Triamcinolone acetonide contains not less than 97.0 per cent and not more than the equivalent of
103.0 per cent of 9-fluoro-11,21-dihydroxy-16,17-(1-methylethylidene-dioxy)pregna-1,4-diene3,20-dione, calculated with reference to the anhydrous substance.
CHARACTERS
alcohol, very slightly soluble in ether.
IDENTIFICATION
obtained with triamcinolone acetonide CRS. If the spectra obtained in the solid state show differences,
volume of methanol R and evaporate to dryness. Using the residues, prepare halogen salt discs or
mulls in liquid paraffin R and record the spectra again.
Prepare the solutions immediately before use and protect from light. Examine the plate in ultraviolet light
the same solvent.
Reference solution (a). Dissolve 20 mg of triamcinolone acetonide CRS in methanol R and dilute to 20 ml
Reference solution (b). Dissolve 10 mg of triamcinolone hexacetonide CRS in reference solution (a) and
solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b)
41-25
Test solution (b). In a separating funnel, dissolve 10 mg of the substance to be examined in 1.5 ml of
glacial acetic acid R, add 0.5 ml of a 20 g/l solution of chromium trioxide R and allow to stand for
60 min. Add 5 ml of water R, 2 ml of methylene chloride R and shake vigorously for 2 min. Allow to
separate and use the lower layer.
Reference solution (a). Dissolve 10 mg of triamcinolone acetonide CRS in methanol R and dilute to 10 ml
Reference solution (b). In a separating funnel, dissolve 10 mg of triamcinolone acetonide CRS in 1.5 ml
of glacial acetic acid R, add 0.5 ml of a 20 g/l solution of chromium trioxide R and allow to stand for
60 min. Add 5 ml of water R, 2 ml of methylene chloride R and shake vigorously for 2 min. Allow to
separate and use the lower layer.
obtained with the corresponding reference solution. The principal spots in the chromatograms
obtained with test solution (b) and reference solution (b) have an Rf value distinctly higher than that
of the principal spots in the chromatograms obtained with test solution (a) and reference solution (a).
blank is red.
TESTS
substance.
Related substances Examine by liquid chromatography (2.2.29). Carry out the test protected from
light.
Reference solution (a). Dissolve 2 mg of triamcinolone acetonide CRS and 2 mg of triamcinolone R in the
as mobile phase at a flow rate of 1.5 ml per minute a mixture prepared as follows: in a 1000 ml
volumetric flask mix 525 ml of methanol R with 400 ml of water R and allow to equilibrate; adjust
the volume to 1000 ml with water R and mix again,
conditions, the retention times are: triamcinolone about 5 min and triamcinolone acetonide about
17 min. The test is not valid unless the resolution between the peaks corresponding to triamcinolone
and triamcinolone acetonide is at least 15; if necessary, adjust the concentration of methanol in the
mobile phase.
Inject separately 20 l of the test solution and 20 l of reference solution (b). Continue the chromatography for 3.5 times the retention time of the principal peak in the chromatogram obtained with
the test solution. In the chromatogram obtained with the test solution: the area of any peak, apart
from the principal peak, is not greater than 0.25 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.25 per cent); the sum of the areas of all the peaks, apart
41-26
peak with an area less then 0.05 times the area of the principal peak in the chromatogram obtained
ASSAY
Protect the solutions from light throughout the assay.
solution to 100.0 ml with alcohol R. Measure the absorbance (2.2.25) at the maximum at 238.5 nm.
STORAGE
IMPURITIES
OH
H
HO
Me H
F
O
Me
OH
OH
H
A. triamcinolone.
__________________________________________________________________________________________________________ Ph Eur
41-27
Triamcinolone Hexacetonide
O
Me
Me
Me
O
O
H
Me
HO
Me
Me
O
H
H
F
Me
O
C30H41FO7
532.6
5611-51-8
Triamcinolone Hexacetonide complies with the requirements of the 3rd edition of the European Pharmacopoeia [0867]. These requirements are reproduced after the heading Definition below.
Preparation
Triamcinolone Hexacetonide Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Triamcinolone hexacetonide contains not less than 97.0 per cent and not more than the equivalent of
103.0 per cent of 9-fluoro-11,21-dihydroxy-16,17-(1-methylethylidenedioxy)pregna-1,4-diene3,20-dione 21-(3,3-dimethylbutanoate), calculated with reference to the anhydrous substance.
CHARACTERS
ethanol and in methanol.
IDENTIFICATION
obtained with triamcinolone hexacetonide CRS.
the same solvent.
Reference solution (a). Dissolve 20 mg of triamcinolone hexacetonide CRS in methanol R and dilute to
Reference solution (b). Dissolve 10 mg of triamcinolone acetonide CRS in reference solution (a) and
solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b)
TESTS
Specific optical rotation (2.2.7). Dissolve 0.100 g in methylene chloride R and dilute to 10.0 ml with
the same solvent. The specific optical rotation is +92 to +98, calculated with reference to the
Related substances Examine by liquid chromatography (2.2.29). Carry out the test protected from
light.
41-28
Reference solution (a). Dissolve 2 mg of triamcinolone hexacetonide CRS and 2 mg of triamcinolone
acetonide CRS in the mobile phase and dilute to 100.0 ml with the mobile phase.
volumetric flask mix 750 ml of methanol R with 200 ml of water R and allow to equilibrate; adjust
the volume to 1000 ml with water R and mix again,
conditions, the retention times are: triamcinolone acetonide about 3 min and triamcinolone
hexacetonide about 12 min. The test is not valid unless the resolution between the peaks corresponding to triamcinolone acetonide and triamcinolone hexacetonide is at least 20.0; if necessary, adjust
the concentration of methanol in the mobile phase.
Inject separately 20 l of the test solution and 20 l of reference solution (b). Continue the chromatography for three times the retention time of the principal peak in the chromatogram obtained with
the test solution. In the chromatogram obtained with the test solution: the area of any peak, apart
with reference solution (b) (1 per cent). Disregard any peak due to the solvent and any peak with an
area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference
solution (b).
of water.
ASSAY
STORAGE
IMPURITIES
A. Triamcinolone acetonide.
__________________________________________________________________________________________________________ Ph Eur
41-29
Triamterene
H2N
NH2
N
N
NH2
C12H11N7
253.3
396-01-0
Triamterene complies with the requirements of the 3rd edition of the European Pharmacopoeia [0058]. These
Preparations
Co-triamterzide Tablets
Triamterene Capsules
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Triamterene contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of 2,4,7-tri-amino-6-phenylpteridine, calculated with reference to the dried substance.
CHARACTERS
A yellow, crystalline powder, very slightly soluble in water and in alcohol, practically insoluble in
ether.
IDENTIFICATION
A. Dissolve 0.1 g in a 10 per cent V/V solution of 1M hydrochloric acid in ethanol R and dilute to
100 ml with the same acid mixture. Dilute 1 ml of this solution to 100 ml with a 10 per cent V/V
solution of 1M hydrochloric acid in ethanol R. Examined between 255 nm and 380 nm, the solution
shows two absorption maxima (2.2.25), at 262 nm and 360 nm, and a shoulder at 285 nm.
B. Examined in ultraviolet light at 365 nm, acid solutions, especially a 1 g/l solution in anhydrous
formic acid R, show an intense blue fluorescence.
TESTS
Acidity Boil 1.0 g with 20 ml of water R for 5 min, cool, filter and wash the filter with three quantities, each of 10 ml, of water R. Combine the filtrate and washings and add 0.3 ml of phenolphthalein
indicator.
Nitrosotriaminopyrimidine Examine by thin-layer chromatography (2.2.27), using silica gel
HF254 R as the coating substance.
Test solution. Dissolve 0.2 g of the substance to be examined in 5 ml of anhydrous formic acid R, stirring with a glass rod if necessary. Prepare immediately before use.
Reference solution. Dissolve 4 mg of nitrosotriaminopyrimidine CRS in anhydrous formic acid R and dilute
Apply separately to the plate as bands 1.5 cm long, 20 l of each solution as two applications of 10 l
each, drying in a current of air after each application. Develop over a path of 5 cm using ether R.
Allow the plate to dry in air and develop over a path of 10 cm using a mixture of 10 volumes of
glacial acetic acid R, 10 volumes of methanol R and 80 volumes of ethyl acetate R, the mixture containing 0.5 g/l of sodium fluoresceinate R. Dry the plate in a current of air and expose to ammonia vapour
for a few seconds. Examine in ultraviolet light at 254 nm and at 365 nm. Any band corresponding to
nitrosotriaminopyrimidine in the chromatogram obtained with the test solution is not more intense
than the band in the chromatogram obtained with the reference solution (0.1 per cent).
coating substance.
Test solution. Dissolve 0.1 g of the substance to be examined in 20 ml of dimethyl sulphoxide R. Dilute
2 ml of the solution to 50 ml with methanol R.
Reference solution. Dilute 1 ml of the test solution to 200 ml with methanol R.
10 volumes of concentrated ammonia R1, 10 volumes of methanol R and 90 volumes of ethyl acetate R.
41-30
Allow the plate to dry in air until the odour of the solvents is no longer detectable and examine in
ultraviolet light at 365 nm. Any spot in the chromatogram obtained with the test solution, apart from
100C to 105C.
ASSAY
with 0.1M perchloric acid determining the end-point potentiometrically (2.2.20).
STORAGE
__________________________________________________________________________________________________________ Ph Eur
41-31
Triclofos Sodium
OH
Cl
O
Cl
Cl
C2H3Cl3NaO4P
ONa
251.4
7246-20-0
Definition Triclofos Sodium is sodium 2,2,2-trichloroethyl hydrogen orthophosphate. It contains

not less than 41.3% and not more than 43.2% of Cl and not less than 97.0% and not more than
102.0% of C2H3Cl3NaO4P, both calculated with reference to the dried substance.
Characteristics A white or almost white powder; odourless or almost odourless; hygroscopic.
Freely soluble in water; slightly soluble in ethanol (96%); practically insoluble in ether.
Identification
triclofos sodium (RS 350).
B. Yields the reactions characteristic of sodium salts, Appendix VI.
C. Heat 0.1 g with 1 g of anhydrous sodium carbonate to a dull red heat and maintain for 10 minutes,
cool, extract the residue with water and filter. The filtrate yields the reactions characteristic of chlorides
and of phosphates, Appendix VI.
Clarity of solution A 2.0% w/v solution is clear, Appendix IV A.
Heavy metals A 10.0% w/v solution complies with limit test A for heavy metals, Appendix VII. Use
lead standard solution (2 ppm Pb) to prepare the standard (20 ppm).
Chloride Dissolve 0.1 g in 50 ml of water. 15 ml of the solution complies with the limit test for
chlorides, Appendix VII (0.17%).
Phosphate Not more than 1.0%, calculated as PO4, when determined by the following method.
Dissolve 25 mg in 10 ml of water, add 4 ml of 1M sulphuric acid, 1 ml of ammonium molybdate solution
and 2 ml of methylaminophenolsulphite reagent and allow to stand for 15 minutes. Add sufficient
water to produce 25 ml, allow to stand for a further 15 minutes and measure the absorbance of a 4-cm
layer of the resulting solution at 730 nm, Appendix II B. Calculate the content of phosphate from a
calibration curve prepared by treating suitable volumes of a 0.00143% w/v solution of potassium
dihydrogen orthophosphate in the same manner.
Assay
For Cl Mix 0.25 g with 1 g of anhydrous sodium carbonate in a nickel crucible about 3 cm in diameter,
fill the crucible completely with anhydrous sodium carbonate and invert into a nickel crucible about 4
cm in diameter; cover the smaller crucible with anhydrous sodium carbonate, well pressed down, using
about 25 g of anhydrous sodium carbonate in all. Heat for 30 minutes at a dull red heat, cool, transfer
to a 400-ml beaker, add 150 ml of water and boil gently for 10 minutes. Filter through absorbent
cotton into a 600-ml beaker, washing the residue thoroughly with hot water, until about 400 ml of
filtrate has been collected. Cool, cautiously add nitric acid until the solution is neutral to litmus paper
and add 3 ml of nitric acid in excess. Add 50 ml of 0.1M silver nitrate VS, allow to stand until
precipitation is complete, filter, wash the precipitate with water and titrate the combined filtrate and
washings with 0.1M ammonium thiocyanate VS using ammonium iron(III) sulphate solution R2 as
indicator. Each ml of 0.1M silver nitrate VS is equivalent to 3.545 mg of Cl.
For C2H3Cl3NaO4P Heat 0.2 g in a Kjeldahl flask with 2 ml of sulphuric acid and 2.5 ml of nitric acid
until brown fumes cease to be evolved, cool, add 1 ml of nitric acid and heat again. Continue adding
nitric acid and heating until brown fumes are no longer evolved and the solution is colourless when
cold. Heat until dense, white fumes are evolved, cool, transfer the solution to a flask with the aid of
150 ml of water, add 50 ml of citricmolybdic acid solution and heat slowly to boiling. Swirling the
flask continuously, add 25 ml of quinoline solution at first dropwise and then in a steady stream, heat
on a water bath for 5 minutes and cool. Filter, wash the precipitate with water until free from acid,
transfer the precipitate to a flask with the aid of 100 ml of water, add 50 ml of 0.5M sodium hydroxide
VS and shake until dissolved. Titrate the excess of alkali with 0.5M hydrochloric acid VS using phenolphthaleinthymol blue solution as indicator. Each ml of 0.5M sodium hydroxide VS is equivalent to
4.835 mg of C2H3Cl3NaO4P. Correct the result for the content of phosphate, as determined by the
test described above; each mg of PO4 is equivalent to 2.65 mg of C2H3Cl3NaO4P.
Storage Triclofos Sodium should be kept in a well-closed container.
Action and use Sedative and hypnotic.
41-32
Preparation
Triclofos Oral Solution
41-33
Triethanolamine
1/01
OH
N
O
OH
C6H15NO3
149.2
102-71-6
Triethanolamine complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Trolamine [1577]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Trolamine contains not less than 99.0 per cent m/m and not more than the equivalent of 103.0 per
cent m/m of total bases, calculated as 2,2,2-nitrilotriethanol with reference to the anhydrous
substance.
CHARACTERS
A clear, viscous, colourless or slightly yellowish liquid, very hygroscopic, miscible with water and with
alcohol, soluble in methylene chloride, slightly soluble in ether.
IDENTIFICATION
A. Relative density (2.2.5): 1.120 to 1.130.
B. Refractive index (2.2.6): 1.482 to 1.485.
C. Examine the chromatograms obtained in the test for related substances. The retention time and
same as those of the principal peak in the chromatogram obtained with the reference solution.
D. To 1 ml add 0.3 ml of copper sulphate solution R. A blue colour develops. Add 2.5 ml of dilute
sodium hydroxide solution R and heat to boiling. The blue colour remains unchanged.
TESTS
Test solution. Dissolve 0.100 g of the substance to be examined in a mixture of equal volumes of acetic
anhydride R and anhydrous pyridine R and dilute to 10.0 ml with the same mixture of solvents. Heat at
50C for 30 min.
Reference solution. Dissolve 50 mg of diethanolamine R, 50 mg of ethanolamine R and 0.400 g of
trolamine CRS in a mixture of equal volumes of acetic anhydride R and anhydrous pyridine R and dilute
to 50.0 ml with the same mixture of solvents. Heat at 50C for 30 min.
a column 2 m long and 2.2 mm in internal diameter packed with silanised diatomaceous earth for gas
chromatography R impregnated with 5 per cent m/m of poly[(cyanopropyl)
(methyl)][(phenyl)(methyl)]siloxane R,
Time
(min)
Column
Temperature
(C)
0 10 140
10 40 140 200
40 45 200
Injection port
250
Detector
250
Rate (C
per min)
2
Comment
isothermal
linear gradient
isothermal
Inject 1 l of the test solution and 1 l of the reference solution. The retention times are about:
7 min for ethanolamine, 19 min for trolamine and 23 min for diethanolamine.
41-34
Calculate the percentage content of related substances from the areas of the peaks in the chromatogram obtained with the test solution by the normalisation procedure, disregarding any peak due to
the solvent. The content of ethanolamine is not greater than 0.2 per cent, the content of diethanolamine is not greater than 1.0 per cent and the total content of all other related substances is not
greater than 0.5 per cent. The test is not valid unless the chromatogram obtained with the reference
solution shows three clearly separated peaks.
N-Nitrosodiethanolamine Not more than 25 ppb, determined by gas chromatography (2.2.28).
Carry out the test under a ventilated hood, wear gloves and safety glasses.
Test solution. In a suitable distillation apparatus mix 100.0 g of the substance to be examined and
100.0 ml of ethylene glycol R and gently distil 10.0 ml under vacuum at a pressure not exceeding
1.3 kPa. From this distillate, distil 1 ml.
Reference solution. Dilute 25.0 mg of N-nitrosodiethanolamine R to 100.0 ml with ethylene glycol R.
Dilute 5.0 ml of this solution to 50.0 ml with ethylene glycol R. Dilute 5.0 ml of this solution to
50.0 ml with ethylene glycol R.
a fused-silica column 30 m long and 0.25 mm in internal diameter coated with macrogol 20,000 R
(film thickness 0.25 m),
helium for chromatography R as the carrier gas at a flow rate of 0.75 ml/min and a split ratio of 1:15,
as detector a mass selective spectrometer set at 72 m/e,
Column
Time
(min)
Temperature
(C)
Rate (C
per min)
02
28
8 25
180
180 240
240
10
Injection port
240
Detector
250
Comment
isothermal
with the test solution, the area of any peak corresponding to N-nitrosodiethanolamine (retention time
of about 20 min) is not greater than the area of the corresponding peak in the chromatogram
Heavy metals (2.4.8). Dilute 5 ml of solution S to 30 ml with water R. The solution complies with
limit test A for heavy metals (10 ppm). Prepare the standard using lead standard solution (1 ppm
Pb) R.
Water (2.5.12). Not more than 1.0 per cent, determined on 1.000 g by the semi-micro determination of water. Open the titration vessel, introduce the substance to be examined directly into the
previously titrated solvent. Stopper the flask immediately.
Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. Do not carry out the
initial heating on a water-bath.
ASSAY
Dissolve 1.200 g in 75 ml of carbon dioxide-free water R. Add 0.3 ml of methyl red solution R. Titrate
1 ml of 1M hydrochloric acid is equivalent to 149.2 mg of C6H15NO3.
STORAGE
IMPURITIES
H2N
OH
A. 2-aminoethanol (ethanolamine),
O
H
N
OH
B. 2,2-iminodiethanol (diethanolamine),
N
O
O
OH
C. 2,2-(nitrosoimino)diethanol (N-nitrosodiethanolamine).
__________________________________________________________________________________________________________ Ph Eur
41-35
Triethyl Citrate
corrected 1/01
HO
tOOC
C12H20O7
COOEt
COOEt
276.3
77-93-0
Triethyl Citrate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1479].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Triethyl citrate contains not less than 98.5 per cent and not more than the equivalent of 101.0 per
cent of triethyl 2-hydroxypropane-1,2,3-tricarboxylate, calculated with reference to the anhydrous
substance.
CHARACTERS
A clear, viscous, colourless or almost colourless liquid, hygroscopic, soluble in water, miscible with
alcohol and with ether, slightly soluble in fatty oils.
IDENTIFICATION
A. It complies with the test for refractive index (see Tests).
B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the Ph. Eur. reference
spectrum of triethyl citrate.
C. It gives the reaction of esters (2.3.1).
D. To 0.5 ml add 5 ml of alcohol R and 4 ml of dilute sodium hydroxide solution R. Boil under reflux for
about 10 min. 2 ml of the solution gives the reaction of citrates (2.3.1).
TESTS
Acidity Dilute 10 g with 10 ml of previously neutralised alcohol R, add 0.5 ml of bromothymol blue
solution R2. Not more than 0.3 ml of 0.1M sodium hydroxide is required to change the colour of the
indicator to blue.
Test solution. Dissolve 1.0 ml of the substance to be examined in methylene chloride R and dilute to
Reference solution (a). Dissolve 1.0 ml of the substance to be examined and 0.5 ml of methyl
tridecanoate R in methylene chloride R and dilute to 50.0 ml with the same solvent.
a fused-silica column 30 m long and 0.32 mm in internal diameter coated with poly(dimethyl)siloxane R (5 m),
helium for chromatography R as the carrier gas with a split ratio of about 1:50 and a linear velocity
of about 26 cm/s,
maintaining the temperature of the column at 200C and that of the injection port and the detector
at 220C.
Inject 1.0 l of each of the solutions. Continue the chromatography for twice the retention time of
triethyl citrate which is about 13.6 min.
The test is not valid unless in the chromatogram obtained with reference solution (a), the resolution between the peaks corresponding to triethyl citrate and methyl tridecanoate is at least 1.5.
Calculate the percentage content of related substances from the areas of the peaks in the chromatogram obtained with the test solution by the normalisation procedure, disregarding any peaks with an
area less than 0.04 per cent of the area of the principal peak. The content of any related substance is
not greater than 0.2 per cent and the sum is not greater than 0.5 per cent.
41-36
Heavy metals (2.4.8). Dissolve 4.0 g in 8 ml of alcohol R and dilute to 20 ml with water R. 12 ml of
the solution complies with limit test B for heavy metals (5 ppm). Prepare the standard using lead
standard solution (1 ppm) obtained by diluting lead standard solution (100 ppm Pb) R with a mixture
of equal volumes of alcohol R and water R.
ASSAY
Introduce 1.500 g into a 250 ml borosilicate-glass flask fitted with a reflux condenser. Add 25 ml of
2-propanol R, 50 ml of water R, 25.0 ml of 1M sodium hydroxide and a few glass beads. Heat under a
reflux condenser for 1 h. Allow to cool. Add 1 ml of phenolphthalein solution R1 and titrate with 1M
hydrochloric acid. Carry out a blank test under the same conditions.
STORAGE
IMPURITIES
COOEt
EtOOC
COOEt
A. triethyl propene-1,2,3-tricarboxylate (triethyl aconitate).

__________________________________________________________________________________________________________ Ph Eur
41-37
Trifluoperazine Hydrochloride
1/01
N
NMe ,2HCl
CF3
C21H24F3N3S,2HCl
480.4
440-17-5
Trifluoperazine Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia [0059]. These requirements are reproduced after the heading Definition below.
Preparation
Trifluoperazine Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Trifluoperazine hydrochloride contains not less than 99.0 per cent and not more than the equivalent
of 101.0 per cent of 10-[3-(4-methylpiperazin-1-yl)propyl]-2-trifluoromethylphenothiazine dihydrochloride, calculated with reference to the dried substance.
CHARACTERS
A white to pale yellow, crystalline powder, hygroscopic, freely soluble in water, soluble in alcohol.
IDENTIFICATION
A. Protect the solutions from bright light and measure the absorbances immediately. Dissolve 50 mg in 0.1M
hydrochloric acid and dilute to 500 ml with the same acid. Examined between 280 nm and 350 nm,
the solution shows an absorption maximum (2.2.25) at 305 nm. Dilute 5 ml of the solution to 100 ml
with 0.1M hydrochloric acid. Examined between 230 nm and 280 nm, this solution shows an absorption maximum at 255 nm. The specific absorbance at this maximum is about 650.
C. Place 0.25 g in a 100 ml separating funnel, add 5 ml of water R and 2 ml of dilute sodium hydroxide
solution R. Shake vigorously with 20 ml of ether R. Wash the ether layer with 5 ml of water R, add
0.15 g of maleic acid R and evaporate the ether. The residue, recrystallised from 30 ml of alcohol R
and dried, melts (2.2.14) at about 192C.
D. Dissolve about 0.5 mg in 1 ml of water R, add 0.1 ml of bromine water R and shake for about
1 min. Add dropwise 1 ml of sulphuric acid R with constant, vigorous agitation. A red colour
develops.
E. Dissolve about 50 mg in 5 ml of water R and add 2 ml of nitric acid R. A dark-red colour develops
which turns to pale yellow. The solution gives reaction (a) of chlorides (2.3.1).
TESTS
Related substances Carry out the test protected from bright light.
Examine by thin-layer chromatography (2.2.27), using a TLC silica gel GF254 plate R.
Test solution. Dissolve 0.2 g of the substance to be examined in a mixture of 5 volumes of diethylamine R and 95 volumes of methanol R and dilute to 10 ml with the same mixture of solvents. Prepare
Reference solution. Dilute 1 ml of the test solution to 200 ml with a mixture of 5 volumes of diethylamine R and 95 volumes of methanol R.
volumes of acetone R, 10 volumes of diethylamine R and 80 volumes of cyclohexane R. Allow the plate
to dry in air and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with
41-38
the test solution, apart from the principal spot, is not more intense than the spot in the chromatogram obtained with the reference solution (0.5 per cent).
100C to 105C.
ASSAY
Dissolve 0.200 g in 50 ml of alcohol R and add 5.0 ml of 0.01M hydrochloric acid. Carry out a potentiometric titration (2.2.20), using 0.1M sodium hydroxide. Read the volume added between the 2
1 ml of 0.1M sodium hydroxide is equivalent to 24.02 mg of C21H26Cl2F3N3S.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
41-39
Triflusal
corrected 1/01
COOH
F3C
C17H7F3O4
OAc
248.2
322-79-2
Triflusal complies with the requirements of the 3rd edition of the European Pharmacopoeia [1377]. These
Action and use Platelet aggregation inhibitor.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Triflusal contains not less than 98.5 per cent and not more than the equivalent of 101.5 per cent of
2-(acetyloxy)-4-(trifluoromethyl)benzoic acid, calculated with reference to the dried substance.
CHARACTERS
A white or almost white powder, practically insoluble in water, very soluble in ethanol, freely soluble
IDENTIFICATION
A. Dissolve 50.0 mg in ethanol R and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml of this
solution to 20.0 ml with ethanol R. Examined between 220 nm and 300 nm (2.2.25), the solution
prepared immediately before use shows two absorption maxima, at 223 nm and 278 nm. The specific
absorbances at the maxima are 63 to 73 and 350 to 370 respectively.
obtained with triflusal CRS. Examine the substances prepared as discs.
C. To 0.2 g add 2.0 ml of dilute sodium hydroxide solution R. Heat to boiling and maintain boiling for
15 min. Allow to cool and add 25.0 ml of dilute sulphuric acid R. A crystalline precipitate is formed.
Filter, wash the precipitate with water R and dry at 100C to 105C. The crystals melt (2.2.14)
between 176C and 178C
blank is red.
TESTS
solution is clear (2.2.1) and not more intensely coloured than reference solution B7 (Method II,
2.2.2).
2-Acetoxyterephthalic acid Examine by liquid chromatography (2.2.29).
Reference solution. Dissolve 40.0 mg of triflusal impurity A CRS in the mobile phase and dilute to
a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with aminopropylsilyl
as mobile phase at a flow rate of 1.2 ml/min a mixture of 25 volumes of 0.05M phosphate buffer
solution pH 4.5 R and 75 volumes of acetonitrile R,
Inject 20 l of the test solution and 20 l of the reference solution. When the chromatograms are
41-40
recorded under the prescribed conditions, the retention time of triflusal is about 2.4 min and the
retention time of 2-acetoxyterephthalic acid (triflusal impurity A) relative to triflusal is about 5.
Continue the chromatography for 20 min. In the chromatogram obtained with the test solution, the
area of the peak due to 2-acetoxyterephthalic acid is not greater than the area of the principal peak in
the chromatogram obtained with the reference solution (0.1 per cent).
4-(Trifluoromethyl)salicylic acid Dissolve 0.10 g in 15 ml of alcohol R. Add 15 ml of cold water R
and 0.5 ml of a 5 g/l solution of ferric ammonium sulphate R. Allow to stand for 1 min. The solution is
not more intensely coloured (Method II, 2.2.2) than that of a reference solution prepared as follows:
dissolve 10.0 mg of triflusal impurity B CRS in 100 ml of alcohol R. To 3 ml of this solution, add
0.1 ml of glacial acetic acid R, 0.5 ml of a 5 g/l solution of ferric ammonium sulphate R, 12 ml of
alcohol R and 15 ml of water R (0.3 per cent).
Heavy metals (2.4.8). Dissolve 2.0 g in 9 ml of alcohol R and dilute to 20 ml with water R. 12 ml
complies with the limit test B for heavy metals (10 ppm). Prepare the standard using lead standard
solution (1 ppm Pb) obtained by diluting lead standard solution (100 ppm Pb) R with a mixture of 6
volumes of water R and 9 volumes of alcohol R.
desiccator in vacuo over diphosphorus pentoxide R.
Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g using a platinum crucible.
ASSAY
Dissolve 0.200 g in 50.0 ml of ethanol R. Titrate with 0.1M sodium hydroxide, determining the endpoint potentiometrically (2.2.20).
1 ml of 0.1M sodium hydroxide is equivalent to 24.82 mg of C10H7F3O4.
STORAGE
Store in an airtight container, at a temperature not exceeding 25C.
IMPURITIES
COOH
HOOC
OAc
A. 2-(acetyloxy)benzene-1,4-dicarboxylic acid (2-acetoxyterephthalic acid),

COOH
F3C
OH
B. 2-hydroxy-4-(trifluoromethyl)benzoic acid (4-(trifluoromethyl)salicylic acid).

__________________________________________________________________________________________________________ Ph Eur
41-41
Medium-chain Triglycerides
1/01
Medium-chain Triglycerides comply with the requirements of the 3rd edition of the European Pharmacopoeia
When Medium-chain Triglycerides are prepared from the endosperm of Cocos nucifera L., the title
Fractionated Coconut Oil may be used.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Medium-chain triglycerides are obtained from the oil extracted from the hard, dried fraction of the
endosperm of Cocos nucifera L. or from the dried endosperm of Elaeis guineensis Jacq. They consist of
a mixture of triglycerides of saturated fatty acids, mainly of caprylic acid (C8H16O2) and of capric
acid (C10H20O2). They contain not less than 95 per cent of saturated fatty acids with 8 and 10
carbon atoms.
CHARACTERS
A colourless or slightly yellowish, oily liquid, practically insoluble in water, miscible with alcohol,
with methylene chloride, with light petroleum and with fatty oils.
IDENTIFICATION
Second identification: A, D.
A. Heat 3.0 g under a reflux condenser for 30 min with 50 ml of a mixture of equal volumes of
alcohol R and 2M alcoholic potassium hydroxide R. Reserve 10 ml of the mixture for identification test
D. To 40 ml of the mixture add 30 ml of water R, evaporate the alcohol and acidify the hot solution
with 25 ml of dilute hydrochloric acid R. After cooling, shake with 50 ml of peroxide-free ether R. Wash
the ether layer with three quantities, each of 10 ml, of sodium chloride solution R, dry over anhydrous
sodium sulphate R and filter. Evaporate the ether and determine the acid value (2.5.1) of the residue,
using 0.300 g. The acid value is 350 to 390.
B. It complies with the test for saponification value (see Tests).
C. It complies with the test for composition of fatty acids (see Tests).
D. Evaporate 10 ml of the alcoholic mixture obtained in identification test A to dryness on a waterbath. Transfer the residue into a test-tube, add 0.3 ml of sulphuric acid R and close the test-tube with
a stopper through which a U-shaped glass tube is inserted. One end of the U-tube is dipped into 3 ml
of a 10 g/l solution of tryptophan R in a mixture of equal volumes of sulphuric acid R and water R.
Heat the test-tube in a silicone-oil bath at 180C for 10 min and collect the liberated fumes in the
tryptophan reagent. Heat the tryptophan reagent on a water-bath for 1 min. A violet colour develops.
TESTS
Alkaline impurities Dissolve 2.00 g in a mixture of 1.5 ml of alcohol R and 3.0 ml of ether R. Add
0.05 ml of bromophenol blue solution R. Not more than 0.15 ml of 0.01M hydrochloric acid is required to
change the colour of the indicator to yellow.
Viscosity (2.2.9). 25 mPas to 33 mPas.
Hydroxyl value (2.5.3, Method A). Not more than 10.
(2.4.22, Method C).
41-42
a fused-silica column 30 m long and 0.32 mm in internal diameter the inner surface of which is
coated with a layer of macrogol 20,000 R (0.5 m thick),
Time
(min)
Column
Temperature
(C)
01
70
1 35 70 240
35 50 240
Injection port
250
Detector
250
Rate (C
per min)
5
Comment
isothermal
linear gradient
isothermal
The fatty acid fraction has the following composition:

myristic acid: not more than 1.0 per cent.
Heavy metals (2.4.8). For medium-chain triglycerides intended for use other than parenteral
nutrition, 2.0 g complies with limit test D for heavy metals (10 ppm). Prepare the standard using
2 ml of lead standard solution (10 ppm Pb) R.
Chromium For medium-chain triglycerides intended for use in parenteral nutrition, not more than
0.05 ppm of Cr, determined by atomic absorption spectrometry (Method II, 2.2.23).
Test solution. Dissolve 2.0 g of the substance to be examined in di-isobutyl ketone R and dilute to
Reference solutions. Prepare three reference solutions by dissolving 2.0 g of the substance to be
examined in the minimum volume of di-isobutyl ketone R, adding 0.5 ml, 1.0 ml and 2.0 ml,
respectively, of chromium standard solution (0.1 ppm Cr) R and diluting to 10.0 ml with di-isobutyl
ketone R.
Measure the absorbance at 357.8 nm using a chromium hollow-cathode lamp as a source of
radiation, a graphite furnace as atomic generator and argon R as the carrier gas.
Copper For medium-chain triglycerides intended for use in parenteral nutrition, not more than
0.1 ppm of Cu, determined by atomic absorption spectrometry (Method II, 2.2.23).
respectively, of copper standard solution (0.1 ppm Cu) R and diluting to 10.0 ml with di-isobutyl
ketone R.
Measure the absorbance at 324.7 nm using a copper hollow-cathode lamp as a source of radiation,
a graphite furnace as atomic generator and argon R as the carrier gas.
Lead For medium-chain triglycerides intended for use in parenteral nutrition, not more than
0.1 ppm of Pb, determined by atomic absorption spectrometry (Method II, 2.2.23).
respectively, of lead standard solution (0.1 ppm Pb) R and diluting to 10.0 ml with di-isobutyl ketone R.
Measure the absorbance at 283.3 nm using a lead hollow-cathode lamp as a source of radiation, a
graphite furnace coated inside with palladium carbide as atomic generator and argon R as the carrier
gas. Calcination is carried out in the presence of oxygen at a temperature below 800C.
Nickel For medium-chain triglycerides intended for use in parenteral nutrition, not more than
0.1 ppm of Ni, determined by atomic absorption spectrometry (Method II, 2.2.23).
41-43
respectively, of nickel standard solution (0.1 ppm Ni) R and diluting to 10.0 ml with di-isobutyl
ketone R.
Measure the absorbance at 232 nm using a nickel hollow-cathode lamp as a source of radiation, a
graphite furnace as atomic generator and argon R as the carrier gas.
Tin For medium-chain triglycerides intended for use in parenteral nutrition, not more than 0.1 ppm
of Sn, determined by atomic absorption spectrometry (Method II, 2.2.23).
respectively, of tin standard solution (0.1 ppm Sn) R and diluting to 10.0 ml with di-isobutyl ketone R.
Measure the absorbance at 286.3 nm using a tin hollow-cathode lamp as a source of radiation, a
graphite furnace coated inside with palladium carbide as atomic generator and argon R as the carrier
gas.
STORAGE
Store in a well-filled container, protected from light.
LABELLING
The label states, where applicable, that the substance is intended for use in parenteral nutrition.
__________________________________________________________________________________________________________ Ph Eur
41-44
Trihexyphenidyl Hydrochloride / Benzhexol Hydrochloride

For the purposes of product labelling in the United Kingdom the pair of names given above shall be used
OH
N
,HCl
and enantiomer
C20H31NO,HCl
337.9
52-49-3
Definition Trihexyphenidyl Hydrochloride is 1-cyclohexyl-1-phenyl-3-piperidinopropan-1-ol

Characteristics A white or creamy white, crystalline powder; odourless or almost odourless.
Slightly soluble in water; soluble in chloroform, in ethanol (96%) and in methanol.
Identification A. The infrared absorption spectrum, Appendix II A, is concordant with the reference
spectrum of trihexyphenidyl hydrochloride (RS 352).
B. Dissolve 0.5 g in 5 ml of warm methanol and make just alkaline to litmus paper with 5M sodium
hydroxide. A precipitate is produced, which, after recrystallisation from methanol, has a melting point of
Acidity Dissolve 1 g in 50 ml of carbon dioxide-free water with the aid of heat. Cool to room
temperature and dilute to 100 ml with the same solvent. The pH of the resulting solution is 5.2 to
6.2, Appendix V L.
Piperidylpropiophenone Dissolve 0.1 g in a mixture of 40 ml of water and 1 ml of 1M hydrochloric
acid with the aid of heat, cool and add sufficient water to produce 100 ml. The absorbance of the
resulting solution at 247 nm is not more than 0.5, calculated with reference to the dried substance,
Appendix II B.
Related substances Carry out the method for liquid chromatography, Appendix III D, using 20 l of
the following solutions in the mobile phase containing (1) 0.20% w/v of the substance being examined, (2) 0.0010% w/v of the substance being examined and (3) 0.0020% w/v of the substance being
examined and 0.0010% w/v of 3-piperidylpropiophenone hydrochloride BPCRS.
(15 cm 4.6 mm) packed with stationary phase C (5 m) (Resolve C18 is suitable), (b) as the mobile
phase with a flow rate of 2 ml per minute a mixture of 800 volumes of acetonitrile, 200 volumes of
water and 0.2 volumes of triethylamine, the pH of the mixture being adjusted to 4.0 with
orthophosphoric acid, and (c) a detection wavelength of 210 nm.
The test is not valid unless the resolution factor between the two principal peaks in the chromatogram obtained with solution (3) is greater than 4.0.
The area of any secondary peak in the chromatogram obtained with solution (1) is not greater than
the area of the peak in the chromatogram obtained with solution (2) (0.5%) and the total area of all
secondary peaks is not greater than twice the area of the peak in the chromatogram obtained with
solution (2) (1%).
1 g.
C20H31NO,HCl.
Preparation
Trihexyphenidyl Tablets/Benzhexol Tablets
41-45
Trimethadione
Me
O
O
O
Me
Me
C6H9NO3
143.1
127-48-0
Trimethadione complies with the requirements of the 3rd edition of the European Pharmacopoeia [0440].
When troxidone is prescribed or demanded, Trimethadione shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Trimethadione contains not less than 98.0 per cent and not more than the equivalent of 102.0 per
cent of 3,5,5-trimethyloxazolidine-2,4-dione, calculated with reference to the dried substance.
CHARACTERS
Colourless or almost colourless crystals, soluble in water, very soluble in alcohol and in ether.
IDENTIFICATION
A. Melting point (2.2.14): 45C to 47C, determined without previous drying.
obtained with trimethadione CRS. Examine the substances prepared as discs using 3 mg of the
substance to be examined and of the reference substance, respectively, in 0.4 g of potassium
bromide R.
C. To 2 ml of solution S (see Tests) add 1 ml of barium hydroxide solution R. A white precipitate is
formed, which dissolves on addition of 1 ml of dilute hydrochloric acid R.
D. Dissolve 0.3 g in a mixture of 5 ml of alcoholic potassium hydroxide solution R and 5 ml of alcohol R
and allow to stand for 10 min. Add 0.05 ml of phenolphthalein solution R1 and neutralise carefully with
hydrochloric acid R. Evaporate to dryness on a water-bath and shake the residue with four quantities,
each of 5 ml, of ether R. Filter the combined ether layers and evaporate to dryness. The residue,
recrystallised from 5 ml of toluene R and dried, melts (2.2.14) at about 80C.
TESTS
indicator.
Loss on drying (2.2.32). Not more than 0.5 per cent, determined on 1.00 g by drying in a desiccator
over anhydrous silica gel R for 6 h.
ASSAY
Examine by gas chromatography (2.2.28), using decanol R as the internal standard.
Internal standard solution. Dissolve 0.125 g of decanol R in ethanol R and dilute to 25 ml with the same
solvent.
dilute to 10.0 ml with the same solution.
Reference solution. Dissolve 0.100 g of trimethadione CRS in the internal standard solution and dilute
to 10.0 ml with the same solution.
41-46
a stainless steel column 0.75 m long and 3 mm in internal diameter packed with styrenedivinylbenzene copolymer R (125 m to 150 m),
Maintain the temperature of the column at 210C, that of the injection port at 240C, and that of the
detector at 270C. Inject 1 l of each solution.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
41-47
Trimethoprim
NH2
OMe
N
H2N
OMe
OMe
C14H18N4O3
290.3
738-70-5
Trimethoprim complies with the requirements of the 3rd edition of the European Pharmacopoeia [0060].
Preparations
Co-trimoxazole Intravenous Infusion
Co-trimoxazole Oral Suspension
Paediatric Co-trimoxazole Oral Suspension
Co-trimoxazole Tablets
Dispersible Co-trimoxazole Tablets
Paediatric Co-trimoxazole Tablets
Trimethoprim Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Trimethoprim contains not less than 98.5 per cent and not more than the equivalent of 101.0 per
cent of 5-(3,4,5-trimethoxybenzyl)pyrimidine-2,4-diamine, calculated with reference to the dried
substance.
CHARACTERS
A white or yellowish-white powder, very slightly soluble in water, slightly soluble in alcohol.
IDENTIFICATION
B. Dissolve 20 mg in 0.1M sodium hydroxide and dilute to 100.0 ml with the same solvent. Dilute
1.0 ml of this solution to 10.0 ml with 0.1M sodium hydroxide. Examined between 230 nm and
350 nm (2.2.25), the solution shows a single absorption maximum, at 287 nm. The specific absorbance at the maximum is about 240 to 250.
obtained with trimethoprim CRS.
D. Dissolve about 25 mg, heating if necessary, in 5 ml of 0.005M sulphuric acid and add 2 ml of a
16 g/l solution of potassium permanganate R in 0.1M sodium hydroxide. Heat to boiling and add to the
hot solution 0.4 ml of formaldehyde R. Mix, add 1 ml of 0.5M sulphuric acid, mix and heat again to
boiling. Cool and filter. To the filtrate add 2 ml of methylene chloride R and shake vigorously. The
organic layer, examined in ultraviolet light at 365 nm, shows a green fluorescence.
TESTS
Appearance of solution Dissolve 0.5 g in 10 ml of a mixture of 1 volume of water R, 4.5 volumes of
methanol R and 5 volumes of methylene chloride R. The solution is not more intensely coloured than
Related substances
A. Examine by liquid chromatography (2.2.29).
Reference solution (b). Dissolve 5.0 mg of trimethoprim CRS and 2.5 mg of trimethoprim impurity E CRS
in the mobile phase and dilute to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to
41-48
a stainless steel column 0.25 m long and 4.0 mm in internal diameter packed with base-deactivated
as mobile phase at a flow rate of 1.3 ml/min, a mixture of 30 volumes of methanol R and 70
volumes of a 1.4 g/l solution of sodium perchlorate R adjusted to pH 3.6 with phosphoric acid R,
Inject 20 l of the test solution and 20 l of reference solution (b). Continue the chromatography of
the test solution for eleven times the retention time of trimethoprim. When the chromatograms are
recorded in the prescribed conditions, the relative retention times are:
Substance
retention time
Correction factor
Trimethoprim
Impurity A
Impurity B
Impurity C
Impurity D
Impurity E
Impurity F
Impurity G
Impurity J
1.0 (Rt = 5.2 min)

1.5
2.3
0.8
2.0
0.9
4.0
2.1
2.7
1
0.43
0.53
0.66
The test is not valid unless: in the chromatogram obtained with reference solution (b), the resolution
between the two principal peaks is at least 2.5. In the chromatogram obtained with the test solution:
the area of any peak apart from the principal peak, is not greater than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.1 per cent), applying the
correction factors indicated in the table for impurities B, E and J; the sum of the areas of all the peaks
apart from the principal peak, is not greater than 0.4 times the area of the principal peak in the
chromatogram obtained with reference solution (a) (0.2 per cent), applying the correction factors
indicated in the table for impurities B, E and J. Disregard any peak with an area less than 0.04 times
the area of the principal peak in the chromatogram obtained with reference solution (a) and any peak
corresponding to impurity H (relative retention time about 10.3).
Reference solution (b). Dissolve 5.0 mg of trimethoprim CRS and 5.0 mg of trimethoprim impurity B CRS
a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with cyanopropylsilyl
silica gel for chromatography R (5 m) with a specific surface area of 350 m2/g and a pore diameter
of 10 nm,
as mobile phase at a flow rate of 0.8 ml/min, a mixture prepared as follows: dissolve 1.14 g of
sodium hexanesulphonate R in 600 ml of a 13.6 g/l solution of potassium dihydrogen phosphate R;
adjust to pH 3.1 using phosphoric acid R and mix with 400 volumes of methanol R,
principal peaks in the chromatogram obtained is at least 50 per cent of the full scale of the recorder.
Inject 20 l of the test solution and 20 l of reference solution (b). Continue the chromatography of
the test solution for six times the retention time of trimethoprim. When the chromatograms are
recorded in the prescribed conditions, the relative retention times are:
Substance
retention time
Correction factor
Trimethoprim
Impurity B
Impurity H
Impurity I
1.0 (Rt = 4.3 min)

1.3
1.8
4.9
1
0.50
0.28
The test is not valid unless, in the chromatogram obtained with reference solution (b), the resolution
between the two principal peaks is at least 2.0. In the chromatogram obtained with the test solution:
41-49
the area of any peak apart from the principal peak, is not greater than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.1 per cent), applying the
correction factors indicated in the table for impurities H and I; the sum of the areas of all the peaks
apart from the principal peak, is not greater than 0.4 times the area of the principal peak in the
chromatogram obtained with reference solution (a) (0.2 per cent), applying the correction factors
indicated in the table for impurities H and I. Disregard any peak with an area less than 0.04 times the
area of the principal peak in the chromatogram obtained with reference solution (a).
100C to 105C.
ASSAY
IMPURITIES
by liquid chromatography A
OMe
MeO
NHMe
N
N
MeO
NH2
A. N2-methyl-5-(3,4,5-trimethoxybenzyl)pyrimidine-2,4-diamine,
OMe
MeO
NH2
N
MeO
O
NH2
B. (2,4-diaminopyrimidin-5-yl)(3,4,5-trimethoxyphenyl)methanone,
OMe
eO
NHMe
and enantiomer
eO
H OH NH2
C. (RS)-(2,4-diaminopyrimidin-5-yl)(3,4,5-trimethoxyphenyl)methanol,
OMe
MeO
NH2
N
MeO
OH
D. 2-amino-5-(3,4,5-trimethoxybenzyl)pyrimidin-4-ol,
OMe
MeO
OH
N
MeO
NH2
E. 4-amino-5-(3,4,5-trimethoxybenzyl)pyrimidine-2-ol,
OMe
MeO
Br
NH2
N
NH2
F. 5-(3-bromo-4,5-dimethoxybenzyl)pyrimidine-2,4-diamine,
41-50
OMe
EtO
NH2
N
MeO
NH2
G. 5-(4-ethoxy-3,5-dimethoxybenzyl)pyrimidine-2,4-diamine,
OMe
MeO
MeO
COOH
J. 3,4,5-trimethoxybenzoic acid.
by liquid chromatography B
OMe
MeO
MeO
COOMe
H. methyl 3,4,5-trimethoxybenzoate (also detected by liquid chromatography A),

OMe
MeO
H
N
MeO
CN
I. 3-(phenylamino)-2-(3,4,5-trimethoxybenzyl)prop-2-enenitrile
__________________________________________________________________________________________________________ Ph Eur
41-51
Trimipramine Maleate
COOH
COOH
N
NMe2
H
Me
and enantiomer
C20H26N2,C4H4O4
410.5
521-78-8
Trimipramine Maleate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Trimipramine Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Trimipramine maleate contains not less than 98.0 per cent and not more than the equivalent of
101.0 per cent of (RS)-5-(3-dimethylamino-2-methylpropyl)-10,11-dihydro-5H-dibenzo[b,f]azepine
maleate, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, slightly soluble in water and in alcohol, practically
insoluble in ether.
IDENTIFICATION
350 nm (2.2.25), the solution shows an absorption maximum at 250 nm and a shoulder at 270 nm.
obtained with trimipramine maleate CRS.
E. Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating substance.
the same solvent.
Reference solution. Dissolve 56 mg of maleic acid R in methanol R and dilute to 10 ml with the same
solvent.
Apply separately to the plate as 10 mm bands 5 l of each solution. Develop over a path of 12 cm
using a mixture of 3 volumes of water R, 7 volumes of anhydrous formic acid R and 90 volumes of diisopropyl ether R. Dry the plate in a current of air for a few minutes and then at 120C for 10 min and
examine in ultraviolet light at 254 nm. The chromatogram obtained with the test solution shows a
zone on the starting line and another zone which is similar in position and size to the principal zone
TESTS
Appearance of solution Dissolve 2.5 g in chloroform R and dilute to 25 ml with the same solvent.
The solution is not more intensely coloured than reference solution BY5 (Method II, 2.2.2).
coating substance.
41-52
Reference solution (a). Dissolve 25 mg of trimipramine maleate CRS in methanol R and dilute to 10 ml
Reference solution (c). Dilute 1 ml of reference solution (a) to 25 ml with methanol R.
Reference solution (d). Dissolve 10 mg of iminodibenzyl R in methanol R and dilute to 100 ml with the
0.7 volumes of concentrated ammonia R, 10 volumes of ethanol R and 90 volumes of toluene R. Allow
the plate to dry in air for 15 min, spray with a 5 g/l solution of potassium dichromate R in a mixture of
1 volume of sulphuric acid R and 4 volumes of water R and examine immediately. In the chromatogram obtained with test solution (a): any spot corresponding to iminodibenzyl is not more intense
than the spot in the chromatogram obtained with reference solution (d) (0.2 per cent); any spot,
apart from the principal spot and the spot corresponding to iminodibenzyl, is not more intense than
the spot in the chromatogram obtained with reference solution (b) (0.5 per cent) and at most three
such spots are more intense than the spot in the chromatogram obtained with reference solution (c)
(0.2 per cent). Disregard any spot at the starting point.
100C to 105C.
ASSAY
STORAGE
__________________________________________________________________________________________________________ Ph Eur
41-53
Triprolidine Hydrochloride
N
H
N
,HCl
Me
C19H22N2,HCl,H2O
332.9
6138-79-0
Definition Triprolidine Hydrochloride is (E)-2-(3-pyrrolidin-1-yl-1-p-tolylprop-1-enyl)pyridine

hydrochloride monohydrate. It contains not less than 98.5% and not more than 101.0% of
C19H22N2,HCl, calculated with reference to the anhydrous substance.
Freely soluble in water; very soluble in chloroform; freely soluble in ethanol (96%); practically
insoluble in ether.
Identification
triprolidine hydrochloride (RS 356).
silica gel F254 precoated plate (Merck silica gel 60 F254 plates are suitable) and a mixture of equal
volumes of butan-2-one and dimethylformamide as the mobile phase. Apply separately to the plate 5 l
of each of three solutions in methanol containing (1) 1.0% w/v of the substance being examined, (2)
0.020% w/v of Z-triprolidine BPCRS and (3) 0.010% w/v of the substance being examined. After
removal of the plate, allow it to dry in air and examine under ultraviolet light (254 nm). In the chromatogram obtained with solution (1) any spot corresponding to Z-triprolidine is not more intense than
the spot in the chromatogram obtained with solution (2) and any other secondary spot is not more
Assay Carry out Method I for non-aqueous titration, Appendix VIII A, using 0.25 g dissolved in a
mixture of 50 ml of anhydrous acetic acid and 0.5 ml of acetic anhydride and crystal violet solution as
indicator. Each ml of 0.1M perchloric acid VS is equivalent to 15.74 mg of C19H22N2,HCl.
Preparation
Triprolidine Tablets
41-54
Trometamol
H2N
HO
C4H11NO3
OH
OH
121.1
77-86-1
Trometamol complies with the requirements of the 3rd edition of the European Pharmacopoeia [1053]. These
Action and use Alkalinising agent.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Trometamol contains not less than 99.0 per cent and not more than the equivalent of 100.5 per cent
of aminomethylidynetri(methanol), calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder, or colourless crystals, freely soluble in water, sparingly soluble in alcohol,
very slightly soluble in ethyl acetate.
IDENTIFICATION
A. Solution S (see Tests) is strongly alkaline (2.2.4).
obtained with trometamol CRS.
TESTS
pH (2.2.3). The pH of freshly prepared solution S is 10.0 to 11.5.
coating substance. Wash the plate with methanol R before applying the solutions.
Test solution (a). Dissolve 0.20 g in 1 ml of water R, with heating, and dilute to 10 ml with
methanol R.
Reference solution (a). Dissolve 20 mg of trometamol CRS in methanol R and dilute to 10 ml with the
same solvent.
Reference solution (b). Dilute 1 ml of test solution (a) to 100 ml with methanol R.
10 volumes of dilute ammonia R1 and 90 volumes of 2-propanol R. Dry the plate at 100C to 105C.
Spray with a 5 g/l solution of potassium permanganate R in a 10 g/l solution of sodium carbonate R.
After about 10 min examine in daylight. Any spot in the chromatogram obtained with test solution
with reference solution (b) (1.0 per cent).
Chlorides (2.4.4). To 10 ml of solution S add 2.5 ml of dilute nitric acid R and dilute to 15 ml with
Heavy metals (2.4.8). Dissolve 2.0 g in 10 ml of water R. Neutralise the solution with hydrochloric
acid R1 and dilute to 20 ml with water R. 12 ml of the solution complies with limit test A for heavy
Iron (2.4.9). Dissolve 1.0 g in water R and dilute to 10 ml with the same solvent. The solution
complies with the limit test for iron (10 ppm).
100C to 105C.
41-55
without a further appropriate procedure for the removal of bacterial endotoxins, not more than 0.03
I.U. of endotoxin per milligram.
ASSAY
Dissolve 0.100 g in 20 ml of water R. Add 0.2 ml of methyl red solution R. Titrate with 0.1M hydrochloric acid until the colour changes from yellow to red.
1 ml of 0.1M hydrochloric acid is equivalent to 12.11 mg of C4H11NO3.
STORAGE
LABELLING
The label states:
IMPURITIES
O2N
HO
OH
OH
A. nitromethylidynetri(methanol).
__________________________________________________________________________________________________________ Ph Eur
41-56
Tropicamide
N
Et
CH2OH
N
O
and enantiomer
C17H20N2O2
284.4
1508-75-4
Tropicamide complies with the requirements of the 3rd edition of the European Pharmacopoeia [1159]. These
Action and use Mydriatic; cycloplegic.
Preparation
Tropicamide Eye Drops
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Tropicamide contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of (RS)-N-ethyl-3-hydroxy-2-phenyl-N-(pyrid-4-ylmethyl)propionamide, calculated with reference to
CHARACTERS
A white or almost white, crystalline powder, slightly soluble in water, freely soluble in alcohol and in
methylene chloride.
IDENTIFICATION
B. Dissolve 20.0 mg in 0.1M hydrochloric acid and dilute to 50.0 ml with the same acid. Dilute 2.0 ml
(2.2.25), the solution shows an absorption maximum at 254 nm. The specific absorbance at the
maximum is 170 to 190.
obtained with tropicamide CRS. Examine the substances prepared as discs.
chromatogram obtained with test solution (b) is similar in position and size to the spot in the chromatogram obtained with reference solution (a).
E. Dissolve about 5 mg in 3 ml of a mixture of 9 ml of acetic anhydride R, 1 ml of acetic acid R and
0.1 g of citric acid R. Heat on a water-bath for 5 min to 10 min. A reddish-yellow colour is produced.
TESTS
Reference solution (a). Dissolve 10 mg of tropicamide CRS in methylene chloride R and dilute to 10 ml
Reference solution (b). Dilute 1 ml of test solution (b) to 10 ml with methylene chloride R.
Reference solution (c). Dilute 2 ml of reference solution (b) to 5 ml with methylene chloride R.
Reference solution (d). Dissolve 20 mg of 4-[(ethylamino)methyl]pyridine R in methylene chloride R and
dilute to 20 ml with the same solvent. Dilute 1 ml of the solution and 1 ml of reference solution (a)
41-57
0.5 volumes of concentrated ammonia R, 5 volumes of methanol R and 95 volumes of methylene
chloride R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Any spot in the
such spot is more intense than the spot in the chromatogram obtained with reference solution (c)
(0.2 per cent). The test is not valid unless the chromatogram obtained with reference solution (d)
Tropic acid To 10.0 mg add 5 mg of sodium tetraborate R and 0.35 ml of a freshly prepared 100 g/l
solution of dimethylaminobenzaldehyde R in a mixture of 1 volume of water R and 9 volumes of
sulphuric acid R. Heat on a water-bath for 3 min. Cool in ice water and add 5 ml of acetic anhydride R.
No violet-red colour develops (0.05 per cent).
Chlorides (2.4.4). Dissolve 1.0 g with heating in 8 ml of acetic acid R, cool and dilute to 10 ml with
the same acid. Dilute 5 ml of the solution to 15 ml with water R. The solution complies with the limit
test for chlorides (100 ppm).
80C at a pressure not exceeding 0.7 kPa for 4 h.
ASSAY
Dissolve 0.200 g in 50 ml of glacial acetic acid R. Add 0.2 ml of naphtholbenzein solution R and titrate
with 0.1M perchloric acid until the colour changes from orange to green.
STORAGE
IMPURITIES
N
NHEt
A. 4-[(ethylamino)methyl]pyridine,
N
Et
CH2
N
O
B. N-ethyl-2-phenyl-N-(pyrid-4-ylmethyl)prop-2-enamide,
H COOH
HO
and enantiomer
C. (RS)-3-hydroxy-2-phenylpropionic acid (tropic acid).

__________________________________________________________________________________________________________ Ph Eur
41-58
Trypsin
9002-07-7
Trypsin complies with the requirements of the 3rd edition of the European Pharmacopoeia [0694]. These
Action and use Proteolytic enzyme.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Trypsin is a proteolytic enzyme obtained by the activation of trypsinogen extracted from the pancreas
of healthy mammals. It has an activity not less than 0.5 microkatal per milligram, calculated with
reference to the dried substance. In solution, it has maximum enzymic activity at pH 8; the activity is
reversibly inhibited at pH 3, at which pH it is most stable.
PRODUCTION
The animals from which trypsin is derived must fulfil the requirements for the health of animals
suitable for human consumption to the satisfaction of the competent authority.
It must have been shown to what extent the method of production allows inactivation or removal
of any contamination by viruses or other infectious agents.
The method of manufacture is validated to demonstrate that the product, if tested, would comply
with the following test:
Histamine (2.6.10). Not more than 1 g of histamine base per 0.2 microkatal of trypsin activity. Use
a 10 g/l solution of the substance to be examined in 0.0015M borate buffer solution pH 8.0 R inactivated
by heating on a water-bath for 30 min. Carry out dilutions with a 9 g/l solution of sodium chloride R.
CHARACTERS
A white or almost white, crystalline or amorphous powder, sparingly soluble in water. The
amorphous form is hygroscopic.
IDENTIFICATION
A. Dilute 1 ml of solution S (see Tests) to 100 ml with water R. In a depression in a white spot-plate,
mix 0.1 ml of this solution with 0.2 ml of tosylarginine methyl ester hydrochloride solution R. A reddishviolet colour develops within 3 min.
B. Dilute 0.5 ml of solution S to 5 ml with water R. Add 0.1 ml of a 20 g/l solution of tosyl-lysylchloromethane hydrochloride R. Adjust to pH 7.0, shake for 2 h and dilute to 50 ml with water R. In
one of the depressions of a white spot-plate, mix 0.1 ml of this solution with 0.2 ml of tosylarginine
methyl ester hydrochloride solution R. No reddish-violet colour develops within 3 min.
TESTS
Appearance of solution Solution S is not more opalescent than reference suspension III (2.2.1).
Absorbance (2.2.25). Dissolve 30.0 mg in 0.001M hydrochloric acid and dilute to 100.0 ml with the
same acid. The solution shows an absorption maximum at 280 nm and a minimum at 250 nm. The
specific absorbance at the maximum is 13.5 to 16.5 and that at the minimum is not greater than 7.0.
Chymotrypsin To 1.8 ml of buffer solution pH 8.0 R add 7.4 ml of water R and 0.5 ml of 0.2M
acetyltyrosine ethyl ester R. While shaking the solution, add 0.3 ml of solution S and start a stop-watch.
After exactly 5 min, measure the pH (2.2.3) (test solution). Prepare a reference solution in the same
manner, replacing solution S by 0.3 ml of a 0.5 g/l solution of chymotrypsin BRP and measure the pH
(2.2.3) exactly 5 min after adding the chymotrypsin. The pH of the test solution is higher than that of
per gram, determined by plate count. It complies with the tests for Escherichia coli and Salmonella
(2.6.13).
ASSAY
The activity of trypsin is determined by comparing the rate at which it hydrolyses benzoylarginine ethyl
ester hydrochloride R with the rate at which trypsin BRP hydrolyses the same substrate in the same
41-59
conditions.
Apparatus. Use a reaction vessel of about 30 ml capacity provided with:
a device that will maintain a temperature of 25.0 0.1C,
a stirring device (for example, a magnetic stirrer),
a lid with holes for the insertion of electrodes, the tip of a burette, a tube for the admission of
nitrogen and the introduction of reagents.
An automatic or manual titration device may be used. For the latter, the burette is graduated in
0.005 ml and the pH meter is provided with a wide-range scale and glass-calomel electrodes.
Test solution. Dissolve sufficient of the substance to be examined in 0.001M hydrochloric acid and dilute
to 25.0 ml with the same acid in order to obtain a solution containing approximately 700 nanokatals
per millilitre.
Reference solution. Dissolve 25.0 mg of trypsin BRP in 0.001M hydrochloric acid and dilute to 25.0 ml
with the same acid.
Store the solutions at 0C to 5C. Warm 1 ml of each solution to about 25C over 15 min and use
50 l of each solution for each titration. Carry out the titration in an atmosphere of nitrogen.
Transfer 10.0 ml of 0.0015M borate buffer solution pH 8.0 R to the reaction vessel and, while stirring,
add 1.0 ml of a freshly prepared 6.86 g/l solution of benzoylarginine ethyl ester hydrochloride R. When
the temperature is steady at 25.0 0.1C (after about 5 min) adjust the pH to exactly 8.0 with 0.1M
sodium hydroxide. Add 50 l of the test solution and start a timer. Maintain the pH at 8.0 by the
addition of 0.1M sodium hydroxide the tip of the microburette being immersed in the solution; note the
volume added every 30 s. Follow the reaction for 8 min. Calculate the volume of 0.1M sodium
hydroxide used per second. Carry out a titration in the same manner using the reference solution and
calculate the volume of 0.1M sodium hydroxide used per second.
Calculate the activity in microkatals per milligram using the expression:
m V
A
m V
m = mass of the substance to be examined, in milligrams,
m = mass of trypsin BRP, in milligrams,
V = volume of 0.1M sodium hydroxide used per second by the test solution,
V = volume of 0.1M sodium hydroxide used per second by the reference solution,
A = activity of trypsin BRP, in microkatals per milligram.
STORAGE
LABELLING
The label states the activity in microkatals per milligram.
__________________________________________________________________________________________________________ Ph Eur
41-60
Tryptophan
H
N
NH2
COOH
C11H12N2O2
204.2
73-22-3
Tryptophan complies with the requirements of the 3rd edition of the European Pharmacopoeia [1272]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Tryptophan contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent
of (S)-2-amino-3-(1H-indol-3-yl)propanoic acid, calculated with reference to the dried substance.
PRODUCTION
When Tryptophan is produced by a process involving fermentation steps, it complies with the
CHARACTERS
A white or almost white, crystalline or amorphous powder, sparingly soluble in water, slightly soluble
in alcohol, practically insoluble in ether. It dissolves in dilute solutions of the alkali hydroxides and
mineral acids.
IDENTIFICATION
B. Examine the substance by infrared absorption spectrophotometry (2.2.24), comparing with the
spectrum obtained with tryptophan CRS. Examine the substances prepared as discs.
D. Dissolve about 20 mg in 10 ml of water R. Add 5 ml of dimethylaminobenzaldehyde solution R6 and
2 ml of hydrochloric acid R1. Heat on a water-bath. A purple-blue colour develops.
TESTS
acid. The solution is clear (2.2.1) and not more intensely coloured than reference solution BY6
(Method II, 2.2.2).
Specific optical rotation (2.2.7). Dissolve 0.25 g in water R, heating on a water-bath, if necessary,
and dilute to 25.0 ml with the same solvent. The specific optical rotation is 30.0 to 33.0, calculated with reference to the dried substance.
gel plate R.
glacial acetic acid R and water R and dilute to 10 ml with the same mixture of solvents.
Test solution (b). Dilute 1 ml of test solution (a) to 50 ml with a mixture of equal volumes of glacial
acetic acid R and water R.
Reference solution (a). Dissolve 10 mg of tryptophan CRS in a mixture of equal volumes of glacial acetic
acid R and water R and dilute to 50 ml with the same mixture of solvents.
glacial acetic acid R and water R.
Reference solution (c). Dissolve 10 mg of tryptophan CRS and 10 mg of tyrosine CRS in a mixture of
equal volumes of glacial acetic acid R and water R and dilute to 25 ml with the same mixture of
solvents.
41-61
plate to dry in air. Spray with ninhydrin solution R and heat at 100C to 105C for 15 min. Any spot
separated spots.
1,1-Ethylidenebistryptophan and other related substances Examine by liquid chromatography
(2.2.29).
Buffer solution pH 2.3. Dissolve 3.90 g of sodium dihydrogen phosphate R in 1000 ml of water R. Add
about 700 ml of a 2.9 g/l solution of phosphoric acid R and adjust to pH 2.3 with the same acidic
solution.
Standard solution. Dissolve 10.0 mg of N-acetyltryptophan R in a mixture of 10 volumes of acetonitrile R and 90 volumes of water R and dilute to 100.0 ml with the same mixture of solvents. Dilute
2.0 ml of the solution to 100.0 ml with the same mixture of solvents.
Test solution (a). Dissolve 0.10 g of the substance to be examined in a mixture of 10 volumes of
acetonitrile R and 90 volumes of water R and dilute to 10.0 ml the same mixture of solvents.
Test solution (b). Dissolve 0.10 g of the substance to be examined in the standard solution and dilute
to 10.0 ml with the same solution.
Reference solution (a). Dissolve 1.0 mg of 1,1-ethylidenebistryptophan R in a mixture of 10 volumes of
Reference solution (b). Dilute 10.0 ml of reference solution (a) to 50.0 ml with the standard solution.
Reference solution (c). Dilute 10.0 ml of reference solution (a) to 50.0 ml with a mixture of 10 volumes
of acetonitrile R and 90 volumes of water R.
Reference solution (d). Dissolve 0.10 g of the substance to be examined in reference solution (c) and
dilute to 10.0 ml with the same solution.
Reference solution (e). Dilute 1.0 ml of reference solution (c) to 10.0 ml with a mixture of 10 volumes
of acetonitrile R and 90 volumes of water R.
as mobile phase at a flow rate of 0.7 ml per minute the following solutions:
Mobile phase A. A mixture of 115 volumes of acetonitrile R and 885 volumes of buffer solution pH
2.3,
Mobile phase B. A mixture of 350 volumes of acetonitrile R and 650 volumes of buffer solution pH
2.3,
a gradient programme using the following conditions:
Interval
(min)
Mobile phase A
(per cent V/V)

(per cent V/V)
010
1045
4565
6566
6680
100
1000
0
0100
100
0
0100
100
1000
0
isocratic
linear gradient
isocratic
linear gradient
equilibration

Inject 20 l of reference solution (b), 20 l of reference solution (d) and 20 l of reference solution
(e).
When the chromatograms are recorded in the prescribed conditions, the retention times are about
8 min for tryptophan, about 29 min for N-acetyltryptophan and about 34 min for 1,1ethylidenebistryptophan. Adjust the sensitivity of the system so that the height of the peak due to
N-acetyltryptophan in the chromatogram obtained with reference solution (b) is at least 50 per cent
of the full scale of the recorder.
The test is not valid unless: in the chromatogram obtained with reference solution (b), the resolution between the peaks corresponding to N-acetyltryptophan and 1,1-ethylidenebistryptophan is at
least 8.0. If necessary, adjust the time programme for the elution gradient. An increase in the duration of elution with mobile phase A produces longer retention times and better resolution; the chromatogram obtained with reference solution (e) has a signal-to-noise ratio of at least 15.
Inject 20 l of test solution (a) and 20 l of test solution (b). Check that in the chromatogram
obtained with test solution (a) there is no peak with the same retention time as N-acetyltryptophan
41-62
(in such case correct the area of the N-acetyltryptophan peak). In the chromatogram obtained with
test solution (b) the area of any peak corresponding to 1,1-ethylidenebistryptophan is not greater
(e) (10 ppm). The sum of the areas of any peaks with retention times less than that of tryptophan is
not greater than 0.5 times the area of the peak corresponding to N-acetyltryptophan in the chromatogram obtained with reference solution (b) (100 ppm). The sum of the areas of any peaks with a
retention time greater than that of tryptophan, apart from N-acetyltryptophan, and up to 1.8 times
the retention time of N-acetyltryptophan, is not greater than 1.5 times the area of the peak due to
N-acetyltryptophan in the chromatogram obtained with reference solution (b) (300 ppm). Disregard
any peak with an area less than 0.02 times that of the peak due to N-acetyltryptophan in the chromatogram obtained with reference solution (b).
The solution without any further addition of nitric acid (200 ppm), complies with the limit test for
chlorides.
Sulphates (2.4.13). Dissolve 0.5 g in a mixture of 5 volumes of dilute hydrochloric acid R and 25
volumes of distilled water R and dilute to 15 ml with the same mixture of solvents. The solution
Heavy metals (2.4.8). 20 g complies with limit test D for heavy metals (10 ppm). Prepare the
100C to 105C.
ASSAY
Dissolve 0.150 g in 3 ml of anhydrous formic acid R. Add 30 ml of anhydrous acetic acid R and 0.1 ml
of naphtholbenzein solution R. Titrate with 0.1M perchloric acid.
STORAGE
IMPURITIES
Me
N
N
H
HOOC
H NH2
COOH
NH2
A. 3,3-[ethylidenebis(1H-indole-1,3-diyl)]bis[(2S)-2-aminopropanoic] acid (1,1ethylidenebistryptophan),

H
N
O
H NH2
*
OH
COOH
and epimer at C*
B. (S)-2-amino-3-[(3RS)-3-hydroxy-2-oxo-2,3-dihydro-1H-indol-3-yl]propanoic acid
(dioxyindolylalanine),
O
H NH2
COOH
NH2
C. (S)-2-amino-4-(2-aminophenyl)-4-oxobutanoic acid (kynurenine),
41-63
H
N
H NH2
COOH
HO
D. (S)-2-amino-3-(5-hydroxy-1H-indol-3-yl)propanoic acid (5-hydroxytryptophan),

O H
NH2
COOH
N
H
CHO
E. (S)-2-amino-4-[2-(formylamino)phenyl]-4-oxobutanoic acid (N-formylkynurenine),

NH2
H H
N
COOH
F. (S)-2-amino-3-(phenylamino)propanoic acid (3-phenylaminoalanine),

H
N
OH
H NH2
COOH
G. (S)-2-amino-3-(2-hydroxy-1H-indol-3-yl)propanoic acid (2-hydroxytryptophan),

H
N
NH
COOH
and enatiomer
H. (3RS)-1,2,3,4-tetrahydro-9H--carboline-3-carboxylic acid,
Me
H
N
NH
COOH
I. 1-methyl-1,2,3,4-tetrahydro-9H--carboline-3-carboxylic acid,
OH
*
H
N
OH
*
N
H
H
COOH
NH2
J. (S)-2-amino-3-[2-[2,3-dihydroxy-1-(1H-indol-3-yl)propyl]-1H-indol-3-yl]propanoic acid,
H
N
N
H
H
COOH
NH2
K. (S)-2-amino-3-[2-(1H-indol-3-ylmethyl)-1H-indol-3-yl]propanoic acid,
41-64
H
N
H
N
NH
COOH
L. 1-(1H-indol-3-ylmethyl)-1,2,3,4-tetrahydro-9H--carboline-3-carboxylic acid.
__________________________________________________________________________________________________________ Ph Eur
42-1
Tubocurarine Chloride
MeO
OH
NMe
O
H
Cl ,HCl
H
+
Me2N
CH2
O
OH
OMe
C 37H41ClN2O6,HCl,5H2O
772
6989-98-6
Tubocurarine Chloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Skeletal muscle relaxant.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Tubocurarine chloride contains not less than 98.0 per cent and not more than the equivalent of
102.0 per cent of (13aR,25aS)-9,19-dihydroxy-18,29-dimethoxy-1,14,14-trimethyl2,3,13a,14,15,16,25,25a-octahydro-13H-4,6:21,24-dietheno-8,12-metheno-1H-[3,2:14,15]
pyrido[1,11-dioxacyclo-icoseno][2,3,4-ij]isoquinolinium chloride hydrochloride, calculated with
CHARACTERS
A white or slightly yellowish, crystalline powder, soluble in water and in alcohol, practically insoluble
in acetone and in ether. It dissolves in solutions of alkali hydroxides.
IDENTIFICATION
A. Dissolve 50.0 mg in water R and dilute to 1000.0 ml with the same solvent. Examined between
230 nm and 350 nm (2.2.25), the solution shows an absorption maximum at 280 nm and a minimum at 255 nm. The specific absorbance at the maximum is 113 to 123, calculated with reference to
obtained with tubocurarine chloride CRS.
C. Dissolve about 25 mg in 1 ml of water R. Add 0.2 ml of ferric chloride solution R2 and heat in a
water-bath for 1 min. The solution is green. Carry out a blank test; after heating in a water-bath, the
colour is brown.
D. To 1 ml of solution S (see Tests) add 1 ml of nitric acid solution of mercury R. A red colour
develops slowly.
F. It gives the reaction of alkaloids (2.3.1).
TESTS
Specific optical rotation (2.2.7). +210 to +222, determined on solution S 3 h after preparation
and calculated with reference to the anhydrous substance.
Chloroform-soluble substances Dissolve 0.25 g in 150 ml of water R. Add 5 ml of a saturated
42-2
solution of sodium bicarbonate R and shake with three quantities, each of 20 ml, of chloroform R. Wash
the combined chloroform layers with 10 ml of water R, filter the chloroform solution into a tared
beaker and wash the filter with two quantities, each of 5 ml, of chloroform R. Add the washings to the
filtrate. Evaporate to dryness on a water-bath and dry at 100C to 105C for 1 h. The residue weighs
not more than 5 mg. The residue does not dissolve in 10 ml of water R, but dissolves on the addition
of 1 ml of dilute hydrochloric acid R.
coating substance.
same solvent.
Reference solution (a). Dilute 1.5 ml of the test solution to 100 ml with water R.
Apply separately to the plate 5 l of each solution. Develop in a non-saturated tank over a path of
15 cm using the lower layer of a mixture of equal volumes of chloroform R, methanol R and a 125 g/l
solution of trichloroacetic acid R. Dry the plate in a current of cold air. Spray with a mixture of 1
volume of potassium ferricyanide solution R, 1 volume of water R and 2 volumes of ferric chloride solution R1, prepared immediately before use. Any spot in the chromatogram obtained with the test
obtained with reference solution (a) (1.5 per cent) and at most one such spot is more intense than the
ASSAY
Dissolve 25.0 mg in water R and dilute to 500.0 ml with the same solvent. In the same manner,
prepare a reference solution using 25.0 mg of tubocurarine chloride CRS. Measure the absorbance
(2.2.25) at the maximum at 280 nm.
Calculate the content of C37H42Cl2N2O6 from the absorbances measured, the concentrations of the
solutions and the declared content of anhydrous tubocurarine chloride in tubocurarine chloride CRS.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
42-3
Javanese Turmeric
corrected 1/01
Javanese Turmeric complies with the requirements of the 3rd edition of the European Pharmacopoeia [1441].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Javanese turmeric consists of the dried rhizome, cut in slices, of Curcuma xanthorrhiza Roxb. (C.
xanthorrhiza D. Dietrich). It contains not less than 50 ml/kg of essential oil and not less than 1.0 per
cent of dicinnamoyl methane derivatives expressed as curcumin (C21H20O6; Mr 368.4), both calculated with reference to the anhydrous drug.
CHARACTERS
Javanese turmeric has an aromatic odour.
IDENTIFICATION
A. The drug consists of orange-yellow to yellowish-brown or greyish-brown slices, mostly peeled
1.5 mm to 6 mm thick and 15 mm to 50 mm, more rarely up to 70 mm, in diameter. Fragments of
the brownish-grey cork are sporadically present. The transverse surface is yellow with dark spots in
the paler centre. The fracture is short and finely grained.
B. Reduce to a powder (355). The powder is reddish-brown. Examine under a microscope, using
chloral hydrate solution R. The powder shows fragments of colourless parenchyma with orange-yellow
to yellowish-brown secretory cells; fragments of reticulate and other vessels; rare fragments of cork
and epidermis and fragments of thick-walled unicellular acute trichomes. Examine under a
microscope using a 50 per cent V/V solution of glycerol R. The powder shows numerous stratified,
ovoid to irregular starch granules, about 30 m to 50 m long and about 10 m to 30 m wide, with
an eccentric hilum and marked, concentric striations.
C. Examine by thin-layer chromatography as described in the test for Curcuma domestica until the
words Allow the plate to dry in the air. Spray the plate with a freshly prepared 0.4 g/l solution of
dichloroquinonechlorimide R in 2-propanol R. Expose the plate to ammonia vapour until the thymol
zone becomes bluish-violet. The chromatogram obtained with the reference solution shows almost in
the middle a bluish-violet zone (thymol) and in the lower part a yellow zone (fluorescein). The
chromatogram obtained with the test solution shows a blue zone (xanthorrhizol) slightly above the
zone due to thymol in the chromatogram obtained with the reference solution and two yellowishbrown to brown zones (curcumin and demethoxycurcumin) between the zones due to thymol and
fluorescein in the chromatogram obtained with the reference solution.
TESTS
Curcuma domestica Examine by thin-layer chromatography (2.2.27), using a TLC silica gel plate R.
Test solution. Shake 0.5 g of the freshly powdered drug (500) with 5 ml of methanol R for 30 min and
filter.
Reference solution. Dissolve 5 mg of fluorescein R and 10 mg of thymol R in 10 ml of methanol R.
mixture of 20 volumes of glacial acetic acid R and 80 volumes of toluene R. Allow the plate to dry in
air. Spray the plate with a mixture of 1 volume of sulphuric acid R and 9 volumes of acetic anhydride R.
Examine the plate in ultraviolet light at 365 nm. In the chromatogram obtained with the test solution, no yellowish-red fluorescent zone (bisdemethoxycurcumin) appears slightly above the greenishblue fluorescent zone of fluorescein in the chromatogram obtained with the reference solution.
Water (2.2.13). Not more than 12.0 per cent, determined by distillation on 20.0 g of powdered drug
(500).
ASSAY
Essential oil Carry out the determination of essential oils in vegetable drugs (2.8.12). Use a 500 ml
round-bottomed flask, 200 ml of water R as the distillation liquid and 0.5 ml of xylene R in the
graduated tube. Reduce the drug to a powder (500) and immediately use 5.0 g for the determination.
Distil at a rate of 3 ml/min to 4 ml/min for 3 h.
Dicinnamoyl methane derivatives To 0.100 g of the powdered drug (180) add 60 ml of glacial
42-4
acetic acid R and heat in a water-bath at 90C for 60 min. Add 2.0 g of boric acid R and 2.0 g of oxalic
acid R and heat in a water-bath at 90C for 10 min. Allow to cool, dilute with glacial acetic acid R to
100.0 ml and shake. Dilute 5.0 ml of the clear supernatant with glacial acetic acid R to 50.0 ml.
Measure the absorbance (2.2.25) at 530 nm, using glacial acetic acid R as the compensation liquid.
Calculate the percentage content of dicinnamoyl methane derivatives, expressed as curcumin, from
the expression:
A 0.426
m
taking the specific absorbance to be 2350.

m = mass of the drug to be examined, in grams.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
42-5
Turpentine Oil
Definition Turpentine Oil is obtained by distillation from the oleoresin obtained from various
species of Pinus and rectified.
Characteristics A clear, bright, colourless liquid, visibly free from water; odour, characteristic.
Solubility in ethanol Soluble, at 20, in 7 volumes of ethanol (90%) and in 3 volumes of ethanol
(96%), Appendix X M.
Residue on evaporation Not more than 1.0% when determined by the method for residue on
evaporation of volatile oils, Appendix X M. Use 2 g and heat for 4 hours.
Storage Turpentine Oil should be kept in a well-filled, well-closed container, protected from light
and stored at a temperature not exceeding 25.
Action and use Rubefacient.
Preparation
White Liniment
42-6
Tyrosine
corrected 1/01
HO
H
NH2
COOH
C9H11NO3
181.2
60-18-4
Tyrosine complies with the requirements of the 3rd edition of the European Pharmacopoeia [1161]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Tyrosine contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of
(S)-2-amino-3-(4-hydroxyphenyl)propanoic acid, calculated with reference to the dried substance.
PRODUCTION
When tyrosine is produced by a process involving fermentation steps, it complies with the requirements of the monograph on Products of fermentation (1468).
CHARACTERS
A white crystalline powder or colourless crystals, very slightly soluble in water, practically insoluble in
alcohol. It dissolves in dilute mineral acids and in dilute solutions of alkali hydroxides.
IDENTIFICATION
obtained with tyrosine CRS. Examine the substances prepared as discs.
D. To about 50 mg add 1 ml of dilute nitric acid R. A dark red colour is produced within 15 min.
E. Dissolve about 30 mg in 2 ml of dilute sodium hydroxide solution R. Add 3 ml of a freshly prepared
mixture of equal volumes of a 100 g/l solution of sodium nitrite R and a solution of 0.5 g of sulphanilic
acid R in a mixture of 6 ml of hydrochloric acid R1 and 94 ml of water R. An orange-red colour is
produced.
TESTS
Appearance of solution Dissolve 0.5 g in dilute hydrochloric acid R and dilute to 20 ml with the
same acid. The solution is clear (2.2.1) and not more intensely coloured than reference solution Y7
(Method II, 2.2.2).
Specific optical rotation (2.2.7). Dissolve 1.25 g in a mixture of equal volumes of dilute hydrochloric acid R and water R and dilute to 25.0 ml with the same mixture of solvents. The specific
optical rotation is 11.0 to 12.3, calculated with reference to the dried substance.
gel plate R.
Test solution (a). Dissolve 0.10 g of the substance to be examined in dilute ammonia R2 and dilute to
Reference solution (a). Dissolve 10 mg of tyrosine CRS in 1 ml of dilute ammonia R2 and dilute to 50 ml
with water R.
Reference solution (c). Dissolve 10 mg of tyrosine CRS and 10 mg of phenylalanine CRS in 1 ml of dilute
ammonia R2 and dilute to 25 ml with water R.
30 volumes of concentrated ammonia R1 and 70 volumes of propanol R. Allow the plate to dry in air,
42-7
spray with ninhydrin solution R and heat at 100C to 105C for 15 min. Any spot in the chromatogram obtained with test solution (a), apart from the principal spot, is not more intense than the spot
in the chromatogram obtained with reference solution (b) (0.5 per cent). The test is not valid unless
the chromatogram obtained with reference solution (c) shows two clearly separated spots.
The solution complies with the limit test for chlorides, without any further addition of nitric acid
(200 ppm).
Sulphates (2.4.13). Dissolve with gentle heating, 0.5 g in 5 ml of dilute hydrochloric acid R and dilute
to 15 ml with distilled water R. The solution complies with the limit test for sulphates (300 ppm).
standard using 0.2 ml of ammonium standard solution (100 ppm NH4) R. Replace the heavy magnesium
oxide R by 2.0 ml of strong sodium hydroxide solution R.
at 100C to 105C.
ASSAY
STORAGE
__________________________________________________________________________________________________________ Ph Eur
42-8
Ubidecarenone
1/01
Me
Me
Me
MeO
8
MeO
Me
Me
O
C59H90O4
863
303-98-0
Ubidecarenone complies with the requirements of the 3rd edition of the European Pharmacopoeia [1578].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ubidecarenone contains not less than 97.0 per cent and not more than the equivalent of 103.0 per
cent of 2-[(all-E)-3,7,11,15,19,23,27,31,35,39-decamethyltetraconta-2,6,10,14,18,22,26,30,34,38decaenyl]-5,6-dimethoxy-3-methylbenzene-1,4-dione.
PRODUCTION
Where applicable, it complies with the requirements of the monograph on Products of fermentation
(1468).
CHARACTERS
A yellow or orange, crystalline powder, practically insoluble in water, soluble in acetone, very slightly
soluble in ethanol.
It gradually decomposes and darkens on exposure to light.
Carry out all operations avoiding exposure to light.
IDENTIFICATION
obtained with ubidecarenone CRS. Examine the substances prepared as discs using potassium bromide R.
B. Examine the chromatograms obtained in the test for related substances. The retention time of the
peak in the chromatogram obtained with reference solution (a).
TESTS
Test solution. Dissolve 25.0 mg of the substance to be examined in 25.0 ml of ethanol R by heating at
about 50C for 2 min. Allow to cool.
Reference solution (a). Dissolve 5 mg of ubidecarenone CRS in 5 ml of ethanol R by heating at about
50C for 2 min. Allow to cool.
Reference solution (b). Dissolve 2 mg of ubidecarenone impurity D CRS in 2 ml of the test solution by
heating at about 50C for 2 min. Allow to cool. Dilute 1 ml to 50 ml with ethanol R.
Reference solution (c). Dilute 1.0 ml of the test solution to 100.0 ml with ethanol R.
as mobile phase at a flow rate of 2 ml/min a mixture of 20 volumes of ethanol R and 80 volumes
of methanol R2,
obtained with 10 l of reference solution (c) is at least 30 per cent of the full scale of the recorder.
Inject 10 l of reference solution (b). When the chromatogram is recorded in the prescribed
conditions, the retention times are: impurity D about 8 min and ubidecarenone about 12 min. The
test is not valid unless the resolution between the peaks corresponding to impurity D and
ubidecarenone is at least 6.5.
Inject 10 l of the test solution and 10 l of reference solution (c). Continue the chromatography of
42-9
the test solution for twice the retention time of ubidecarenone. In the chromatogram obtained with
the test solution: the area of any peak, apart from the principal peak, is not greater than 0.5 times the
area of the principal peak in the chromatogram obtained with reference solution (c) (0.5 per cent);
the sum of the areas of all the peaks, apart from the principal peak, is not greater than the area of the
principal peak in the chromatogram obtained with reference solution (c) (1.0 per cent). Disregard
Impurity F Examine by liquid chromatography (2.2.29).
Test solution. Dissolve 25.0 mg of the substance to be examined in 25.0 ml of hexane R.
Reference solution (a). Dissolve 10.0 mg of ubidecarenone for syste suitability CRS in 10.0 ml of
hexane R.
Reference solution (b). Dilute 1 ml of the test solution to 100.0 ml with hexane R.
as mobile phase at a flow rate of 2 ml/min a mixture of 3 volumes of ethyl acetate R and 97
volumes of hexane R,
principal peak in the chromatogram obtained with reference solution (b) is at least 50 per cent of the
conditions, the retention times are: impurity F about 8.5 min and ubidecarenone about 10 min. The
test is not valid unless there is a baseline separation between the peaks corresponding to impurity F
and ubidecarenone.
Inject 20 l of the test solution. The area of the peak corresponding to impurity F is not greater
(b) (0.1 per cent).
ASSAY
Dissolve 50.0 mg in 1.0 ml of hexane R and dilute to 50.0 ml with ethanol R. Dilute 2.0 ml of the
STORAGE
IMPURITIES
OH
MeO
MeO
Me
OH
A. 2,3-dimethoxy-5-methylbenzene-1,4-diol,
O
Me
Me
Me
eO
n
eO
Me
Me
O
B. n = 5: 2-[(all-E)-3,7,11,15,19,23,27-heptamethyloctadocosa-2,6,10,14,18,22,26heptaenyl]-5,6-dimethoxy-3-methylbenzene-1,4-dione (ubiquinone-7),
C. n = 6: 5,6-dimethoxy-3-methyl-2-[(all-E)-3,7,11,15,19,23,27,31-octamethyldotriaconta2,6,10,14,18,22,26,30-octaenyl]benzene-1,4-dione (ubiquinone-8),
D. n = 7: 5,6-dimethoxy-3-methyl-2-[(all-E)-3,7,11,15,19,23,27,31,35nonamethylhexatriaconta-2,6,10,14,18,22,26,30,34-nonaenyl]benzene-1,4-dione
(ubiquinone-9),
42-10
Me
Me
8
Me
eO
eO
Me
Me
and enantiomer
OH
E. (2RS)-7,8-dimethoxy-2,5-dimethyl-2-[(all-E)-4,8,12,16,20,24,28,32,36nonamethylheptatriaconta-3,7,11,15,19,23,27,31,35-nonaenyl]-2H-1-benzopyran-6-ol
(ubicromenol),
Me
O
Me
8
eO
Me
Me
eO
Me
O
F. 2-[(2Z,6E,10E,14E,18E,22E,26E,30E,34E,38E)-3,7,11,15,19,23,27,31,35,39-decamethyl2,6,10,14,18,22,26,30,34,38-tetracontadecaenyl]-5,6-dimethoxy-3-methylbenzene-1,4dione (ubidecarenone (Z)-isomer).

__________________________________________________________________________________________________________ Ph Eur
42-11
Undecenoic Acid
H
2C
C 11H20O2
COOH
184.3
112-38-9
Undecenoic Acid complies with the requirements of the 3rd edition of the European Pharmacopoeia for
Undecylenic Acid [0461]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Undecylenic acid contains not less than 97.0 per cent and not more than the equivalent of 102.0 per
cent of undec-10-enoic acid.
CHARACTERS
A white or very pale yellow, crystalline mass or colourless or pale yellow liquid, practically insoluble
in water, freely soluble in alcohol, in ether and in fatty and essential oils.
IDENTIFICATION
A. Refractive index (2.2.6): 1.447 to 1.450, determined at 25 0.5C.
B. Freezing point (2.2.18): 21C to 24C.
C. To 2.0 g add 2 ml of freshly distilled aniline R and boil under a reflux condenser for 10 min. Allow
to cool and add 30 ml of ether R. Shake with three quantities, each of 20 ml, of dilute hydrochloric
acid R and then with 20 ml of water R. Evaporate the organic layer to dryness on a water-bath. The
residue, after recrystallisation twice from alcohol (70 per cent V/V) R and drying in vacuo for 3 h, melts
(2.2.14) at 66C to 68C.
D. Dissolve 0.1 g in a mixture of 2 ml of dilute sulphuric acid R and 5 ml of glacial acetic acid R. Add
dropwise 0.25 ml of potassium permanganate solution R. The colour of the potassium permanganate is
discharged.
TESTS
Fixed and mineral oils To 1.0 g add 5 ml of sodium carbonate solution R and 25 ml of water R and
boil for 3 min. The hot solution is not more opalescent than reference suspension II (2.2.1).
Water-soluble acids To 1.0 g add 20 ml of water R heated to 35C to 45C and shake for 2 min.
Cool and filter the aqueous layer through a moistened filter. To 10 ml of the filtrate add 0.1 ml of
phenolphthalein solution R. Not more than 0.1 ml of 0.1M sodium hydroxide is required to change the
Degree of unsaturation Dissolve 85.0 mg in a mixture of 5 ml of dilute hydrochloric acid R and
30 ml of glacial acetic acid R. Using 0.05 ml of indigo carmine solution R1, added towards the end of
the titration, as indicator, titrate with 0.0167M bromide-bromate until the colour changes from blue to
yellow. 8.9 ml to 9.4 ml of 0.0167M bromide-bromate is required. Carry out a blank titration.
ASSAY
Dissolve 0.750 g in 10 ml of alcohol R. Using 0.1 ml of phenolphthalein solution R as indicator, titrate
with 0.5M sodium hydroxide until a pink colour is obtained.
STORAGE
Store in a well-closed, non-metallic container, protected from light, in a cool place.
__________________________________________________________________________________________________________ Ph Eur
42-12
Urea
O
H2N
CH4N2O
NH2
60.1
57-13-6
Urea complies with the requirements of the 3rd edition of the European Pharmacopoeia [0743]. These requirements are reproduced after the heading Definition below.
Preparation
Urea Cream
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Urea contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of
CH4N2O, calculated with reference to the dried substance.
CHARACTERS
A white, crystalline powder or transparent crystals, slightly hygroscopic, very soluble in water, soluble
in alcohol, practically insoluble in methylene chloride.
IDENTIFICATION
obtained with urea CRS. Examine the substances prepared as discs.
C. Dissolve 0.1 g in 1 ml of water R. Add 1 ml of nitric acid R. A white, crystalline precipitate is
formed.
D. Heat 0.5 g in a test tube until it liquefies and the liquid becomes turbid. Cool, dissolve in a
mixture of 10 ml of water R and 1 ml of dilute sodium hydroxide solution R and add 0.05 ml of copper
sulphate solution R. A reddish-violet colour is produced.
TESTS
Appearance of solution To 2.5 ml of solution S add 7.5 ml of water R. The solution is clear (2.2.1)
and colourless (Method II, 2.2.2).
Alkalinity To 2.5 ml of solution S add 7.5 ml of water R, 0.1 ml of methyl red solution R and 0.4 ml
of 0.01M hydrochloric acid. The solution is red to orange.
Biuret To 10 ml of solution S add 5 ml of water R, 0.5 ml of a 5 g/l solution of copper sulphate R and
0.5 ml of strong sodium hydroxide solution R. Allow to stand for 5 min. Any reddish-violet colour in the
using 10 ml of a 0.2 g/l solution of biuret R (0.1 per cent).
Ammonium (2.4.1). 0.1 ml of solution S complies with the limit test for ammonium (500 ppm).
100C to 105C for 1 h.
ASSAY
Dissolve 0.500 g in a 10 per cent V/V solution of sulphuric acid R and dilute to 100.0 ml with the
same acid. Introduce 5.0 ml of this solution into a long-necked combustion flask, add 10 ml of
sulphuric acid R and heat gently until gas is no longer evolved. Boil gently for 10 min, cool and add
cautiously 40 ml of water R. Cool again and place in a steam-distillation apparatus. Add 50 ml of
strong sodium hydroxide solution R and distil immediately by passing steam through the mixture. Distil
for 1 h, collecting about 50 ml of distillate in 40 ml of a 40 g/l solution of boric acid R. Add 0.25 ml of
42-13
methyl red mixed solution R and titrate with 0.1M hydrochloric acid. Carry out a blank assay.
1 ml of 0.1M hydrochloric acid is equivalent to 3.003 mg of CH4N2O.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
42-14
Urofollitropin
97048-13-0
Urofollitropin complies with the requirements of the 3rd edition of the European Pharmacopoeia [0958]. These
Action and use Gonadotrophic hormone.
Preparation
Urofollitropin Injection
When urofollitrophin is prescribed or demanded, Urofollitropin shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Urofollitropin is a dry preparation containing menopausal gonadotrophin obtained from the urine of
post-menopausal women. It has follicle-stimulating activity and no or virtually no luteinising activity.
The potency is not less than 90 International Units of follicle-stimulating hormone (hFSH) per
milligram. The ratio of units of luteinising hormone (interstitial-cell-stimulating hormone)
[hLH(ICSH)] to units of follicle-stimulating hormone is not more than 1/60.
PRODUCTION
It may be prepared by a suitable fractionation procedure followed by immunoaffinity chromatography. It is prepared in conditions designed to minimise microbial contamination. The manufacturing process must have been shown to reduce any viral contamination such as hepatitis virus or HIV
by appropriate validated methods.
CHARACTERS
An almost white or slightly yellowish powder, soluble in water.
IDENTIFICATION
When administered as prescribed in the assay it causes enlargement of the ovaries of immature female
rats.
TESTS
Hepatitis virus antigens Examined by a suitably sensitive immunochemical method (2.7.1),
hepatitis virus antigens are not detected.
HIV antigen Examined by a suitably sensitive immunochemical method (2.7.1), HIV antigen is not
detected.
Residual luteinising activity The International Units of FSH and LH are the activities contained
in stated amounts of the International Standard of human urinary follicle-stimulating hormone and
luteinising hormone (interstitial-cell-stimulating hormone) which consists of a mixture of a freezedried extract of urine of post-menopausal women with lactose. The equivalence in International
Units of the International Standard is stated by the World Health Organisation. Use immature
female rats approximately 21 days old and having masses such that the difference between the
heaviest and the lightest rat is not more than 10 g. Assign the rats at random to four equal groups of
at least six animals. If sets of four litter mates are available, assign one litter mate at random from
each set to each group and mark according to litter.
Inject subcutaneously into each rat 50 I.U. of serum gonadotrophin R on the first day and 25 I.U. of
chorionic gonadotrophin R on the fourth day, each in 0.5 ml of phosphate-albumin buffered saline pH
7.2 R.
Choose three doses of the reference preparation such that the smallest dose produces a depletion of
the ovarian ascorbic acid content in all the rats and the largest dose does not produce a maximal
depletion in all the rats. Use doses in geometric progression; as an initial approximation, total doses
of 0.5 I.U., 1.0 I.U. and 2.0 I.U. may be tried although the dose to be used will depend on the
sensitivity of the animals.
Choose a dose of the preparation to be examined expected to contain 60X I.U. of follicle-stimulating hormone (hFSH), in which X = the number of I.U. of hLH in the middle dose of the reference
preparation.
Dissolve separately the total quantities of the preparation to be examined and of the reference
preparation in 1.0 ml of phosphate-albumin buffered saline pH 7.2 R. Inject into a tail vein to each
separate group of rats 6 days after the injection of chorionic gonadotrophin. Exactly 4 h after the
injection, kill the rats and remove the ovaries from each animal. Remove any extraneous fluid and
tissue from the ovaries and weigh the ovaries immediately.
Treat the combined ovaries from each rat separately, as follows. Crush and homogenise within
2 min in a freshly prepared 25 g/l solution of metaphosphoric acid R at a temperature of 4C and dilute
42-15
to 7 ml with the same solution. Allow the homogenate to stand for 30 min at 4C and centrifuge at
4C at approximately 2500 g. Filter the supernatant liquid, if necessary, through a 0.22 m filter.
Prepare a fresh solution consisting of a mixture of 2 ml of a 45.3 g/l solution of sodium acetate R
adjusted to pH 7 with acetic acid R, 3 ml of water R and 2 ml of 2,6-dichlorophenolindophenol standard
solution R. Mix 2 ml of this solution with 2 ml of the clear supernatant liquid. 30 s after mixing,
measure the absorbance (2.2.25) of the solution at the maximum at about 520 nm. Use as reference a
solution with a known content of ascorbic acid CRS in a 25 g/l solution of metaphosphoric acid R, treated
by the same process.
Calculate the amount of ascorbic acid from the ascorbic acid standard curve obtained and express in
milligrams per 0.1 g of ovary to obtain the ascorbic acid content of the ovaries. Calculate the mean and
its variance of the ascorbic acid content of the ovaries of the rats treated with the preparation to be
examined.
For each dose-group of the reference preparation plot the mean ascorbic acid content of the ovaries
as a function of the logarithm of the dose and analyse the regression of the ascorbic acid content on the
logarithm of the dose injected, using standard methods of analysis (the method of least squares).
The test is not valid unless:
The slope constant b is significant at the 5 per cent level of significance,
for the groups treated with the reference preparation, the sum of squares due to linear regression
is equal to at least 95 per cent of the total sum of squares of the ascorbic acid content.
the within-group variance of the ascorbic acid content of the group receiving the preparation to be
examined is not significantly different at the 5 per cent level of significance from the within-group
variance of the ascorbic acid content of the groups receiving the reference preparation.
The mean ascorbic acid content of the ovaries of the rats treated with the preparation to be examined is not significantly lower than that of the rats treated with the middle dose of the reference
preparation (calculated from the regression equation) at the 5 per cent level of significance.
Water Not more than 5.0 per cent m/m, determined by gas chromatography (2.2.28), using anhydrous
methanol R as the internal standard.
Use dry glassware (which may be silicone-treated).
Internal standard solution. Dilute 15 l of anhydrous methanol R to 100 ml with 2-propanol R1.
Test solution (a). Dissolve 4 mg of the substance to be examined in 0.5 ml of 2-propanol R1.
Test solution (b). Dissolve 4 mg of the substance to be examined in 0.5 ml of the internal standard
solution.
Reference solution. Add 10 l of water R to 50 ml of the internal standard solution.
a stainless steel column 1 m long and 2 mm in internal diameter packed with styrene-divinylbenzene
helium for chromatography R as the carrier gas,
a thermal-conductivity detector,
Inject the chosen volumes of each solution. Calculate the content of water assuming its density
(2.2.5) at 20C to be 0.9972 g/ml and taking into account any water detectable in the internal standard
solution.
Pyrogens (2.6.8). Inject per kilogram of the rabbits mass a quantity equivalent to 5 I.U. of folliclestimulating hormone, dissolved in not more than 1 ml of a sterile pyrogen-free 9 g/l solution of sodium
chloride R.
ASSAY
The follicle-stimulating activity of urofollitropin is estimated by comparing under given conditions its
effect in enlarging the ovaries of immature rats treated with chorionic gonadotrophin with the same
effect of the International Standard preparation of human urinary follicle-stimulating hormone and
luteinising hormone or of a reference preparation calibrated in International Units. The International
Units of FSH and LH are the activities contained in stated amounts of the International Standard of
human urinary follicle-stimulating hormone and luteinising hormone (interstitial-cell-stimulating
hormone) which consists of a mixture of a freeze-dried extract of urine of post-menopausal women
with lactose. The equivalence in International Units of the International Standard is stated by the
World Health Organisation.
Use immature female rats of the same strain, 19 to 28 days old, differing in age by not more than 3
days and having masses such that the difference between the heaviest and the lightest rat is not more
than 10 g. Assign the rats at random to six equal groups of at least five animals. If sets of six litter
mates are available, assign one litter mate from each set to each group and mark according to litter.
Choose three doses of the reference preparation and three doses of the preparation to be examined
42-16
such that the smallest dose produces a positive response in some of the rats and the largest dose does
not produce a maximal response in all the rats. Use doses in geometric progression and as an initial
approximation total doses of 1.5 I.U., 3.0 I.U. and 6.0 I.U. may be tried although the dose will
depend on the sensitivity of the animals used, which may vary widely.
Dissolve separately the total quantities of the preparation to be examined and of the reference
preparation corresponding to the daily doses to be used in sufficient phosphate-albumin buffered saline
pH 7.2 R such that the daily dose is administered in a volume of about 0.5 ml. The buffer solution
shall contain in the daily dose not less than 14 I.U. of chorionic gonadotrophin to ensure complete
luteinisation. Add a suitable antimicrobial preservative such as 4 g/l of phenol or 0.02 g/l of thiomersal. Store the solutions at 5 3C.
Inject subcutaneously into each rat the daily dose allocated to its group. Repeat the injection of
each dose 24 h and 48 h after the first injection. About 24 h after the last injection, kill the rats and
remove the ovaries from each animal. Remove any extraneous fluid and tissue from the ovaries and
weigh the two combined ovaries of each animal immediately. Calculate the results by the usual
statistical methods, using the mass of the two combined ovaries as the response. (The precision of the
assay may be improved by a suitable correction of the organ mass with reference to the mass of the
animal from which it was taken; an analysis of covariance may be used).
STORAGE
Store in an airtight, tamper-proof container, protected from light, at a temperature of 2C to 8C. If
the substance is sterile, store in a sterile, airtight, tamper-proof container.
LABELLING
The label states:
the activity expressed in International Units of follicle-stimulating hormone per container,
the potency expressed in International Units of follicle-stimulating hormone per milligram,
where applicable, that the substance is sterile.
__________________________________________________________________________________________________________ Ph Eur
42-17
Urokinase
9039-53-6
Urokinase complies with the requirements of the 3rd edition of the European Pharmacopoeia [0695]. These
Action and use Fibrinolytic enzyme.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Urokinase is an enzyme, obtained from human urine, that activates plasminogen. It consists of a
mixture of low-molecular-mass (LMM) (Mr 33,000) and high-molecular-mass (HMM) (Mr 54,000)
forms, the high-molecular-mass form being predominant. The potency is not less than 70,000
International Units per milligram of protein.
PRODUCTION
It is produced by validated methods of manufacturing designed to minimise or eliminate microbial
and viral contamination and vasoactive substances; in particular, adequate measures to inactivate
viruses are taken, such as heating of the substance in solution at 60C for 10 h.
CHARACTERS
A white or almost white, amorphous powder, soluble in water.
IDENTIFICATION
A. Place separately in two haemolysis tubes 0.5 ml of citrated human plasma and 0.5 ml of citrated
bovine plasma and maintain in a water-bath at 37C. To each tube add 0.1 ml of a solution containing a quantity of the substance to be examined equivalent to 1000 I.U. per millilitre in phosphate
buffer solution pH 7.4 R and 0.1 ml of a solution containing a quantity of human thrombin R equivalent
to 20 I.U. per millilitre in phosphate buffer solution pH 7.4 R. Shake immediately. In both tubes, a clot
forms and lyses within 30 min.
B. Carry out identification by a suitable immunodiffusion test.
TESTS
Appearance of solution Dissolve 10 mg in 10 ml of water R. The solution is clear (2.2.1) and
Hepatitis B surface antigen Examine by a suitably sensitive method such as radioimmunoassay.
Hepatitis B surface antigen is not detected.
Thromboplastic contaminants
Test solutions. Dissolve suitable quantities of the substance to be examined in barbital buffer solution pH
7.4 R to obtain solutions with activities of 5000 I.U., 2500 I.U., 1250 I.U., 625 I.U. and 312 I.U.
per millilitre.
To each of six haemolysis tubes 1 cm in internal diameter add 0.1 ml of citrated rabbit plasma R.
Allocate the test solutions one to each of five of the tubes; add to each tube 0.1 ml of the solution
allocated to it and to the sixth tube add 0.1 ml of barbital buffer solution pH 7.4 R (blank). Incubate
the tubes at 25 0.5C for 5 min and add 0.1 ml of a 3.675 g/l solution of calcium chloride R.
Measure with a stop-watch the coagulation time for each tube. Plot the shortening of the recalcification time (clotting time of the blank minus clotting time measured) against log concentration in
International Units. Extrapolate the best-fitting straight line through the five points until it reaches
the log-concentration axis. The urokinase activity at the intersection point, which represents the limit
concentration for coagulant activity (zero coagulant activity), is not less than 150 I.U. per millilitre.
Molecular fractions Examine by size-exclusion chromatography (2.2.30).
Test solution. Dissolve about 1 mg of the substance to be examined in 1.0 ml of 0.02M phosphate buffer
pH 8.0 R. Prepare immediately before use.
a column 0.9 m long and 16 mm in internal diameter packed with cross-linked dextran for chromatography R3,
as mobile phase at a flow rate of 6 ml per hour a 17.5 g/l solution of sodium chloride R in 0.02M
phosphate buffer solution pH 8.0 R,
equilibrating the column and operating at 5C. Apply the test solution to the head of the column
rinsing twice with 0.5 ml portions of the buffer and carry out the elution. The eluate may be collected
in fractions of 1 ml. Measure the absorbance (2.2.25) of the eluate at the maximum at 280 nm and
plot the individual values on a graph. Draw perpendicular lines towards the axis of the abscissae from
the minima before the HMM peak, between the HMM and the LMM peaks, and after the LMM
42-18
peak, thus identifying the fractions to be considered in calculating the HMM/LMM activity ratio.
Pool the HMM fractions and, separately, the LMM fractions. Determine separately the urokinase
activity in International Units of each of the fraction pools by the method prescribed under Assay.
The ratio of the urokinase activity in the HMM fraction pool to that in the LMM fraction pool is not
less than 2.0.
Total protein Determine the nitrogen content, using 10 mg, by the method of sulphuric acid digestion (2.5.9) and calculate the quantity of protein by multiplying by 6.25.
further appropriate procedure for the removal of pyrogen, it complies with the test for pyrogens.
Inject per kilogram of the rabbits mass 1.0 ml of a sterile 9 g/l solution of sodium chloride R containing a quantity of the substance to be examined equivalent to 20,000 I.U. per millilitre.
ASSAY
The potency of urokinase is determined by comparing its capacity to activate plasminogen to form
plasmin with the same capacity of a reference preparation of urokinase calibrated in International
Units; the formation of plasmin is measured by the determination of the lysis time of a fibrin clot in
given conditions.
The International Unit is the activity contained in a stated amount of the International Reference
Preparation, which consists of freeze-dried urokinase with lactose. The equivalence in International
Units of the International Reference Preparation is stated by the World Health Organisation.
Unless otherwise prescribed, use phosphate buffer solution pH 7.4 R containing 30 g/l of bovine albumin R for
the preparation of the solutions and dilutions used in the assay.
Test solution. Prepare a solution of the substance to be examined expected to have an activity of
1000 I.U. per millilitre.
Reference solution. Prepare a solution of a reference preparation having an activity of 1000 I.U. per
millilitre.
Keep the test solution and the reference solution in iced water and use within 6 h. Prepare three
serial 1.5-fold dilutions of the reference preparation such that the longest clot-lysis time is less than
20 min and the shortest clot-lysis time is greater than 3 min. Prepare three similar dilutions of the test
solution. Keep the solutions in iced water and use within 1 h. Use twenty-four tubes 8 mm in diameter. Label the tubes T1, T2 and T3 for the dilutions of the test solution and S1, S2 and S3 for the
dilutions of the reference solution, allocating four tubes to each dilution. Place the tubes in iced
water. Into each tube, introduce 0.2 ml of the appropriate dilution, 0.2 ml of phosphate buffer solution
pH 7.4 R containing 30 g/l of bovine albumin R and 0.1 ml of a solution of human thrombin R having
an activity of not less than 20 I.U. per millilitre. Place the tubes in a water-bath at 37C and allow to
stand for 2 min to attain temperature equilibrium. Using an automatic pipette, introduce into the
bottom of the first tube 0.5 ml of a 10 g/l solution of bovine euglobulins R, ensuring mixing. At
intervals of 5 s, introduce successively into the remaining tubes 0.5 ml of a 10 g/l solution of bovine
euglobulins R. Using a stop-watch, measure for each tube the time in seconds that elapses between the
addition of the euglobulins solution and the lysis of the clot. Plot the logarithms of the lysis times for
the substance to be examined and for the reference preparation against the logarithms of the concentration and calculate the activity of the substance to be examined using the usual statistical methods.
STORAGE
Store in an airtight container, protected from light, at a temperature not exceeding 8C. If the
substance is sterile, store in a sterile, airtight, tamper-proof container.
LABELLING
The label states:
the potency in International Units per milligram of protein,
where applicable, that the contents are sterile,
where applicable, that the contents are apyrogenic.
__________________________________________________________________________________________________________ Ph Eur
42-19
Ursodeoxycholic Acid
H
Me
COOH
Me
H
Me
H
H
HO
H
C24H40O4
OH
392.6
128-13-2
Ursodeoxycholic Acid complies with the requirements of the 3rd edition of the European Pharmacopoeia
Action and use Used to dissolve cholesterol gallstones.
Preparations
Ursodeoxycholic Acid Capsules
Ursodeoxycholic Acid Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Ursodeoxycholic acid contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of 3,7-dihydroxy-5-cholan-24-oic acid, calculated with reference to the dried
substance.
CHARACTERS
A white or almost white powder, very slightly soluble in water, freely soluble in alcohol, slightly
soluble in acetone and in methylene chloride.
It melts at about 202C
IDENTIFICATION
obtained with ursodeoxycholic acid CRS. Examine the substances as discs prepared using potassium
bromide R.
C. Dissolve about 10 mg in 1 ml of sulphuric acid R. Add 0.1 ml of formaldehyde solution R and allow
to stand for 5 min. Add 5 ml of water R. The suspension obtained is greenish-blue.
TESTS
substance.
Test solution (a). Dissolve 0.40 g of the substance to be examined in a mixture of 1 volume of water R
and 9 volumes of acetone R and dilute to 10 ml with the same mixture of solvents.
Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with a mixture of 1 volume of water R and 9
volumes of acetone R.
Reference solution (a). Dissolve 40 mg of ursodeoxycholic acid CRS in a mixture of 1 volume of water R
Reference solution (b). Dissolve 20 mg of lithocholic acid CRS in a mixture of 1 volume of water R and 9
volumes of acetone R and dilute to 10 ml with the same mixture of solvents. Dilute 2 ml of the solution to 100 ml with a mixture of 1 volume of water R and 9 volumes of acetone R.
42-20
Reference solution (c). Dissolve 20 mg of chenodeoxycholic acid CRS in a mixture of 1 volume of water R
Reference solution (d). Dissolve 20 mg of cholic acid CRS in a mixture of 1 volume of water R and 9
volumes of acetone R and dilute to 100 ml with the same mixture of solvents.
Reference solution (e). Dilute 0.5 ml of test solution (a) to 20 ml with a mixture of 1 volume of water R
and 9 volumes of acetone R. Dilute 1 ml of the solution to 10 ml with a mixture of 1 volume of
water R and 9 volumes of acetone R.
Reference solution (f). Dissolve 10 mg of ursodeoxycholic acid CRS in reference solution (c) and dilute
15 cm using a mixture of 1 volume of glacial acetic acid R, 30 volumes of acetone R and 60 volumes of
methylene chloride R. Dry the plate at 120C for 10 min. Spray the plate immediately with a 47.6 g/l
solution of phosphomolybdic acid R in a mixture of 1 volume of sulphuric acid R and 20 volumes of
glacial acetic acid R and heat again at 120C until blue spots appear on a lighter background. In the
chromatogram obtained with test solution (a): any spot corresponding to lithocholic acid is not more
intense than the principal spot in the chromatogram obtained with reference solution (b) (0.1 per
cent); any spot corresponding to chenodeoxycholic acid is not more intense than the principal spot in
the chromatogram obtained with reference solution (c) (1 per cent); any spot corresponding to cholic
acid is not more intense than the principal spot in the chromatogram obtained with reference solution
(d) (0.5 per cent); any spot, apart from the principal spot and any spots corresponding to lithocholic
acid, chenodeoxycholic acid and cholic acid, is not more intense than the principal spot in the chromatogram obtained with reference solution (e) (0.25 per cent). The test is not valid unless the chromatogram obtained with reference solution (f) shows two clearly separated principal spots.
100C to 105C.
ASSAY
Dissolve 0.350 g in 50 ml of alcohol R, previously neutralised to 0.2 ml of phenolphthalein solution R.
Add 50 ml of water R and titrate with 0.1M sodium hydroxide until a pink colour is obtained.
IMPURITIES
Me
R3
Me
H
Me
OR
H
H
H
HO
R1
H
R2
A. R = H; R1 = H; R2 = OH; R3 = H: chenodeoxycholic acid,

B. R = H: R1 = H; R2 = OH; R3 = OH: 3,7,12-trihydroxy-5-cholan-24-oic acid (cholic acid),
C. R = H; R1 = H; R2 = H; R3 = H: 3-hydroxy-5-cholan-24-oic acid (lithocholic acid),
D. R = H; R1 = OH; R2 = H; R3 = OH: 3,7,12-trihydroxy-5-cholan-24-oic acid (ursocholic acid),
E. R = H; R1 = H; R2 = H; R3 = OH: 3, 12-dihydroxy-5-cholan-24-oic acid (deoxycholic acid),
F. R = H; R1,R2 = O; R3 = H: 3-hydroxy-7-oxo-5-cholan-24-oic acid,
G. R = CH3; R1 = OH; R2 = H; R3 = H: methyl 3,7-dihydroxy-5-cholan-24-oate.
__________________________________________________________________________________________________________ Ph Eur
42-21
Valerian
1/01
Valerian complies with the requirements of the 3rd edition of the European Pharmacopoeia for Valerian Root
Action and use Sedative.
When Powdered Valerian is prescribed or demanded, material complying with the appropriate
requirements below shall be dispensed or supplied.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Valerian root consists of the dried, whole or fragmented underground parts of Valeriana officinalis L.
s.l., including the rhizome surrounded by the roots and stolons. It contains not less than 5 ml/kg of
essential oil for the whole drug and not less than 3 ml/kg of essential oil for the cut drug, both
calculated with reference to the dried drug and not less than 0.17 per cent of sesquiterpenic acids
expressed as valerenic acid (C15H22O2; 234), calculated with reference to the dried drug.
CHARACTERS
Valerian root has a characteristic odour.
IDENTIFICATION
A. The rhizome is yellowish-grey to pale brownish-grey, obconical to cylindrical, up to about 50 mm
long and 30 mm in diameter; the base is elongated or compressed, usually entirely covered by
numerous roots. The apex usually exhibits a cup-shaped scar from the aerial parts; stem bases are
rarely present. When cut longitudinally, the pith exhibits a central cavity transversed by septa. The
roots are numerous, almost cylindrical, of the same colour as the rhizome, 1 mm to 3 mm in
diameter and sometimes more than 100 mm long. A few filiform fragile secondary roots are present.
The fracture is short. The stolons show prominent nodes separated by longitudinally striated
internodes, each 20 mm to 50 mm long, with a fibrous fracture.
B. Reduce to a powder (355). The powder is pale yellowish-grey to pale greyish-brown. Examine
under a microscope using chloral hydrate solution R. The powder shows cells containing a pale brown
resin or droplets of essential oil; isolated rectangular sclereids with pitted walls 5 m to 15 m thick;
reticulately-thickened xylem vessels; occasional fragments of cork cells and epidermal cells, some with
root hairs. Examine under a microscope using a 50 per cent V/V solution of glycerol R. The powder
shows numerous fragments of parenchyma with cells, containing single or compound starch granules;
the single granules are rounded or elongated, 5 m to 15 m in diameter and have sometimes a cleft
or radiate hilum; the compound granules have two to six components with an overall diameter of up
to 20 m.
Test solution. Shake 1.0 g of the powdered drug (355) with 6.0 ml of methanol R in a 25 ml flask for
15 min and filter. Rinse the flask and the filter with the aid of a small portion of methanol R to obtain
5 ml of filtrate. Evaporate the filtrate to about 2 ml and add 3 ml of a 100 g/l solution of potassium
hydroxide R. Shake with two quantities, each of 5 ml, of methylene chloride R. Allow to separate and
discard the lower layer. Heat the aqueous layer in a water-bath at 40C for 10 min. Cool and add
dilute hydrochloric acid R until it gives an acid reaction. Shake with two quantities, each of 5 ml, of
methylene chloride R. Filter the combined lower layers over anhydrous sodium sulphate R. Evaporate the
filtrate to dryness. Dissolve the residue in 1.0 ml of methylene chloride R.
Reference solution. Dissolve 5 mg of fluorescein R and 5 mg of Sudan red G R in 20.0 ml of methanol R.
0.5 volumes of glacial acetic acid R, 35 volumes of ethyl acetate R and 65 volumes of hexane R. Allow
the plate to dry in air. Examine in daylight. The chromatogram obtained with the reference solution
shows in its middle part the red zone of Sudan red G and in its lower part the greenish-yellow zone of
fluorescein. Spray the plate with anisaldehyde solution R. Examine in daylight while heating at 100C
to 105C for 5 min to 10 min. The chromatogram obtained with the test solution shows a violet-blue
zone corresponding to hydroxyvalerenic acid at about the same height as the zone of fluorescein in
the chromatogram obtained with the reference solution and also shows a violet zone corresponding to
valerenic acid at about the same height as that of the zone due to Sudan red G in the chromatogram
obtained with the reference solution. The chromatogram obtained with the test solution shows in the
upper half other, mostly less intense, pink to violet zones.
42-22
TESTS
Foreign matter (2.8.2). Not more than 5 per cent of stem bases and not more than 2 per cent of
other foreign matter.
Loss on drying (2.2.32). Not more than 12.0 per cent, determined on 1.000 g of well homogenised
powdered drug (355) by drying in an oven at 100C to 105C, for 2 h.
ASSAY
Essential oil Carry out the determination of essential oils in vegetable drugs (2.8.12). Use 40.0 g of
freshly powdered drug (500), a 2000 ml flask, 500 ml of water R as the distillation liquid and 0.50 ml
of xylene R in the graduated tube. Distil at a rate of 3 ml/min to 4 ml/min for 4 h.
Sesquiterpenic acids Examine by liquid chromatography (2.2.29).
Test solution. Place 1.50 g of the powdered drug (710) in a 100 ml round-bottomed flask with a
ground-glass neck. Add 20 ml of anhydrous methanol R. Mix and heat on a water-bath under a reflux
condenser for 30 min. Allow to cool and filter. Place the filter with the residue in the 100 ml roundbottomed flask. Add 20 ml of anhydrous methanol R and heat on a water-bath under the reflux
condenser for 15 min. Allow to cool and filter. Combine the filtrates and dilute to 50.0 ml with
anhydrous methanol R rinsing the round-bottomed flask and the filter.
Reference solution. Prepare the solution immediately before use, protected from bright light. Dissolve 30 mg
of dantron R in anhydrous methanol R and dilute to 100.0 ml with the same solvent. Dilute 5.0 ml of
the solution to 50.0 ml with anhydrous methanol R.
Mobile phase A. Mix 20 volumes of acetonitrile R and 80 volumes of a 5 g/l solution of phosphoric
acid R,
Mobile phase B. Mix 80 volumes of acetonitrile R and 20 volumes of a 5 g/l solution of phosphoric
acid R,
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
Comment
05
55
45
Isocratic
5 18
55 20
45 80
Linear gradient
18 20
20
80
Isocratic
20 22
20 55
80 45
Linear gradient

Inject the test solution and the reference solution.
When the chromatograms are recorded in the prescribed conditions, the relative retention times (to
dantron) are: acetoxyvalerenic acid about 0.7 and valerenic acid about 1.2.
Calculate the percentage content of acetoxyvalerenic acid using the expression:
A2 m 1 11.51
A1 m2
Calculate the percentage content of valerenic acid using the expression:
A3 m1 8.09
A1 m2
Or calculate the percentage content of both sesquiterpenic acids using the expression:
A2 11.51 A3 8.09
+
m1
A1
A1
m2
A1 = area of the peak due to dantron in the chromatogram obtained with the reference solution,
A2 = area of the peak due to acetoxyvalerenic acid in the chromatogram obtained with the test
solution,
A3 = area of the peak due to valerenic acid in the chromatogram obtained with the test solu-
42-23
tion,
m1 = mass of dantron, in grams, used to prepare the reference solution,
m2 = mass of the drug to be examined, in grams.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
42-24
Valine
H
H3C
H3C
NH2
COOH
C 5H11NO2
117.1
72-18-4
Valine complies with the requirements of the 3rd edition of the European Pharmacopoeia [0796]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Valine contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of
(S)-2-amino-3-methylbutanoic acid, calculated with reference to the dried substance.
PRODUCTION
When Valine is produced by a process involving fermentation steps, it complies with the requirements of the monograph on Products of fermentation (1468).
CHARACTERS
White or almost white, crystalline powder or colourless crystals, soluble in water, very slightly soluble
in alcohol, practically insoluble in ether.
IDENTIFICATION
obtained with valine CRS. Examine the substances prepared as discs.
TESTS
substance.
gel plate R.
Reference solution (a). Dissolve 10 mg of valine CRS in 0.1M hydrochloric acid and dilute to 50 ml with
the same acid.
Reference solution (c). Dissolve 10 mg of phenylalanine CRS and 10 mg of valine CRS in 0.1M hydrochloric acid and dilute to 25 ml with the same acid.
spots.
42-25
To the inner wall of the upper watch glass stick a piece of red litmus paper R 5 mm square and wetted
watch-glass and dissolve or suspend in 0.5 ml of water R. To the solution or the suspension add
0.30 g of heavy magnesium oxide R. Briefly triturate with a glass rod. Immediately close the cell by
putting the two watch glasses together. Heat at 40C for 15 min. The litmus paper is not more
intensely blue coloured than a standard prepared at the same time and in the same manner using
0.1 ml of ammonium standard solution (100 ppm NH4) R, 0.5 ml of water R and 0.30 g of heavy
magnesium oxide R (200 ppm).
100C to 105C.
ASSAY
Dissolve 0.100 g in 3 ml of anhydrous formic acid R. Add 30 ml of anhydrous acetic acid R. Using
0.1 ml of naphtholbenzein solution R as indicator, titrate with 0.1M perchloric acid until the colour
changes from brownish- yellow to green.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
42-26
Valproic Acid
H 3C
H 3C
C8H16O2
COOH
H
144.2
99-66-1
Valproic Acid complies with the requirements of the 3rd edition of the European Pharmacopoeia [1378].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Valproic acid contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent
of 2-propylpentanoic acid.
CHARACTERS
A colourless or very slightly yellow, clear liquid, slightly viscous, very slightly soluble in water,
miscible with alcohol and with methylene chloride. It dissolves in dilute solutions of alkali hydroxides.
IDENTIFICATION
A. Refractive index (2.2.5): 1.422 to 1.425.
obtained with valproic acid CRS.
the same solvent.
Reference solution. Dissolve 50 mg of valproic acid CRS in methanol R and dilute to 5 ml with the same
solvent.
Apply to the plate 2 l of each solution. Develop over a path of 15 cm using a mixture of equal
volumes of ether R and methylene chloride R. Allow the plate to dry in air. Spray with bromocresol green
solution R. The principal spot in the chromatogram obtained with the test solution is similar in
position, colour and size to the principal spot in the chromatogram obtained with the reference
solution.
D. To 1 ml add 3 ml of dilute sodium hydroxide solution R. Add 3 ml of water R and 1 ml of a 100 g/l
solution of cobalt nitrate R. A violet precipitate is formed. Filter; the precipitate dissolves in methylene
chloride R.
TESTS
Appearance of solution. Dissolve 2.0 g in dilute sodium hydroxide solution R and dilute to 10 ml with
the same alkaline solution. The solution is clear (2.2.1) and not more intensely coloured than
Related substances. Examine by gas chromatography (2.2.28), using butyric acid R as the internal
standard.
Internal standard solution. Dissolve 10 mg of butyric acid R in heptane R and dilute to 200 ml with the
same solvent.
dilute to 5.0 ml with the same solution. Dilute 1.0 ml of the solution to 10.0 ml with heptane R.
Reference solution. Dissolve 20 mg of the substance to be examined and 20 mg of 2-(1-methylethyl)pentanoic acid CRS in heptane R and dilute to 10 ml with the same solvent. Dilute 1 ml of the solution
to 10 ml with heptane R.
a wide-bore fused-silica column 30 m long and 0.53 mm in internal diameter coated with
macrogol 20,000 2-nitroterephthalate R (film thickness 0.5 m),
42-27
Time
(min)
Temperature
(C)
Rate
(C/min)
Comment
010
1030
130
130190
Injection port
220
isothermal
linear
gradient
Detector
220
Column
Inject 1 l of each solution. The test is not valid unless, in the chromatogram obtained with the
reference solution, the resolution between the peaks corresponding to 2-(1-methylethyl)pentanoic
acid and valproic acid is at least 3.0. In the chromatogram obtained with the test solution: the sum of
the area of the peaks, apart from the principal peak, is not greater than three times the area of the
peak due to the internal standard (0.3 per cent); none of the peaks, apart from the principal peak, has
an area greater than that of the peak due to the internal standard (0.1 per cent). Disregard any peak
with an area less than 0.1 times that of the peak due to the internal standard.
Heavy metals (2.4.8). Dissolve 2.0 g in alcohol (80 per cent V/V) R and dilute to 20 ml with the same
solvent. 12 ml of the solution complies with limit test B for heavy metals (20 ppm). Prepare the
standard using lead standard solution (2 ppm Pb) obtained by diluting lead standard solution (100 ppm
Pb) R with alcohol (80 per cent V/V) R.
ASSAY
Dissolve 0.100 g in 25 ml of alcohol R. Add 2 ml of water R. Titrate with 0.1M sodium hydroxide,
STORAGE
IMPURITIES
H3C
COOH
R R1
A. R = R1 = H: pentanoic acid (valeric acid),

B. R = H, R1 = CH2-CH3: (2RS)-2-ethylpentanoic acid,
C. R = H, R1 = CH(CH3)2: (2RS)-2-(1-methylethyl)pentanoic acid,
D. R = R1 = CH2-CH2-CH3 = 2,2-dipropylpentanoic acid,
H3C
CONH2
R R1
E. R = R1 = H: pentanamide (valeramide),
F. R = H, R1 = CH2-CH2-CH3: 2-propylpentanamide,
G. R = R1 = CH2-CH2-CH3: 2,2-dipropylpentanamide,
H3C
CN
R R1
H. R = R = H: pentanenitrile (valeronitrile),
I. R = H, R = CH2-CH2-CH3: 2-propylpentanenitrile,
J. R = R = CH2-CH2-CH3: 2,2-dipropylpentanenitrile.
__________________________________________________________________________________________________________ Ph Eur
42-28
Vancomycin Hydrochloride
NH2
HO
Me
Me
O O
HO
OH
O
HOCH2
Cl
Cl
,HCl
HO
OH
O
O
H
HN
H
N
O
H
N
N
H H
COOH
O
O
NH2
N
H
CH3
H
NH CH
3
Me
OH
HO
OH
C66H75Cl2N9O24,HCl
1486
1404-93-9
Vancomycin Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Vancomycin Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Vancomycin hydrochloride is the hydrochloride of a mixture of related glycopeptides, consisting
principally of the monohydrochloride of (S)-(3S,6R,7R,22R,23S,26S,36R,38aR)-44-[2-O-(3-amino2,3,6-trideoxy-3-C-methyl--L-lyxo-hexo-pyranosyl)--D-glucopyranosyl]oxy-3-(carbamoylmethyl)10,19-dichloro-2,3,4,5,6,7,23,24,25,26,36,37,38,38a-tetradecahydro-7,22,28,30,32-pentahydroxy-6[(2R)-4-methyl-2-(methylamino)valeramido]-2,5,24,38,39-pentaoxo-22H-8,11:18,21-dietheno23,36-(iminomethano)-13,16:31,35-dimetheno-1H,16H-[1,6,9]oxadiazacyclohexadecino[4,5-m][10,2,16]benzoxadiazacyclotetracosine-26-carboxylic acid (vancomycin B), a substance produced by
certain strains of Amycolatopsis orientalis or obtained by any other means. The potency is not less than
1050 I.U. per milligram, calculated with reference to the anhydrous substance.
CHARACTERS
A white or almost white powder, hygroscopic, freely soluble in water, slightly soluble in alcohol.
IDENTIFICATION
A. Examine the chromatograms obtained in the test for vancomycin B. The retention time of the
principal peak in the chromatogram obtained with test solution (a) is similar to that of the principal
TESTS
Appearance of solution Dissolve 2.50 g in water R and dilute to 25.0 ml with the same solvent. The
solution is clear (2.2.1). The absorbance (2.2.25) of the solution measured at 450 nm is not greater
than 0.10.
Vancomycin B Not less than 93.0 per cent, determined by liquid chromatography (2.2.29).
Test solution (a). Dissolve 10.0 mg of the substance to be examined in mobile phase A and dilute to
Test solution (b). Dilute 2.0 ml of test solution (a) to 50.0 ml with mobile phase A.
Test solution (c). Dilute 0.5 ml of test solution (b) to 20.0 ml with mobile phase A.
42-29
Reference solution. Dissolve 5 mg of vancomycin hydrochloride CRS in 4 ml of water R and dilute to
10 ml with the same solvent. Heat at 65C for 24 h. Allow to cool.
Use the solutions within 4 h of preparation.
as mobile phases at a flow rate of 1.0 ml per minute the following solutions:
Mobile phase A. To 4 ml of triethylamine R add 1996 ml of water R and adjust to pH 3.2 with
phosphoric acid R; to 920 ml of this solution add 10 ml of tetrahydrofuran R and 70 ml of acetonitrile R,
Mobile phase B. To 4 ml of triethylamine R add 1996 ml of water R and adjust to pH 3.2 with
phosphoric acid R; to 700 ml of this solution add 10 ml of tetrahydrofuran R and 290 ml of acetonitrile R,
Record the chromatograms under the following conditions. Elute initially with mobile phase A.
After 13 min, use gradient elution increasing the concentration of mobile phase B by 11 per cent V/V
per minute. Finally, elute for 4 min using mobile phase B.
Inject test solution (c). The test is not valid unless the principal peak in the chromatogram
obtained has a signal-to-noise ratio of at least five. Inject test solution (b). The test is not valid unless
the symmetry factor of the vancomycin peak is at most 1.6. Inject the reference solution. The test is
not valid unless the resolution between the two principal peaks is at least 5.0. Inject test solution (a).
Calculate the percentage content of vancomycin B hydrochloride using the expression:
A b 100
A b + ( A t / 25 )
Ab = area of the peak corresponding to vancomycin B in the chromatogram obtained with test
solution (b),
At = sum of the areas of the peaks corresponding to impurities in the chromatogram obtained
with test solution (a).
Related substances Examine by liquid chromatography (2.2.29), as described under the test for
vancomycin B.
Inject separately test solution (a), test solution (b) and test solution (c). Calculate the percentage
content of each impurity using the expression:
( Ai / 25 ) 100
A b + ( At / 25 )
Ai = area of an impurity peak in the chromatogram obtained with test solution (a),
Ab = area of the peak corresponding to vancomycin B in the chromatogram obtained with test
solution (b),
At = sum of the areas of the peaks corresponding to impurities in the chromatogram obtained
with test solution (a).
The content of no impurity is greater than 4.0 per cent and the sum of the contents of impurities is
not greater than 7.0 per cent. Disregard any peak with an area less than that of the principal peak in
the chromatogram obtained with test solution (c).
without a further appropriate procedure for removal of bacterial endotoxins, not more than 0.25 I.U.
of endotoxin per milligram.
ASSAY
Carry out the microbiological assay of antibiotics (2.7.2). Use vancomycin hydrochloride CRS as the
reference substance.
STORAGE
42-30
LABELLING
The label states:
IMPURITIES
O
N
H
CH3
H NH2 CH3
A. N-demethylvancomycin,
O
H
N
N
H
CH3
N
H H
H NH CH3
Me
COOH
B. desamidovancomycin,
Cl
OH
O
Cl
O
OH
C. aglucovancomycin,
OH
HO
OH
O
HOCH2
Cl
O
O
Cl
O
OH
D. desvancosaminylvancomycin.
__________________________________________________________________________________________________________ Ph Eur
42-31
Vanillin
CHO
HO
OMe
C 8 H 8 O3
152.1
121-33-5
Vanillin complies with the requirements of the 3rd edition of the European Pharmacopoeia [0747]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Vanillin contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of
4-hydroxy-3-methoxybenzaldehyde, calculated with reference to the dried substance.
CHARACTERS
White or slightly yellowish, crystalline powder or needles, slightly soluble in water, freely soluble in
alcohol and in methanol, soluble in ether. It dissolves in dilute solutions of alkali hydroxides.
IDENTIFICATION
obtained with vanillin CRS. Examine the substances prepared as discs.
C. Examine the chromatograms obtained in the test for related substances in daylight after spraying.
The principal spot in the chromatogram obtained with test solution (b) is similar in position, colour
D. To 5 ml of a saturated solution of the substance to be examined add 0.2 ml of ferric chloride solution R1. A blue colour is produced. Heat to 80C. The solution becomes brown. On cooling, a white
TESTS
solution is clear (2.2.1) and not more intensely coloured than reference solution B6 (Method II,
2.2.2).
coating substance.
Test solution (a). Dissolve 0.1 g of the substance to be examined in methanol R and dilute to 5 ml with
the same solvent.
Reference solution (a). Dissolve 10 mg of vanillin CRS in methanol R and dilute to 5 ml with the same
solvent.
Reference solution (b). Dilute 0.5 ml of test solution (a) to 100 ml with methanol R.
10 cm using a mixture of 0.5 volumes of anhydrous acetic acid R, 1 volume of methanol R and 98.5
volumes of methylene chloride R. Dry the plate in a current of cold air. Examine in ultraviolet light at
cent). Spray with dinitrophenylhydrazine-aceto-hydrochloric solution R and examine in daylight. Any
spot in the chromatogram obtained with test solution (a), apart from the principal spot, is not more
intense than the spot in the chromatogram obtained with reference solution (b) (0.5 per cent).
Reaction with sulphuric acid Dissolve 50 mg in 5 ml of sulphuric acid R. After 5 min, the solution
is not more intensely coloured than a mixture of 4.9 ml of yellow primary solution and 0.1 ml of red
primary solution or a mixture of 4.9 ml of yellow primary solution and 0.1 ml of blue primary solution (Method I, 2.2.2).
42-32
desiccator for 4 h.
ASSAY
Dissolve 0.120 g in 20 ml of alcohol R and add 60 ml of carbon dioxide-free water R. Titrate with 0.1M
sodium hydroxide, determining the end-point potentiometrically (2.2.20).
STORAGE
__________________________________________________________________________________________________________ Ph Eur
42-33
Vegetable Fatty Oils

1/01
Vegetable Fatty Oils comply with the requirements of the 3rd edition of the European Pharmacopoeia [1579].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Vegetable fatty oils are mainly solid or liquid triglycerides of fatty acids. They may contain small
amounts of other lipids such as waxes, free fatty acids, partial glycerides or unsaponifiable matters.
Vegetable fatty oils are obtained from the seeds, the fruit or the pit/stone/kernel of various plants by
expression and/or solvent extraction, then possibly refined and hydrogenated. A suitable antioxidant
may be added if necessary.
Virgin oil: an oil obtained from raw materials of special quality by mechanical procedures (e. g. by
cold expression or centrifugation).
Refined oil: an oil obtained by expression and/or solvent extraction, and subsequently, either alkali
refining (followed by bleaching and deodorisation) or physical refining.
Hydrogenated oil: an oil obtained by expression and/or solvent extraction, and subsequently, either
alkali refining or physical refining, then possible bleaching, followed by drying, hydrogenation and
subsequent bleaching and deodorisation.
Only phosphoric acid and alkali refined oils are used in the preparation of parenteral dosage forms.
PRODUCTION
OBTAINING OF A CRUDE OIL
When the plant has a high oil content, the oil is generally obtained by expression under heating
followed by an extraction; when the plant has a low oil content, the oil is generally obtained by direct
extraction.
Mechanical procedures
A. Expression
High-pressure screw-pressing. It consists of some or all of the following steps: cleaning, drying, dehulling or decorticating, grinding, cooking and flaking.
During cleaning the foreign matter is eliminated. Drying may be necessary if the seed moisture content
is higher than desirable for downstream processing. Decorticating is useful to obtain a high-protein
meal by reduction of fibre and to reduce impurities in the oil. Cooking serves various purposes:
completion of the breakdown of oil cells, lowering of the viscosity of the oil, coagulation of the
protein in the meal, adjustment of the moisture level, sterilisation of the seed, detoxifying undesirable
seed constituents (gossypol for cottonseed) and fixing certain phosphatides in the cake thus lowering
subsequent refining losses. The efficacy of the expression process is such that only 3 per cent to 6 per
cent of the oil is left in the cake.
Wet screw-pressing. The bunches are loaded into cages (for palm fruit) and moved into a horizontal
steriliser with application of live steam and heating. The purposes of this steriliser are inactivation of
enzymes, loosening of the fruit on the bunch, coagulation of proteins, etc. After heating in a digester,
the pulp is fed to a screw-press. The oil is centrifugally clarified and vacuum-dried.
Pre-pressing followed by solvent extraction. The same sequence of steps is performed as above. The main
function of pre-pressing is to obtain a cake of excellent permeability for the following solvent extraction stage. The extraction is performed either in a percolation type or in an immersion type
apparatus. The efficacy of the solvent extraction process is such that residual oil levels in meal are
generally below 1 per cent.
B. Centrifugation
Centrifugation separates the oily phase from the aqueous phase which contains water, water-soluble
components and residual solid particles. This operation can be carried out using:
self-cleaning bowl or disc centrifuges,
super-decanters, which are horizontal turbines equipped with a cylindrical bowl that tapers
slightly at one end and which contains a continuously turning screw that scrapes the sides of the
bowl. The screw and the bowl rotate at different speeds. The solid particles are discarded from
the tapered end of the bowl and the oil flows out from the other end.
Solvent extraction Prior to extraction, the following steps are carried out: the seeds are tempered
for about a week at a temperature below 24C in order to loosen the hull from the seed and allow the
seed moisture to attain equilibrium. Then the seeds are cleaned, ground, dehulled and flaked. The
most widely used solvent is a mixture of mainly n-hexane and methylpentanes (bp: 65-70C)
42-34
commonly referred to as hexane. Due to the major fire and explosive risks of this mixture, liquified
gases and supercritical gases may also be used.
REFINING
The objective of refining is to remove impurities and contaminants of the oil with the least possible
damage to the triglycerides and with minimal loss of oil. The contents of the following substances are
reduced:
free fatty acids which may cause deterioration of the oil by oxidation, smoked taste when heated
and sharp flavour (by alkali refining),
water which favours the enzymatic hydrolysis reactions (by alkali refining, drying),
partial glycerides which may cause foaming and bitter taste (by neutralisation, washing),
phosphatides and phosphorous compounds which have emulsifying properties, may cause
deposits, a darkening of the oil when heated, a cloudy appearance and bad organoleptic stability
(by alkali refining),
colouring matters such as chlorophyll (by alkali refining), carotenoids (by bleaching),
glycolipids which may form colloidal solutions with water,
free hydrocarbons, paraffin, waxes and resinous materials,
metals (Fe, Cu, Pb, Sn, Pt, Pd, etc.) which are strong oxidation catalysts,
pigments such as gossypol (in cottonseed oil) or mycotoxins such as aflatoxin (mainly in arachis
oil),
pesticides,
oxidation products (aldehydes, peroxides),
proteins having possible allergic reactions,
unsaponifiable matters (e. g. lignins, sterols, tocopherols and other vitamins),
polycyclic aromatic hydrocarbons.
Alkali refining It involves the following steps: degumming, neutralisation using alkali, washing and
drying.
Degumming. During this step of the refining, i.e. treatment with water and/or phosphoric acid, and/or
sodium chloride, the phosphatides, phosphorous compounds and metals are eliminated. The use of
this step depends on the nature of the oil.
Neutralisation with alkali. This step reduces the free fatty acid content below 0.1 per cent; the fatty
acids are converted into oil-insoluble soaps, also called soapstocks. Other substances may be
removed by adsorption on these soaps: mucilaginous substances, phosphatides, oxidation products,
colouring matters, etc. All substances that become insoluble in the oil on hydration are removed.
Neutralisation with alkali has the disadvantage of saponifying a portion of neutral oil if the neutralisation is not well conducted.
Washing. This operation consists in removing the excess of soaps and alkali as well as the remaining
traces of metals, phosphatides and other impurities, using hot water.
Drying. The remaining water is eliminated under vacuum before any further steps, such as bleaching.
Physical refining It involves a steam treatment of the oil under high vacuum at a temperature
greater than 235C. This technique must be applied to oils naturally low in phosphatides and metals
(palm, coconut, olive) or from which phosphatides and metals have been removed by an acid
treatment using concentrated phosphoric acid followed by an adsorptive treatment with activated
bleaching earth (for sunflower, rapeseed, soya-bean). Moreover it cannot be used for heat sensitive
oils (cottonseed oil) which darken.
Bleaching The common method of bleaching is by adsorption treatment of the oil, which is
generally heated at 90C for 30 min under vacuum, with bleaching earth (natural or activated) or
carbon (activated or not); synthetic silica adsorbents may also be added. Substances which have not
been totally removed during refining are eliminated, for example carotenoids and chlorophyll.
Deodorisation Deodorisation eliminates odours, volatile substances and residual extraction solvents;
it involves injecting dry vapour into the oil which is kept under vacuum at a high temperature.
Different temperatures are used according to the oil: 200C to 235C for 1 h 30 min to 3 h or greater
than 240C for 30 min.
One of the main side reactions is thermic decolourisation due to the destruction of carotenoids
when the temperature is greater than 150C. This technique provokes a loss of substances which may
be distilled (free fatty acids, sterols, tocopherols, part of the refined oil) and may cause cis-trans
isomerisation of the unsaturated fatty-acid double bonds.
WINTERISATION
Elimination of solids and waxes by filtration at low temperature (also called dewaxing). These solids
and waxes could affect the appearance of the oil and cause deposits.
HYDROGENATION
The hydrogenation of the dried and/or bleached oil is performed using a catalyst (e.g. Ni, Pt, Pd), at
42-35
a temperature of about 100C to 200C under hydrogen pressure. The catalyst is then removed by
filtration at 90C. The hydrogen must be pure: free of poisons for the catalyst, water-free, low in
carbon dioxide, methane and nitrogen contents. Small amounts of trans-fatty acids or polymers may
be obtained.
CHROMATOGRAPHIC PURIFICATION
In high purity applications, mainly for parenteral uses, the oil may be further purified by passing the
oil through a column containing an activated earth. A solvent may sometimes be used to improve the
efficiency. High polarity molecules such as oxidised materials, acids, alcohols, partial glycerides and
free sterols are preferentially removed.
When the oil is used in the preparation of parenteral dosage forms, the limits set in the monograph
for the acid value, the peroxide value and the water content may be different.
LABELLING
The label states:
where applicable, that the oil was obtained by expression or extraction,
where applicable, that the oil is suitable for use in the manufacture of parenteral dosage forms,
__________________________________________________________________________________________________________ Ph Eur
42-36
Hydrogenated Vegetable Oil

68334-00-9
Definition Hydrogenated Vegetable Oil is a mixture of triglycerides of fatty acids of vegetable origin.
Characteristics An almost white, fine powder at room temperature and a pale yellow, oily liquid
above its melting point.
Practically insoluble in water; soluble in chloroform, in hexane and in hot propan-2-ol.
Identification Complies with the tests for Acid value, Iodine value and Saponification value.
Iodine value Not more than 5 (iodine monochloride method), Appendix X E. Dissolve 1 g in 20 ml of
dichloromethane.
Saponification value 175 to 205, Appendix X G, Method II.
Unsaponifiable matter Not more than 0.8% w/w, Appendix X H.
Storage Hydrogenated Vegetable Oil should be kept in a well-closed container and stored at a
temperature of 8 to 25.
42-37
Verapamil Hydrochloride
Pr i
CN
Me
N
OMe
,HCl
MeO
OMe
OMe
and enantiomer
C 27H38N 2O4,HCl
491.1
152-11-4
Verapamil Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparations
Verapamil Injection
Verapamil Tablets
Prolonged-release Verapamil Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Verapamil hydrochloride contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of (2RS)-2-(3,4-dimethoxyphenyl)-5-[[2-(3,4-dimethoxyphenyl)ethyl] (methyl)amino]-2-(1-methylethyl)pentanenitrile hydrochloride, calculated with reference to the dried
substance.
CHARACTERS
A white, crystalline powder, soluble in water, sparingly soluble in alcohol, freely soluble in methanol.
IDENTIFICATION
5.0 ml of this solution to 50.0 ml with 0.01M hydrochloric acid. Examined between 210 nm and and
340 nm (2.2.25), the solution shows two absorption maxima, at 229 nm and 278 nm, and a shoulder
at 282 mn. The ratio of the absorbance at the maximum at 278 nm to that at the maximum at
229 nm is 0.35 to 0.39.
obtained with verapamil hydrochloride CRS. Examine the substances prepared as discs.
Reference solution (a). Dissolve 20 mg of verapamil hydrochloride CRS in methylene chloride R and dilute
Reference solution (b). Dissolve 5 mg of papaverine hydrochloride CRS in reference solution (a) and
dilute to 5 ml with the same reference solution.
15 volumes of diethylamine R and 85 volumes of cyclohexane R. Allow the plate to dry in air and
(b) shows two clearly separated principal spots.
TESTS
Solution S Dissolve 1.0 g in carbon dioxide-free water R while gently heating and dilute to 20.0 ml
42-38
+0.10.
Test solution. Dissolve 25.0 mg of the substance to be examined in the initial mobile phase composition and dilute to 10.0 ml with the same mobile phase.
Reference solution (a). Dissolve 5 mg of verapamil hydrochloride CRS, 5 mg of verapamil impurity I CRS
and 5 mg of verapamil impurity M CRS in the initial mobile phase composition and dilute to 20 ml
with the same mobile phase. Dilute 1 ml of this solution to 10 ml with the initial mobile phase
composition.
Reference solution (b). Dilute 1.0 ml of the test solution to 100.0 ml with the initial mobile phase
composition. Dilute 1.0 ml of this solution to 10.0 ml with the same mobile phase.
chromatography, octadecanoylaminopropylsilyl R (5 m),
as mobile phase at a flow rate of 1.5 ml/min a double isocratic programme using the following
conditions:
Mobile phase A. A 6.97 g/l solution of dipotassium hydrogen phosphate R adjusted to pH 7.20 with
phosphoric acid R,
Time
(min)
Mobile phase A
(per cent V/V)

(per cent V/V)
022
2227
63
6335
37
3765
2735
3536
35
3563
65
6537
3650
63
37
first isocratic step

switch to second
isocratic step
second isocratic step
switch to initial
mobile phase
equilibration

Equilibrate the column with the initial mobile phase composition for about 60 min.
conditions the retention times are: verapamil, about 16 min; verapamil impurity I, about 21 min and
verapamil impurity M, about 32 min, eluting as a doublet. The test is not valid unless the resolution
between the peaks corresponding to verapamil and verapamil impurity I is at least 5.0 and verapamil
impurity M elutes from the column.
Adjust the sensitivity of the system so that the height of principal peak in the chromatogram obtained
with 10 l of the reference solution (b) is at least 15 per cent of the full scale of the recorder.
Inject separately 10 l of the test solution and 10 l of reference solution (b). In the chromatogram
cent); the sum of the areas of the peaks, apart from the principal peak, is not greater than three times
cent). Disregard any peak with an area less than 0.1 times the area of the principal peak in the
100C to 105C.
ASSAY
Dissolve 0.400 g in 50 ml of ethanol R and add 5.0 ml of 0.01M hydrochloric acid. Titrate with 0.1M
sodium hydroxide, determining the end-point potentiometrically (2.2.20). Measure the volume of
titrant required between the two points of inflection.
STORAGE
42-39
IMPURITIES
MeO
Ar =
MeO
Ar
Me
Me
Ar
A. N,N-bis[2-(3,4-dimethoxyphenyl)ethyl]-N,N-dimethylpropane-1,3-diamine,
NHMe
Ar
B. 2-(3,4-dimethoxyphenyl)-N-methylethanamine,
NMe2
Ar
C. 2-(3,4-dimethoxyphenyl)-N,N-dimethylethanamine,
Me
N
Ar
Cl
D. 3-chloro-N-[2-(3,4-dimethoxyphenyl)ethyl]-N-methylpropan-1-amine,
E. Ar-CH2OH: (3,4-dimethoxyphenyl)methanol,
Pr i
CN
NHMe
Ar
and enantiomer
F. (2RS)-2-(3,4-dimethoxyphenyl)-5-(methylamino)-2-(1-methylethyl)pentanenitrile,
G. Ar-CHO: 3,4-dimethoxybenzaldehyde,
Et
Me
CN
Ar
Ar
and enantiomer
H. (2RS)-5-[[2-(3,4-dimethoxyphenyl)ethyl](methyl)amino]-2-(3,4-dimethoxyphenyl)-2ethylpentanenitrile,
Pr i
CN
Ar
Ar
and enantiomer
Me
I. (2RS)-2-(3,4-dimethoxyphenyl)-2-[2-[[2-(3,4-dimethoxyphenyl)ethyl](methyl)amino]ethyl]-3-methylbutane nitrile
Pr i
CN
H
N
Ar
Ar
and enantiomer
J. (2RS)-2-(3,4-dimethoxyphenyl)-5-[[2-(3,4-dimethoxyphenyl)ethyl]amino]-2-(1-methylethyl)pentanenitrile (N-norverapamil),
Pr i
CN
and enantiomer
Ar
K. (2RS)-2-(3,4-dimethoxyphenyl)-3-methylbutanenitrile,
O
Ar
CH3
CH3
L. 1-(3,4-dimethoxyphenyl)-2-methylpropan-1-one,
42-40
Ar
Pr i
CN Pr i
CN
N
Ar
Ar
M. 5,5-[[2-(3,4-dimethoxyphenyl)ethyl]imino]bis[2-(3,4-dimethoxyphenyl)-2-(1-methylethyl)pentanenitrile],
Pr i
CN
Me
CN Pr i
Ar
Ar
N. 5,5-(methylimino)bis[2-(3,4-dimethoxyphenyl)-2-(1-methylethyl)pentanenitrile],
Prn CN
Me
N
Ar
and enantiomer
Ar
O. (2RS)-2-(3,4-dimethoxyphenyl)-5-[2-[[2-3,4-dimethoxyphenyl)ethyl](methyl)amino]ethyl]-2-propylpentanenitrile,
Pr i
Ar
CN
CN Pr i
Ar
P. 2,6-bis(3,4-dimethoxyphenyl)-2,6-bis(1-methylethyl)heptane-1,7-dinitrile.
__________________________________________________________________________________________________________ Ph Eur
42-41
Vigabatrin
H
NH2
H2C
COOH
and enantiomer
C6H11NO2
129.2
60643-86-9
Definition Vigabatrin is (RS)-4-aminohex-5-enoic acid. It contains not less than 98.0% and not
more than 102.0% of C6H11NO2, calculated with reference to the anhydrous, ethanol-free substance.
Characteristics A white to almost white powder.
Identification
vigabatrin (RS 360).
B. In the Assay, the retention time of the principal peak in the chromatogram obtained with solution
(1) is the same as that of the principal peak in the chromatogram obtained with solution (2).
Optical rotation +0.5 to 0.5, determined in a 20% w/v solution, Appendix V F.
Related substances The sum of the impurities determined by methods A and B below is not more
than 0.5%.
A. Not more than 0.1% of any single impurity determined by the following method. Carry out the
method for liquid chromatography, Appendix III D, using the following solutions in the mobile phase.
Solution (1) contains 0.4% w/v of the substance being examined. Solution (2) contains 0.0004% w/v
of 3-aminopent-4-ene-1,1-dicarboxylic acid BPCRS. Solution (3) contains 0.0004% w/v of 5-vinyl-2pyrrolidone BPCRS. Solution (4) contains 0.0004% w/v of (E)-4-amino-2-ethylidenebutyric acid hydrochloride BPCRS. Solution (5) contains 0.002% w/v of 5-vinyl-2-pyrrolidone BPCRS and 0.4% w/v of
vigabatrin BPCRS.
The chromatographic procedure may be carried out using (a) two stainless steel columns in series;
the first (25 cm 4.6 mm) packed with particles of silica, the surface of which has been modified
with chemically-bonded hexylsilyl groups (5 m) (Spherisorb C6 is suitable) and the second
(25 cm 4.6 mm) packed with cation exchange resin (10 m) (Partisil-10 SCX is suitable), (b) as
the mobile phase at a flow rate of 1.0 ml per minute, a mixture of 25 volumes of acetonitrile, 25
volumes of a phosphate buffer solution prepared by dissolving 58.5 g of sodium dihydrogen orthophosphate monohydrate in water, adding 23 ml of orthophosphoric acid and sufficient water to produce
1000 ml, and 950 volumes of water and (c) a detection wavelength of 210 nm.
Inject 20 l of solution (5). When the chromatograms are recorded under the prescribed conditions, the retention times are 5-vinyl-2-pyrrolidone, about 18 minutes and vigabatrin, about 21
minutes. The test is not valid unless the resolution factor between the peaks corresponding to 5-vinyl2-pyrrolidone and vigabatrin is at least 1.5; if necessary, adjust the concentration of the mobile phase
(reduce the concentration of the phosphate buffer solution to increase the retention time of vigabatrin
and increase the concentration of acetonitrile to decrease the retention time of 5-vinyl-2-pyrrolidone).
Inject separately 20 l of each of solutions (1) to (4). For solution (1) continue the chromatography
for twice the retention time of the principal peak. In the chromatogram obtained with solution (1) the
areas of any peaks corresponding to 5-vinyl-2-pyrrolidone and (E)-4-amino-2-ethylidenebutyric acid
are not greater than the areas of the peaks in the chromatograms obtained with solutions (3) and (4),
respectively (0.1% of each). The area of any other secondary peak is not greater than the area of the
peak in the chromatogram obtained with solution (2) (0.1% ). Calculate the percentage content of
5-vinyl-2-pyrrolidone and (E)-4-amino-2-ethylidenebutyric acid using the areas of the peaks in the
chromatograms obtained with solutions (3) and (4) respectively and of any other impurity from the
peak in the chromatogram obtained with solution (2) taking, for the purposes of calculation, that this
peak is equivalent to 0.1%, and hence determine the sum of the contents of the impurities.
B. Not more than 0.2% of 4-aminobutyric acid when determined by the following method. Carry out
the method for liquid chromatography, Appendix III D, using the following solutions. For solution (1)
add to 1 ml of a 0.20% w/v solution of the substance being examined 2 ml of a solution prepared by
dissolving 7.7 g of boric acid in water, adjusting to pH 7.7 with a 50% w/v solution of sodium hydroxide
and diluting to 250 ml with water and mix. Add 3 ml of a 0.16% w/v solution of (9-fluorenyl)methyl
chloroformate in acetone, mix and allow to stand for 5 minutes. Add 3 ml of ethyl acetate, shake
vigorously for a few seconds, and allow to separate; use the lower layer within 8 hours of preparation.
Prepare solution (2) in the same manner using 1 ml of a solution containing 0.20% w/v of the
substance being examined and 0.002% w/v of 4-aminobutyric acid in place of the solution of the
substance being examined.
42-42
(15 cm 4.6 mm) packed with particles of silica the surface of which has been modified by
chemically-bonded phenyl groups (5 m) (Vydac Phenyl is suitable), (b) as the mobile phase at a flow
rate of 1.0 ml per minute, a mixture of 25 volumes of acetonitrile and 75 volumes of a solution
prepared by dissolving 8.2 g of anhydrous sodium acetate in water, adjusting the pH to 4.2 with glacial
acetic acid and diluting to 2000 ml with water and (c) a detection wavelength of 263 nm.
Inject 25 l of solution (2). When the chromatogram is recorded under the prescribed conditions,
the retention times for the derivatives of the following are (9-fluorenyl)methanol, about 6 minutes;
4-aminobutyric acid, about 9 minutes and vigabatrin, about 14 minutes. The test is not valid unless
the resolution factor between the peaks corresponding to the derivatives of 4-aminobutyric acid and
(9-fluoreny)lmethanol is at least 2; if necessary, adjust the proportion of the components of the
mobile phase.
Inject separately 25 l of each of solutions (1) and (2) and calculate the content of 4-aminobutyric
acid in the substance being examined from the chromatograms obtained.
Ethanol Not more than 0.6% w/w of ethanol when determined by the following method. Carry out
the method for head-space gas chromatography, Appendix III B. Solution (1) contains 2 g of the
substance being examined in 10 ml of a solution containing 0.0025% w/v of 1,2-dichloroethane
(internal standard) in water in a 20-ml headspace vial. Heat the vial at 60 for 30 minutes. Solution
(2) contains 0.025% w/v of absolute ethanol and 0.0025% w/v of 1,2-dichloroethane in water. Use water
as the blank.
The chromatographic procedure may be carried out using a fused-silica column (60 m 0.32 mm)
coated with a 1.0-m film of bonded methylsilicone (SPB-1 is suitable) at an initial temperature of
35 for 12 minutes, increasing to 175 at a constant rate of 10 per minute, maintaining the injector
at 150 and the detector at 250. Use helium as the carrier gas. Calculate the content of ethanol in the
substance being examined from the areas of the peaks in the chromatograms obtained with solutions
(1) and (2).
Water Not more than 0.5% w/w, Appendix IX C. Use 0.3 g dissolved in 50 ml of anhydrous
methanol.
Assay Carry out the method for liquid chromatography, Appendix III D, using the following solutions
in the mobile phase. Solution (1) contains 0.2% w/v of the substance being examined. Solution (2)
contains 0.2% w/v of vigabatrin BPCRS. Solution (3) contains 0.002% w/v of 5-vinyl-2-pyrrolidone
BPCRS and 0.2% w/v of vigabatrin BPCRS.
(25 cm 4.6 mm) packed with cation exchange resin (10 m) (Whatman Partisil 10 SCX is suitable), (b) as the mobile phase with a flow rate of 1.5 ml per minute a mixture of 4 volumes of acetonitrile, 40 volumes of methanol and 1000 volumes of a 0.34% w/v solution of potassium dihydrogen
orthophosphate, the mixture adjusted to pH 2.8 with orthophosphoric acid and (c) a detection wavelength of 210 nm.
Inject 20 l of solution (3). When the chromatogram is recorded under the prescribed conditions,
the retention times are 5-vinyl-2-pyrrolidone, about 5 minutes and vigabatrin, about 8 minutes. The
test is not valid unless the resolution factor between the peaks corresponding to 5-vinyl-2-pyrrolidone
and vigabatrin is at least 1.5.
Inject separately 20 l of solutions (1) and (2) and calculate the percentage content of vigabatrin
from the areas of the peaks using the declared content of C6H11NO2 in vigabatrin BPCRS.
Storage Vigabatrin should be kept in a well-closed container.
Preparations
Vigabatrin Oral Powder
Vigabatrin Tablets
IMPURITIES
O
HN
H2C
A. 5-vinyl-2-pyrrolidone
H3C
H2N
COOH
B. (E)-4-amino-2-ethylidenebutyric acid
42-43
O
HN
H2C
CONH2
C. 2-oxo-5-vinylpyrrolidine-3-carboxamide
H2N
COOH
D. 4-aminobutyric acid
NH2 COOH
H2C
COOH
E. 3-aminopent-4-ene-1,1-dicarboxylic acid
42-44
Vinblastine Sulphate
OH
Et
N
H
COOMe
,H2SO4
N
Et
H
MeO
N
H
Me
OAc
H
COOMe
HO
C 46H58N4O9,H2SO4
909
143-67-9
Vinblastine Sulphate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Vinblastine Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Vinblastine sulphate contains not less than 95.0 per cent and not more than the equivalent of
104.0 per cent of methyl (3aR,4R,5S,5aR,10bR,13aR)-4-acetoxy-3a-ethyl-9-[(5S,7R,9S)-5-ethyl-5hydroxy-9-(methoxycarbonyl)-1,4,5,6,7,8,9,10-octahydro-2H-3,7-methano-azacyclo-undecino[5,4b]indol-9-yl]-5-hydroxy-8-methoxy-6-methyl-3a,4,5,5a,6,11,12,13a-octahydro-1H-indolizino[8,1cd]carbazole-5-carboxylate sulphate, calculated with reference to the dried substance.
CHARACTERS
A white or slightly yellowish, crystalline powder, very hygroscopic, freely soluble in water, practically
insoluble in alcohol and in ether.
IDENTIFICATION
spectrum of vinblastine sulphate.
B. Examine the chromatograms obtained in the assay. The principal peak in the chromatogram
obtained with the test solution is similar in position and approximate size to the principal peak in the
TESTS
Solution S Dissolve 50.0 mg in carbon dioxide-free water R and dilute to 10.0 ml with the same
solvent.
pH (2.2.3). Dilute 3 ml of solution S to 10 ml with carbon dioxide-free water R. The pH of this solution is 3.5 to 5.0.
Related substances Examine the chromatograms obtained in the assay. In the chromatogram
obtained with the test solution, the area of any peak apart from the principal peak is not greater than
the area of the principal peak in the chromatogram obtained with reference solution (c) (2.0 per cent)
and the sum of the areas of any such peaks is not greater than 2.5 times the area of the principal peak
in the chromatogram obtained with reference solution (c) (5.0 per cent). Disregard any peak with an
area less than that of the peak in the chromatogram obtained with reference solution (d).
Loss on drying Not more than 15.0 per cent, determined on 3 mg by thermogravimetry (2.2.34).
42-45
Heat to 200C at a rate of 5C per minute, under a stream of nitrogen for chromatography R, at a flow
rate of 40 ml per minute.
ASSAY
Keep the solutions in iced water before use.
Test solution. Dilute 1.0 ml of solution S (see Tests) to 5.0 ml with water R.
Reference solution (a). Dissolve 5.0 mg of vinblastine sulphate CRS in water R and dilute to 5.0 ml with
the same solvent.
Reference solution (b). Dissolve 1.0 mg of vincristine sulphate CRS in 1.0 ml of reference solution (a).
Reference solution (c). Dilute 1.0 ml of reference solution (a) to 50.0 ml with water R.
Reference solution (d). Dilute 1.0 ml of reference solution (c) to 20.0 ml with water R.
gel for chromatography R (5 m). Place between the injector and the column a guard column
packed with suitable silica gel,
as mobile phase at a flow rate of 1.0 ml per minute a mixture of 38 volumes of a 1.5 per cent
V/V solution of diethylamine R adjusted to pH 7.5 with phosphoric acid R, 12 volumes of acetonitrile R and 50 volumes of methanol R,
a loop injector.
Inject 10 l of each solution and record the chromatograms for three times the retention time of the
peak corresponding to vinblastine. The assay is not valid unless: in the chromatogram obtained with
reference solution (b) the resolution between the peaks corresponding to vincristine and vinblastine is
not less than four; the peak in the chromatogram obtained with reference solution (d) has a signal-tonoise ratio not less than five. Calculate the percentage content of C46H60N4O13S from the area of the
principal peak in each of the chromatograms obtained with the test solution and reference solution
(a) and from the declared content of vinblastine sulphate CRS.
STORAGE
Store in an airtight, glass container, protected from light, at a temperature not exceeding 20C. If
the substance is sterile, store in an sterile, airtight, tamper-proof glass container.
LABELLING
__________________________________________________________________________________________________________ Ph Eur
42-46
Vincristine Sulphate
OH
Et
N
H
COOMe
,H2SO4
N
Et
H
MeO
N
H
OHC
OAc
H
COOMe
HO
C 46H56N4O10,H 2SO 4
923.1
2068-78-2
Vincristine Sulphate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Vincristine Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Vincristine sulphate contains not less than 95.0 per cent and not more than the equivalent of
104.0 per cent of methyl (3aR,4R,5S,5aR,10bR,13aR)-4-acetoxy-3a-ethyl-9-[(5S,7R,9S)-5-ethyl-5hydroxy-9-(methoxycarbonyl)-1,4,5,6,7,8,9,10-octahydro-2H-3,7-methano-azacyclo-undecino[5,4b]indol-9-yl]-5-hydroxy-8-methoxy-6-oxo-3a,4,5,5a,6,11,12,13a-octahydro-1H-indolizino[8,1cd]carbazole-5-carboxylate sulphate, calculated with reference to the dried substance.
CHARACTERS
A white or slightly yellowish, crystalline powder, very hygroscopic, freely soluble in water, slightly
IDENTIFICATION
spectrum of vincristine sulphate.
B. Examine the chromatograms obtained in the assay. The principal peak in the chromatogram
obtained with the test solution is similar in position and approximate size to the principal peak in the
TESTS
Solution S Dissolve 50.0 mg in carbon dioxide-free water R and dilute to 10.0 ml with the same
solvent.
Related substances Examine the chromatograms obtained in the assay. In the chromatogram
obtained with the test solution, the area of any peak apart from the principal peak is not greater than
the area of the principal peak in the chromatogram obtained with reference solution (c) (2.0 per cent)
and the sum of the areas of any such peaks is not greater than 2.5 times the area of the principal peak
in the chromatogram obtained with reference solution (c) (5.0 per cent). Disregard any peak with an
area less than that of the peak in the chromatogram obtained with reference solution (d).
Loss on drying Not more than 12.0 per cent, determined on 3 mg by thermogravimetry (2.2.34).
Heat to 200C at a rate of 5C per minute, under a stream of nitrogen for chromatography R, at a flow
rate of 40 ml per minute.
42-47
ASSAY
Examine by liquid chromatography (2.2.29). Keep the solutions in iced water before use.
Test solution. Dilute 1.0 ml of solution S (see Tests) to 5.0 ml with water R.
Reference solution (a). Dissolve 5.0 mg of vincristine sulphate CRS in water R and dilute to 5.0 ml with
the same solvent.
Reference solution (b). Dissolve 1.0 mg of vinblastine sulphate CRS in 1.0 ml of reference solution (a).
Reference solution (d). Dilute 1.0 ml of reference solution (c) to 20.0 ml with water R.
gel for chromatography R (5 m). Place between the injector and the column a guard column
packed with suitable silica gel,
as mobile phase at a flow rate of 1.0 ml per minute a mixture of 30 volumes of a 1.5 per cent
V/V solution of diethylamine R adjusted to pH 7.5 with phosphoric acid R and 70 volumes of
methanol R,
a loop injector.
Inject 10 l of each solution. Record the chromatograms for three times the retention time of the
peak corresponding to vincristine. The assay is not valid unless: in the chromatogram obtained with
reference solution (b) the resolution between the peaks corresponding to vincristine and vinblastine is
at least four; the peak in the chromatogram obtained with reference solution (d) has a signal-to-noise
ratio of at least five. Calculate the percentage content of C46H58N4O14S from the area of the principal
peak in each of the chromatograms obtained with the test solution and reference solution (a) and
from the declared content of vincristine sulphate CRS.
STORAGE
Store in an airtight, glass container, protected from light, at a temperature not exceeding 20C. If
the substance is sterile, store in a sterile, airtight, tamper-proof glass container.
LABELLING
__________________________________________________________________________________________________________ Ph Eur
42-48
Vindesine Sulphate
Et
OH
N
H
H3COOC
N
MeO
,H2SO4
C43H57N5O11S
Et
H
OH
OH
H CONH2
Me
852.0
59917-39-4
Vindesine Sulphate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1276].
Preparation
Vindesine Injection
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Vindesine sulphate contains not less than 96.0 per cent and not more than the equivalent of
103.0 per cent of methyl (5S,7R,9S)-9-[(3aR,4R,5S,5aR,10bR,13aR)-5-carbamoyl-3a-ethyl-4,5dihydroxy-8-methoxy-6-methyl-3a,4,5,5a,6,11,12,13a-octahydro-1H-indolizino[8,1-cd]carbazol-9yl]-5-ethyl-5-hydroxy-1,4,5,6,7,8,9,10-octahydro-2H-3,7-methanoazacycloundecino[4,5-b]indole-9carboxylate sulphate, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, amorphous substance, hygroscopic, freely soluble in water and in methanol
and practically insoluble in cyclohexane.
IDENTIFICATION
spectrum of vindesine sulphate.
TESTS
Solution S Dissolve 50 mg in carbon dioxide-free water R and dilute to 10 ml with the same solvent.
Related substances Examine by liquid chromatography (2.2.29). Keep the solutions in iced water
before use.
the same solvent.
Reference solution (a). Dilute 1.0 ml of the test solution to 50.0 ml with water R.
Reference solution (b). Dissolve 1.0 mg of desacetylvinblastine CRS in water R, add 1.0 ml of the test
solution and dilute to 50.0 ml with water R.
Mobile phase A. A 1.5 per cent V/V solution of diethylamine R adjusted to pH 7.4 with phosphoric
acid R,
42-49
Time
(min)
Mobile phase A
(per cent V/V)

(per cent V/V)
040
4049
49end
49
49
4930
30
51
51
5170
70
equilibration
isocratic
linear gradient
isocratic

Inject separately 200 l of the test solution, 200 l of reference solution (a), 200 l of reference
solution (b) and 200 l of reference solution (c). Maintain the final concentration of the mobile phase
until the total run time is twice the retention time of the principal peak in the chromatogram obtained
with the test solution. The test is not valid unless: in the chromatogram obtained with reference
solution (b) the retention time of vindesine is less than 40 min; the symmetry factor of the vindesine
peak is not more than 2.0; and the resolution of the vindesine and desacetylvinblastine peaks is at
least 2.0. In the chromatogram obtained with the test solution: the area of any peak apart from the
principal peak is not greater than half of the area of the principal peak in the chromatogram obtained
with reference solution (a) (1 per cent); and the sum of the areas of any such peaks is not greater than
the area of the principal peak in the chromatogram obtained with reference solution (a) (2 per cent).
Disregard any peak with an area less than the area of the principal peak in the chromatogram
Acetonitrile. Not more than 1.5 per cent m/m of acetonitrile, determined by gas chromatography
(2.2.28).
Internal standard solution (a). Dilute 0.500 g of propanol R to 100 ml with water R.
Internal standard solution (b). Dilute 10.0 ml of internal standard solution (a) to 50.0 ml with water R.
Reference solution. Dilute 10.0 g of acetonitrile R to 1000 ml with water R. To 3.0 ml of this solution
add 10.0 ml of internal standard solution (a) and dilute to 50.0 ml with water R.
Test solution. Dissolve 40 mg of the substance to be examined in 1.0 ml of internal standard solution
(b).
a glass column 1.25 m long and 3 mm in internal diameter packed with ethylvinylbenzenedivinylbenzene copolymer R,
detector at 250C.
Inject 3 l of the reference solution. The test is not valid unless the resolution between acetonitrile
and propanol is greater than 1.5 and the symmetry factor for the acetonitrile peak is not greater than
1.6.
Inject 3 l of the reference solution and 3 l of the test solution.
Loss on drying. Not more than 10.0 per cent, determined on 9.00 mg by thermogravimetry
(2.2.34). Heat to 200C at a rate of 5C per minute, under a current of nitrogen for chromatography R
at a flow rate of 40 ml/min.
ASSAY
Examine by liquid chromatography (2.2.29). Keep the solutions in iced water before use.
the same solvent.
Reference solution (a). Dissolve and dilute the entire contents of a vial of vindesine sulphate CRS with
water R to yield a concentration of approximately 0.50 mg/ml.
Reference solution (b). Add 1.0 mg of desacetylvinblastine CRS to 2.0 ml of reference solution (a).
as mobile phase at a flow rate of 1 ml/min a mixture of 38 volumes of a 1.5 per cent V/V solution
of diethylamine R, previously adjusted to pH 7.4 with phosphoric acid R, and 62 volumes of
methanol R,
Inject 20 l of reference solution (b) five times. The test is not valid unless: the resolution between
the peaks corresponding to vindesine sulphate and desacetylvinblastine sulphate is greater than 1.5;
the symmetry factor of the vindesine peak is not more than 2.0; and the coefficient of variation of the
area of the vindesine peak, calculated on the five injections is not greater than 1.5 per cent.
42-50
Inject separately 20 l of the test solution and 20 l of reference solution (a).
Calculate the content of vindesine sulphate (C43H57N5O11S) using the stated content of vindesine
sulphate CRS.
STORAGE
Store in an airtight polypropylene container with a polypropylene cap, at a temperature not exceeding
50C. If the substance is sterile, store in a sterile, airtight, tamper-proof container.
LABELLING
IMPURITIES
O
Et
OH
N
H
N
H
H3COOC
Et
H
OH
OH
N H CONH2
Me
H
MeO
A. vindesine 3-N-oxide,
B. vinblastine,
Et
OH
N
H
H3COOC
MeO
N
H
N
Me
Et
H
OH
OH
CONHNH2
C. desacetylvinblastine hydrazide.
__________________________________________________________________________________________________________ Ph Eur
42-51
Vitamin A
1/01
e
Me
Me
Me
OR
Me
R=H
R = CO-CH3
R = CO-C2H5
R = CO-C15H31
C20H30O
C22H32O2
C23H34O2
C36H60O2
286.5
328.5
342.5
524.9
Vitamin A complies with the requirements of the 3rd edition of the European Pharmacopoeia [0217]. These
In the monographs below, the term Retinol is used within titles for preparations containing
synthetic ester(s) and the term Vitamin A within the title for the preparation containing material of
natural origin.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Vitamin A refers to a number of substances of very similar structure (including (Z)-isomers) found in
animal tissues and possessing similar activity. The principal and biologically most active substance is
all-(E)-retinol (all -(E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-enyl)nona-2,4,6,8-tetraen-1-ol;
C20H30O). Vitamin A is generally used in the form of esters such as the acetate, propionate and
palmitate.
Synthetic retinol ester refers to an ester of synthetic retinol (acetate, propionate or palmitate) or a
mixture of synthetic retinol esters.
The activity of vitamin A is expressed in retinol equivalents (R.E.) 1 mg R.E. corresponds to the
activity of 1 mg of all-(E)-retinol. The activity of the other retinol esters is calculated using the
stoichiometry, so that 1 mg R.E. of vitamin A corresponds to the activity of:
1.147 mg of all-(E)-retinol acetate,
1.195 mg of all-(E)-retinol propionate,
1.832 mg of all-(E)-retinol palmitate.
International units (I.U.) are also used. 1 I.U. of vitamin A is equivalent to the activity of 0.300 g of
all -(E)-retinol. The activity of the other retinol esters is calculated using the stoichiometry, so that 1
I.U. of vitamin A is equivalent to the activity of:
0.344 g of all-(E)-retinol acetate,
0.359 g of all-(E)-retinol propionate,
0.550 g of all-(E)-retinol palmitate,
1 mg of retinol equivalent is equivalent to 3333 I.U.
CHARACTERS
Retinol acetate occurs as pale-yellow crystals (melting point about 60C). Once melted retinol acetate
tends to yield a supercooled melt.
Retinol propionate occurs as a reddish-brown oily liquid.
Retinol palmitate is a fat-like, light yellow solid or a yellow oily liquid, if melted (melting point about
26C).
All retinol esters are practically insoluble in water, soluble or partly soluble in ethanol and miscible
with organic solvents. Vitamin A and its esters are very sensitive to the action of air, oxidising agents,
acids, light and heat.
Carry out the assay and all tests as rapidly as possible, avoiding exposure to actinic light and air, oxidising
agents, oxidation catalysts (e.g. copper, iron), acids and heat; use freshly prepared solutions.
IDENTIFICATION
A. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel F254 plate R.
Test solution. Prepare a solution containing about 3.3 I.U. of vitamin A per microlitre in cyclohexane R, stabilised with a 1 g/l solution of butylhydroxytoluene R.
42-52
Reference solution. Prepare a reference solution containing about 0.01 mg of retinol esters CRS per
microlitre (i.e. 3.3 I.U. of each ester per microlitre) in cyclohexane R, stabilised with a 1 g/l solution of
butylhydroxytoluene R.
Apply to the plate 3 l of each solution. Develop immediately over 2/3 of the plate using a mixture of
20 volumes of ether R and 80 volumes of cyclohexane R. Allow the plate to dry in air and examine in
ultraviolet light at 254 nm. The identification is not valid unless the chromatogram obtained with the
reference solution shows the individual spots of the corresponding esters. The elution order from
bottom to top is: retinol acetate, retinol propionate and retinol palmitate. The composition of the test
solution is confirmed by the correspondence of the principal spot or spots with those obtained with
B. It complies with the test for related substances.
TESTS
Retinol Examine by thin-layer chromatography (2.2.27), using a TLC silica gel F254 plate R.
Test solution. Prepare a solution in cyclohexane R, stabilised with a 1 g/l solution of
butylhydroxytoluene R, containing about 330 I.U. of vitamin A per microlitre.
Reference solution. Shake 1 ml of the test solution with 20 ml of 0.1M tetrabutylammonium hydroxide in
2-propanol for 2 min and dilute to 100 ml with cyclohexane R, stabilised with a 1 g/l solution of
butylhydroxytoluene R.
Apply to the plate 3 l of each solution. Develop immediately over a path of 15 cm using a mixture of
ultraviolet light at 254 nm. In the chromatogram obtained with the reference solution no or only
traces of the retinol ester are seen. Any spot corresponding to retinol in the chromatogram obtained
with the test solution is not more intense than the spot in the chromatogram obtained with the
reference solution (1.0 per cent).
Related substances Examine by ultraviolet absorption spectrophotometry (2.2.25). Determine the
absorption maximum of the solution for Activity. The solution shows an absorption maximum at
325 nm to 327 nm. Measure the absorbances at 300 nm, 350 nm and 370 nm and calculate the ratio
A/A326 for each wavelength. None of the ratios A /A326 exceed:
0.593 at 300 nm,
0.537 at 350 nm,
0.142 at 370 nm.
ACTIVITY
The activity of the substance is determined in order to be taken into account for the production of
concentrates.
Examine by ultraviolet absorption spectrophotometry (2.2.25). Dissolve 25 mg to 100 mg, weighed
with an accuracy of 0.1 per cent, in 5 ml of pentane R and dilute with 2-propanol R1 to a presumed
concentration of 10 I.U. to 15 I.U. per millilitre. This solution is also used for the test for related
substances.
Determine the absorbance of the maximum at 326 nm. Calculate the activity of vitamin A in
International Units per gram from the expression:
A326 V 1900
100 m
A326 = absorbance at 326 nm,

m = mass of the substance to be examined, in grams,
V = total volume to which the substance to be examined is diluted to give 10 I.U. to 15 I.U.
per millilitre,
1900 = factor to convert the specific absorbance of esters of retinol into International Units per
gram.
STORAGE
Once the container has been opened, its contents are to be used as soon as possible; any part of the
contents not used at once should be protected by an atmosphere of inert gas.
LABELLING
The label states:
the number of International Units per gram,
the name of the ester or esters.
42-53
IMPURITIES
RO
e
Me
Me
OR
Me
Me
Me
Me
Me
Me
Me
A. R = H, CO-CH3: kitols (Diels-Alder dimers of vitamin A),

e
Me
Me
Me
CH2
Me
B. (3E,5E,7E)-3,7-dimethyl-9-[(1Z)-2,6,6-trimethylcyclohex-2-enylidene)nona-1,3,5,7tetraene (anhydro-vitamin A),

e
Me
Me
Me
OH
Me
C. (3E,5E,7E)-3,7-dimethyl-9-[(1Z)-2,6,6-trimethylcyclohex-2-enylidene)nona-3,5,7-trien-1ol (retro-vitamin A),

D. oxidation products of vitamin A.
__________________________________________________________________________________________________________ Ph Eur
42-54
Natural Vitamin A Ester Concentrate

Definition Natural Vitamin A Ester Concentrate consists of a natural ester or a mixture of natural
esters of retinol or of a solution of the ester or mixture of esters in Arachis Oil or other suitable
vegetable oil. It contains in 1 g not less than 485,000 IU of vitamin A and not less than 97.0% of
the number of IU of vitamin A stated on the label. It may contain a suitable antioxidant or mixture
of antioxidants.
Characteristics A yellow oil or a mixture of oil and crystalline material which yields a
homogeneous yellow oil on warming; odour, faint.
Solubility Practically insoluble in water; soluble or partly soluble in ethanol (96%); miscible with
chloroform, with ether and with petroleum spirit.
Identification
A. Dissolve a quantity containing 10 IU in a mixture of 100 parts of absolute ethanol and 1 part of
hydrochloric acid. The light absorption, Appendix II B, of the solution immediately after preparation
exhibits a single maximum at 326 nm. Heat the solution in a water bath for 30 seconds and cool
rapidly. The light absorption in the range 300 to 400 nm exhibits a low maximum or inflection at
332 nm and sharp maxima at 348, 367 and 389 nm.
B. Dissolve a quantity containing 30 IU in 1 ml of chloroform and add 10 ml of antimony trichloride
solution. A transient bright blue colour is produced immediately.
Peroxide value Place 1 g in a boiling tube (20 cm 2.5 cm) and dissolve in 20 ml of a mixture of
2 volumes of glacial acetic acid and 1 volume of ethanol-free chloroform. Add 1 g of finely powdered
potassium iodide and pass a rapid current of oxygen-free nitrogen through the mixture for 1 minute.
Stopper the tube loosely, partly immerse in boiling water for 30 seconds and then in water at 80
for 2 minutes, tighten the stopper and cool rapidly. Transfer the contents to a flask containing
25 ml of a freshly prepared 1% w/v solution of potassium iodide, rinse the tube with a further 25 ml
of the potassium iodide solution, shake the combined solution and rinsings and titrate with 0.01M
sodium thiosulphate VS. Repeat the procedure without the concentrate. The difference between the
titrations does not exceed 1.4 ml.
Retinol Carry out the method for descending paper chromatography, Appendix III E, using a mixture
of 70 volumes of 1,4-dioxan, 15 volumes of methanol and 15 volumes of water containing 1% w/v of
butylated hydroxyanisole in the bottom of the tank and as the mobile phase. Saturate the paper with a
10% w/v solution of liquid paraffin in petroleum spirit (boiling range, 40 to 60) and dry without the
aid of heat. Apply separately to the impregnated paper 5 l and 10 l of each of the following
freshly prepared solutions. For solution (1) dissolve sufficient of the concentrate in petroleum spirit
(boiling range, 40 to 60) to yield a solution containing 16,150 to 17,850 IU per ml. For solution
(2) saponify a portion of the concentrate by the method described under the assay of vitamin A,
other vitamin A, Appendix VIII K, and dissolve the residue in sufficient petroleum spirit (boiling
range, 40 to 60) to produce a solution of retinol containing about 340 IU per ml.
Develop until the solvent front approaches the bottom of the paper. Examine the dried paper
under ultraviolet light (365 nm). The fluorescence of any spot corresponding to retinol in the
chromatograms obtained with solution (1) is not more intense than that of the spot in the
corresponding chromatogram obtained with solution (2).
Assay Carry out the test as rapidly as possible, avoiding exposure to actinic light and air, oxidising agents,
oxidation catalysts (e.g. copper and iron) and acids.
Examine by ultraviolet absorption spectrophotometry, Appendix II B (Method A). If method A is
found not to be valid, examine by liquid chromatography, Appendix III D (Method B).
Method A
Test solution To a quantity of the substance being examined containing 50,000 IU in a roundbottomed flask, add 3 ml of a freshly prepared 50% w/w solution of potassium hydroxide and 30 ml of
absolute ethanol. Boil under a reflux condenser in a current of nitrogen for 30 minutes. Cool rapidly
and add 30 ml of water. Extract with four 50-ml quantities of ether discarding the lower layer after
complete separation. Wash the combined upper layers with four 50-ml quantities of water and
evaporate to dryness under a gentle current of nitrogen at a temperature not exceeding 30 or in a
rotary evaporator at a temperature not exceeding 30 under reduced pressure (water ejector).
Dissolve the residue in sufficient propan-2-ol to give an expected concentration of vitamin A
equivalent to 10 to 15 IU per ml.
Measure the absorbances, Appendix II B, of the solution at 300 nm, 310 nm, 325 nm and 334 nm
and at the wavelength of maximum absorption with a suitable spectrophotometer in 1-cm specially
matched cells, using propan-2-ol as the compensation liquid.
Calculate the content of vitamin A, as all-trans-retinol, in IU per gram from the expression:
A325
1830
V
100m
42-55
where
m = mass of the substance to be examined in grams,
V = total volume of solution containing 10 IU to 15 IU of vitamin A per ml,
1830 = conversion factor for the specific absorbance of all-trans-retinol in IU.
The above expression can be used only if A325 has a value of not greater than A325,corr/0.970 where
A325,corr is the corrected absorbance at 325 nm and is given by the equation:
A325, corr = 6.815A325 2.555A310 4.260A334
where A designates the absorbance at the wavelength indicated by the subscript.
If A325 has a value greater than A325,corr/0.970, calculate the content of vitamin A from the
expression:
A325,corr
1830
V
100m

(a) the wavelength of maximum absorption lies between 323 nm and 327 nm and
(b) the absorbance at 300 nm relative to that at 325 nm is at most 0.73.
Method B
Carry out the method for liquid chromatography, Appendix III D, using the following solutions.
Prepare solution (1) as follows. To a quantity of the substance being examined containing 50,000 IU
in a round-bottomed flask, add 5 ml of a freshly prepared 10% w/v solution of ascorbic acid and 10 ml
of a freshly prepared 80% w/v solution of potassium hydroxide and 100 ml of ethanol (96%). Boil
under a reflux condenser on a water bath for 15 minutes. Add 100 ml of a 1% w/v solution of sodium
chloride and cool. Transfer the solution to a 500 ml separating funnel rinsing the round-bottomed
flask with about 75 ml of a 1% w/v solution of sodium chloride and then with 150 ml of a mixture of
equal volumes of petroleum spirit (boiling range, 40 to 60) and ether. Shake for 1 minute. When the
layers have separated completely, discard the lower layer and wash the upper layer, first with 50 ml of
a 3% w/v solution of potassium hydroxide in a 10% v/v solution of ethanol (96%) and then with three
50-ml quantities of a 1% w/v solution of sodium chloride. Filter the upper layer through 5 g of
anhydrous sodium sulphate on a fast filter paper into a 250 ml flask suitable for a rotary evaporator.
Wash the funnel with 10 ml of fresh extraction mixture, filter and combine the upper layers. Distil
them at a temperature not exceeding 30 under reduced pressure (water ejector) and fill with nitrogen
when evaporation is completed. Alternatively evaporate the solvent under a gentle current of nitrogen
at a temperature not exceeding 30. Dissolve the residue in propan-2-ol, transfer to a 25-ml
volumetric flask and dilute to 25 ml with propan-2-ol. Gentle heating in an ultrasonic bath may be
required. For solution (2) prepare a solution of retinyl acetate EPCRS in propan-2-ol R1 so that 1 ml
contains about 1000 IU of all-trans-retinol.
The exact concentration of solution (2) is assessed by ultraviolet absorption spectrophotometry,
Appendix II B. Dilute solution (2) with propan-2-ol R1 to a presumed concentration of 10 IU per ml
to 15 IU per ml and measure the absorbance at 326 nm in matched 1-cm cells using propan-2-ol R1 as
the compensation liquid.
Calculate the content of vitamin A in IU per ml of solution (2) from the following expression,
taking into account the assigned content of retinyl acetate EPCRS:
A326
1900 V2
100 V1
where
V2 = volume of the diluted solution,
V1 = volume of solution (2) used,
1900 =conversion factor for the specific absorbance of retinyl acetate EPCRS in IU.
For solution (3) proceed as described for solution (1) but use 2 ml of solution (2) in place of the
substance being examined.
The exact concentration of solution (3) is assessed by ultraviolet absorption spectrophotometry,
Appendix II B. Dilute solution (3) with propan-2-ol R1 to a presumed concentration of 10 IU per ml
to 15 IU per ml of all-trans-retinol and measure the absorbance at 325 nm in matched 1 cm cells
using propan-2-ol R1 as the compensation liquid.
Calculate the content of all-trans-retinol in IU per millilitre of solution (3) from the expression:
A325
where
A325 =absorbance at 325 nm,
1830 V4
100 V3
42-56
V3 = volume of the diluted solution,
V4 = volume of solution (3) used,
1830 = conversion factor for the specific absorbance of all-trans-retinol in IU.
4.6 mm) packed with octadecylsilyl silica gel for chromatography (5 m to 10 m), (b) as mobile phase
at a flow rate of 1 ml per minute a mixture of 3 volumes of water and 97 volumes of methanol, (c) a
detection wavelength of 325 nm, (d) a 10 l loop injector and (e) an electronic integrator.
Inject in triplicate solution (1) and solution (3). The retention time of all-trans-retinol is 5 1
minute.
The assay is not valid unless (a) the chromatogram obtained with solution (1) shows a peak
corresponding to that of all-trans-retinol in the chromatogram obtained with solution (3), (b) when
using the method of standard additions to solution (1) there is greater than 95% recovery of the
added retinyl acetate EPCRS, and (c) the recovery of all-trans-retinol in solution (3) as assessed by
direct absorption spectrophotometry is greater than 95%.
Calculate the content of vitamin A using the following expression:
A1
C V 1
A2
m
where A1 = area of the peak corresponding to all-trans-retinol in the chromatogram obtained with
solution (1),
A2 = area of the peak corresponding to all-trans-retinol in the chromatogram obtained with
solution (3),
C = concentration of retinyl acetate EPCRS in solution (2) as assessed prior to the saponification in International Units per ml (= 1000 IU per ml),
V = volume of solution (2) treated,
m = mass of the substance being examined in solution (1).
Storage Vitamin A Ester Concentrate (Natural) should be kept in an airtight container, protected
from light and stored at a temperature of 8 to 15. Once the container has been opened its
contents should be used as soon as possible; any part of the contents not used at once should be
protected by an atmosphere of an inert gas.
Labelling The label states (1) the number of IU (Units) of vitamin A per g; (2) the name of the
ester or esters; (3) the name and proportion of the principal excipients; (4) the method of restoring
the solution if partial crystallisation has occurred.
42-57
Synthetic Retinol Concentrate (Oily Form)

1/01
Synthetic Retinol Concentrate (Oily Form) complies with the requirements of the 3rd edition of the European
Pharmacopoeia for Synthetic Vitamin A Concentrate (Oily Form) [0219]. These requirements are
reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Synthetic vitamin A concentrate (oily form) is prepared from synthetic retinol ester (0217) as is or by
dilution with a suitable vegetable oil. The concentrate may contain suitable stabilisers such as
antioxidants.
The declared content of vitamin A is not less than 500,000 I.U. per gram and the concentrate
contains not less than 95.0 per cent and not more than 110.0 per cent of the content stated on the
label.
CHARACTERS
A yellow or brownish-yellow, oily liquid, practically insoluble in water, soluble or partly soluble in
ethanol, miscible with organic solvents. Partial crystallisation may occur in highly concentrated
solutions.
IDENTIFICATION
Examine by thin-layer chromatography (2.2.27), using a TLC silica gel F254 plate R.
Test solution. Prepare a solution in cyclohexane R, stabilised with a 1 g/l solution of
butylhydroxytoluene R, containing about 3.3 I.U. of vitamin A per microlitre.
Reference solution. Prepare a reference solution in cyclohexane R, stabilised with a 1 g/l solution of
butylhydroxytoluene R, containing about 0.01 mg of retinol esters CRS per microlitre (i.e. 3.3 I.U. of
each ester per microlitre).
TESTS
ASSAY
Carry out the assay as rapidly as possible, avoiding exposure to actinic light and air, oxidising agents,
oxidation catalysts (e.g. copper, iron), acids and prolonged heat; use freshly prepared solutions. If partial
crystallisation has occurred, homogenise the material at a temperature of about 65C, but avoid prolonged
heating.
Carry out the assay according to Method A. If the assay is not shown to be valid, use Method B.
Method A Examine by ultraviolet absorption spectrophotometry (2.2.25). Dissolve 25 mg to
100 mg, weighed with an accuracy of 0.1 per cent, in 5 ml of pentane R and dilute with 2-propanol R1
to a presumed concentration of 10 I.U. to 15 I.U. per millilitre.
Verify that the absorption maximum of the solution lies between 325 nm and 327 nm and measure
the absorbances at 300 nm, 326 nm, 350 nm and 370 nm. Repeat the readings at each wavelength
and take the mean values. Calculate the ratio A/A326 for each wavelength.
If the ratios do not exceed:
0.593 at 300 nm,
0.537 at 350 nm,
0.142 at 370 nm.
calculate the content of vitamin A in International Units per gram from the expression:
A326 V 1900
100 m
42-58
m = mass of the preparation to be examined, in grams,
V = total volume to which the preparation to be examined is diluted to give 10 I.U. to 15 I.U.
per millilitre,
gram.
If one or more of the ratios A/A326 exceeds the values given, or if the wavelength of the absorption
maximum does not lie between 325 nm and 327 nm, use Method B.
Method B Examine by liquid chromatography (2.2.29).
Test solution (a). Introduce into a 50 ml volumetric flask, an amount of the preparation to be examined, weighed with an accuracy of 0.1 per cent and equivalent to about 120,000 I.U. of vitamin A
and dissolve immediately in 5 ml of pentane R. Add 20 mg to 30 mg of butylhydroxytoluene R and
20 ml of 0.1M tetrabutylammonium hydroxide in 2-propanol. Swirl gently for 5 min (an ultrasonic bath is
recommended). Dilute to 50.0 ml with 2-propanol R and homogenise carefully to avoid air-bubbles.
Test solution (b). Introduce 20 mg to 30 mg of butylhydroxytoluene R into a 50 ml volumetric flask,
add 5 ml of 2-propanol R, 5.0 ml of test solution (a) and dilute to 50.0 ml with 2-propanol R.
Homogenise carefully to avoid air-bubbles.
Reference solution (a). Introduce into a 50 ml volumetric flask about 120 mg of retinol acetate CRS,
weighed with an accuracy of 0.1 per cent and proceed as described for test solution (a).
Reference solution (b). Introduce 20 mg to 30 mg of butylhydroxytoluene R into a 50 ml volumetric
flask, add 5 ml of 2-propanol R, 5.0 ml of reference solution (a) and dilute to 50.0 ml with 2-propanol R. Homogenise carefully to avoid air-bubbles.
methanol R,
a loop injector.
the chromatogram obtained with reference solution (b) shows a principal peak corresponding to
that of all-(E)-retinol, the retention time of all-(E)-retinol being about 3 min,
there is no peak corresponding to the unsaponified retinol acetate in the chromatogram obtained
with reference solution (b) at a retention time of about 6 min.
Inject a suitable volume of reference solution (b) in order to obtain an absorbance in the range of
0.5 to 1.0 at 325 nm and record the chromatogram using an attenuation so that the height of the
peak corresponding to vitamin A is not less than 50 per cent of the full scale of the recorder.
Make a total of six such injections. The relative standard deviation of the response for reference
solution (b) is not greater than 1 per cent.
Inject the same volume of test solution (b) and record the chromatogram in the same manner.
Calculate the content of vitamin A from the expression:
A1 C m 2
A2 m1
A1 = area of the peak corresponding to all-(E)-retinol in the chromatogram obtained with test
solution (b),
A2 = area of the peak corresponding to all-(E)-retinol in the chromatogram obtained with
reference solution (b),
C = concentration of retinol acetate CRS in International Units per gram, determined by
method A; the absorption ratios A/A326 must conform,
m1 = mass of the substance to be examined in test solution (a), in milligrams,
m2 = mass of retinol acetate CRS in reference solution (a), in milligrams.
STORAGE
LABELLING
The label states:
the name of the ester or esters,
the name of any added stabilisers,
the method of restoring the solution if partial crystallisation has occurred.
__________________________________________________________________________________________________________ Ph Eur
42-59
Synthetic Retinol Concentrate (Powder Form)

1/01
Synthetic Retinol Concentrate (Powder Form) complies with the requirements of the 3rd edition of the
European Pharmacopoeia for Synthetic Vitamin A Concentrate (Powder Form) [0218]. These requirements
are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Synthetic vitamin A concentrate (powder form) is obtained by dispersing a synthetic retinol ester
(0217) in a matrix of Gelatin (0330) or Acacia (0307) or other suitable material.
The declared content of vitamin A is not less than 250,000 I.U. per gram and the concentrate
contains not less than 95.0 per cent and not more than 115.0 per cent of the content stated on the
label. The concentrate may contain suitable stabilisers such as antioxidants.
CHARACTERS
A yellowish powder usually in the form of particles of almost uniform size which, depending on their
formulation, may be practically insoluble in water or may swell or form an emulsion.
IDENTIFICATION
Test solution. Introduce into a 20 ml glass-stoppered test tube a quantity of the preparation to be
examined containing about the equivalent of 17,000 I.U. of vitamin A. Add about 20 mg of
bromelains R, 2 ml of water R and about 150 l of 2-propanol R, swirling gently for 2 to 5 min in a
water-bath at 60C to 65C. Cool to below 30C and add 5 ml of 2-propanol R, stabilised with a 1 g/l
solution of butylhydroxytoluene R. Shake vigorously for 1 min, allow to stand for a few minutes and
use the supernatant solution.
Reference solution. Prepare a reference solution in 2-propanol R, stabilised with a 1 g/l solution of
ASSAY
oxidation catalysts (e.g. copper, iron), acids and prolonged heat.
Test solution (a). Introduce into a 50 ml volumetric flask, an amount of the preparation to be examined, weighed with an accuracy of 0.1 per cent and equivalent to about 120,000 I.U. of vitamin A.
Add 20 mg to 30 mg of bromelains R, 20 mg to 30 mg of butylhydroxytoluene R, 2.0 ml of water R and
0.15 ml of 2-propanol R. Heat gently in a water-bath at 60C to 65C for 2 to 5 min. Cool to below
30C and add 20 ml of 0.1M tetrabutylammonium hydroxide in 2-propanol. Swirl gently for 5 min (an
ultrasonic bath is recommended). Dilute to 50.0 ml with 2-propanol R and homogenise carefully to
avoid air-bubbles. Residue of the matrix may cause more or less cloudiness of the solution.
Homogenise carefully to avoid air-bubbles. Filter before injection.
weighed with an accuracy of 0.1 per cent and dissolve immediately in 5 ml of pentane R. Add 20 mg
to 30 mg of butylhydroxytoluene R and 20 ml of 0.1M tetrabutylammonium hydroxide in 2-propanol. Swirl
gently for 5 min (an ultrasonic bath is recommended) and dilute to 50.0 ml with 2-propanol R.
Reference solution (b). Place 20 mg to 30 mg of butylhydroxytoluene R in a 50 ml volumetric flask, add
5 ml of 2-propanol R, 5.0 ml of reference solution (a) and dilute to 50.0 ml with 2-propanol R.
42-60
methanol R,
a loop injector.
there is no peak corresponding to unsaponified retinol acetate in the chromatogram obtained
Make a total of six injections. The relative standard deviation of the response for reference solution
(b) should not be greater than 1 per cent.
A1 C m 2
A2 m1
solution (b),
C = concentration of retinol acetate CRS in International Units per gram, determined by the
method below,
The exact concentration of retinol acetate CRS is assessed by ultraviolet absorption spectrophotometry
(2.2.25). Dissolve 25 mg to 100 mg, weighed with an accuracy of 0.1 per cent, in 5 ml of pentane R
and dilute with 2-propanol R1 to a presumed concentration of 10 I.U. to 15 I.U. per millilitre.
0.593 at 300 nm,
0.537 at 350 nm,
0.142 at 370 nm,
A326 V 1900
100 m

m = mass of the CRS, in grams,
V = total volume to which the CRS is diluted to give 10 I.U. to 15 I.U. per millilitre,
gram.
The absorption ratios A/A326 must conform.
STORAGE
LABELLING
The label states:
the name of the principal excipient or excipients used and the name of any added stabilisers.
__________________________________________________________________________________________________________ Ph Eur
42-61
Synthetic Retinol Concentrate, Solubilisate/Emulsion

1/01
Synthetic Retinol Concentrate (Water-dispersible Form)
Synthetic Retinol Concentrate, Solubilisate/Emulsion complies with the requirements of the 3rd edition of the
European Pharmacopoeia for Synthetic Vitamin A Concentrate, Solubilisate/Emulsion [0220]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Synthetic Vitamin A concentrate solubilisate/emulsion is a liquid form (water is generally used as
solvent) of a synthetic retinol ester (0217) and a suitable solubiliser. The declared content of vitamin
A is not less than 100,000 I.U. per gram and the concentrate contains not less than 95.0 per cent and
not more than 115.0 per cent of the content stated on the label. The concentrate may contain
suitable stabilisers such as antimicrobial preservatives and antioxidants.
CHARACTERS
A yellow or yellowish liquid of variable opalescence and viscosity. Highly concentrated solutions may
become cloudy at low temperature or take the form of a gel.
A mixture of 1 g with 10 ml of water R previously warmed to 50C gives after cooling to 20C, a
uniform, slightly opalescent and slightly yellow dispersion.
IDENTIFICATION
Test solution. Introduce into a 20 ml glass-stoppered test-tube a quantity of the preparation to be
examined containing about the equivalent of 17,000 I.U. of vitamin A. Add 5 ml of 2-propanol R,
stabilised with a 1 g/l solution of butylhydroxytoluene R and mix thoroughly.
Reference solution. Prepare a reference solution in 2-propanol R, stabilised with a 1 g/l solution of
ASSAY
oxidation catalysts (e.g. copper, iron), acids and prolonged heat.
Test solution (a). Introduce into a 50 ml volumetric flask, an amount of the preparation to be examined, weighed with an accuracy of 0.1 per cent and equivalent to about 120,000 I.U. of vitamin A
and dissolve immediately in 5 ml of 2-propanol R. Add 20 mg to 30 mg of butylhydroxytoluene R and
20 ml of 0.1M tetrabutylammonium hydroxide in 2-propanol. Swirl gently for 5 min (an ultrasonic bath is
recommended). Dilute to 50.0 ml with 2-propanol R and homogenise carefully to avoid air-bubbles.
weighed with an accuracy of 0.1 per cent and proceed as described for test solution (a).
Reference solution (b). Place 20 mg to 30 mg of butylhydroxytoluene R into a 50 ml volumetric flask,
add 5 ml of 2-propanol R, 5.0 ml of reference solution (a) and dilute to 50.0 ml with 2-propanol R.
methanol R,
42-62
a loop injector.
there is no peak corresponding to the unsaponified retinol acetate in the chromatogram obtained
Make a total of six injections. The relative standard deviation of the response for reference solution
(b) should not be greater than 1 per cent.
A1 C m 2
A2 m1
solution (b),
C = concentration of retinol acetate CRS in International Units per gram, determined by the
method below,
The exact concentration of retinol acetate CRS is assessed by ultraviolet absorption spectrophotometry
(2.2.25). Dissolve 25 mg to 100 mg, weighed with an accuracy of 0.1 per cent, in 5 ml of pentane R
and dilute with 2-propanol R1 to a presumed concentration of 10 I.U. to 15 I.U. per millilitre.
0.593 at 300 nm,
0.537 at 350 nm,
0.142 at 370 nm,
A326 V 1900
100 m

m = mass of the CRS, in grams,
V = total volume to which the CRS is diluted to give 10 I.U. to 15 I.U. per millilitre,
gram.
The absorption ratios A/A326 must conform.
STORAGE
Store in an airtight container, protected from light, at the temperature stated on the label.
LABELLING
The label states:
the name of the principal solubiliser or solubilisers used and the name of any added stabilisers,
the storage temperature.
__________________________________________________________________________________________________________ Ph Eur
43-1
Warfarin Sodium
O
NaO
H
CH 3
O
and enantiomer
C 19H15NaO4
330.3
129-06-6
Warfarin Sodium complies with the requirements of the 3rd edition of the European Pharmacopoeia [0698].
Preparation
Warfarin Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Warfarin sodium contains not less than 98.0 per cent and not more than the equivalent of 102.0 per
cent of sodium (RS)-2-oxo-3-(3-oxo-1-phenylbutyl)-2H-chromen-4-olate, calculated with reference
CHARACTERS
A white powder, hygroscopic, very soluble in water and in alcohol, soluble in acetone, very slightly
soluble in ether and in methylene chloride.
IDENTIFICATION
First identification: B, D, E.
A. Dissolve 1 g in 25 ml of water R, add 2 ml of dilute hydrochloric acid R and filter. Reserve the
filtrate for identification test E. The precipitate, washed with water R and dried at 100C to 105C,
melts (2.2.14) at 159C to 163C.
B. Dissolve 1 g in 25 ml of water R, add 2 ml of dilute hydrochloric acid R and filter. Reserve the filtrate
for identification test E. Examine the precipitate by infra-red absorption spectrophotometry (2.2.24)
comparing with the spectrum obtained with the precipitate prepared in the same manner from
warfarin sodium CRS.
D. Dissolve 1 g in 10 ml of water R, add 5 ml of nitric acid R and filter. To the filtrate add 2 ml of
potassium dichromate solution R1 and shake for 5 min. Allow to stand for 20 min. The solution is not
greenish-blue when compared with a blank.
E. The filtrate obtained in identification test A or B gives reaction (b) of sodium (2.3.1).
TESTS
coating substance.
Reference solution (b). Dissolve 40 mg of warfarin sodium CRS in acetone R and dilute to 10 ml with
the same solvent.
43-2
Reference solution (c). Dissolve 10 mg of acenocoumarol CRS in acetone R, add 1 ml of test solution (a)
20 volumes of glacial acetic acid R, 50 volumes of chloroform R and 50 volumes of cyclohexane R. Allow
chromatogram obtained with reference solution (c) shows two clearly separated spots and the chromatogram obtained with reference solution (a) shows a clearly visible spot.
Phenolic ketones Dissolve 1.25 g in a 50 g/l solution of sodium hydroxide R and dilute to 10.0 ml
with the same solvent. The absorbance (2.2.25), measured at 385 nm within 15 min of preparing the
solution, is not greater than 0.20.
ASSAY
Dissolve 0.1000 g in 0.01M sodium hydroxide and dilute to 100.0 ml with the same solvent. Dilute
10.0 ml of this solution to 100.0 ml with 0.01M sodium hydroxide. Dilute 10.0 ml of the latter solution
to 100.0 ml with 0.01M sodium hydroxide. Measure the absorbance (2.2.25) at the maximum at
308 nm.
Calculate the content of C19H15NaO4 taking the specific absorbance to be 431.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
43-3
Warfarin Sodium Clathrate

O
O
H
NaO
H3C
CH3
O
OH
CH 3
and enantiomer
Warfarin Sodium Clathrate complies with the requirements of the 3rd edition of the European Pharmacopoeia
Preparation
Warfarin Tablets
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Warfarin sodium clathrate contains not less than 98.0 per cent and not more than the equivalent of
102.0 per cent of sodium (RS)-2-oxo-3-(3-oxo-1-phenylbutyl)-2H-chromen-4-olate, calculated with
reference to the anhydrous, 2-propanol-free substance. Warfarin sodium clathrate contains
approximately 92 per cent of warfarin sodium. It consists of warfarin sodium and 2-propanol
(molecular proportions 2:1) in the form of a clathrate. It contains not less than 8.0 per cent and not
more than 8.5 per cent of 2-propanol.
CHARACTERS
A white powder, very soluble in water, freely soluble in alcohol, soluble in acetone, very slightly
soluble in ether and in methylene chloride.
IDENTIFICATION
First identification: B, D, E.
A. Dissolve 1 g in 25 ml of water R, add 2 ml of dilute hydrochloric acid R and filter. Reserve the
filtrate for identification test E. The precipitate, washed with water R and dried at 100C to 105C,
melts (2.2.14) at 159C to 163C.
B. Dissolve 1 g in 25 ml of water R, add 2 ml of dilute hydrochloric acid R and filter. Reserve the filtrate
for identification test E. Examine the precipitate by infrared absorption spectrophotometry (2.2.24)
comparing with the spectrum obtained with the precipitate prepared in the same manner from
warfarin sodium CRS.
D. Dissolve 1 g in 10 ml of water R, add 5 ml of nitric acid R and filter. To the filtrate add 2 ml of
potassium dichromate solution R1 and shake for 5 min. Allow to stand for 20 min. The solution is
greenish-blue when compared with a blank.
E. The filtrate obtained in identification test A or B gives reaction (b) of sodium (2.3.1).
TESTS
coating substance.
43-4
Reference solution (b). Dissolve 40 mg of warfarin sodium CRS in acetone R and dilute to 10 ml with
the same solvent.
Reference solution (c). Dissolve 10 mg of acenocoumarol CRS in acetone R, add 1 ml of test solution (a)
20 volumes of glacial acetic acid R, 50 volumes of chloroform R and 50 volumes of cyclohexane R. Allow
chromatogram obtained with reference solution (c) shows two clearly separated spots and the chromatogram obtained with reference solution (a) shows a clearly visible spot.
Phenolic ketones Dissolve 1.25 g in a 50 g/l solution of sodium hydroxide R and dilute to 10.0 ml
with the same solvent. The absorbance (2.2.25), measured at 385 nm within 15 min of preparing the
solution, is not greater than 0.20.
2-Propanol 8.0 per cent m/m to 8.5 per cent m/m, determined by gas chromatography (2.2.28) using
propanol R as internal standard.
Internal standard solution. Dilute 1.0 ml of propanol R to 200.0 ml with water R.
Test solution (a). Dissolve 0.250 g of the substance to be examined in water R and dilute to 5.0 ml
Test solution (b). Dissolve 0.50 g of the substance to be examined in the internal standard solution
and dilute to 10.0 ml with the internal standard solution.
Reference solution. Dilute 0.50 ml of 2-propanol R to 100.0 ml with the internal standard solution.
a column 1.5 m long and 4 mm in internal diameter packed with ethylvinylbenzene-divinylbenzene
the detector at 200C. Inject the selected volumes of the test solutions and the reference solution.
Calculate the content of 2-propanol taking its density at 20C to be 0.785 g/ml.
ASSAY
Dissolve 0.1000 g in 0.01M sodium hydroxide and dilute to 100.0 ml with the same solvent. Dilute
10.0 ml of this solution to 100.0 ml with 0.01M sodium hydroxide. Dilute 10.0 ml of the latter solution
to 100.0 ml with 0.01M sodium hydroxide. Measure the absorbance (2.2.25) at the maximum at
308 nm.
Calculate the content of C19H15NaO4 taking the specific absorbance to be 431.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
43-5
Purified Water
H 2O
18.02
7732-18-5
Purified Water complies with the requirements of the 3rd edition of the European Pharmacopoeia [0008].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Purified water is water for the preparation of medicinal products other than those that are required to
be both sterile and apyrogenic, unless otherwise justified and authorised.
PURIFIED WATER IN BULK
PRODUCTION
Purified water is prepared by distillation, by ion exchange or by any other suitable method from water
that complies with the regulations on water intended for human consumption laid down by the
competent authority.
During production and subsequent storage, appropriate measures are taken to ensure that the total
viable aerobic count is adequately controlled and monitored. Appropriate alert and action limits
should be set so as to detect adverse trends. Under normal conditions, an appropriate action limit is a
total viable aerobic count (2.6.12) of 100 micro-organisms per millilitre, determined by membrane
filtration using agar medium B. The size of the sample is to be chosen in relation to the expected
result.
In addition, the test for total organic carbon (2.2.44) with a limit of 0.5 mg/l or alternatively the
following test for oxidisable substances is carried out: To 100 ml add 10 ml of dilute sulphuric acid R
and 0.1 ml of 0.02M potassium permanganate and boil for 5 min. The solution remains faintly pink.
Conductivity (2.2.38) at 20C not more than 4.3 S.cm-1) is also controlled.
Purified water in bulk is stored and distributed in containers designed to prevent growth of microorganisms and to avoid any other contamination.
CHARACTERS
A clear, colourless, odourless and tasteless liquid.
TESTS
Nitrates Place 5 ml in a test-tube immersed in iced water, add 0.4 ml of a 100 g/l solution of
potassium chloride R, 0.1 ml of diphenylamine solution R and, dropwise with shaking, 5 ml of nitrogenfree sulphuric acid R. Transfer the tube to a water-bath at 50C. After 15 min, any blue colour in the
using a mixture of 4.5 ml of nitrate-free water R and 0.5 ml of nitrate standard solution (2 ppm NO3) R
(0.2 ppm).
Heavy metals (2.4.8). Heat 200 ml in a glass evaporating dish on a water-bath until the volume is
reduced to 20 ml. 12 ml of the concentrated solution complies with limit test A for heavy metals
(0.1 ppm). Prepare the standard using lead standard solution (1 ppm Pb) R.
Aluminium (2.4.17). If intended for use in the manufacture of dialysis solutions, it complies with
the test for aluminium. To 400 ml add 10 ml of acetate buffer solution pH 6.0 R and 100 ml of distilled
water R. The solution complies with the limit test for aluminium (10 g/l). Use as the reference
pH 6.0 R and 98 ml of distilled water R. To prepare the blank, use a mixture of 10 ml of acetate buffer
solution pH 6.0 R and 100 ml of distilled water R.
Bacterial endotoxins (2.6.14). If intended for use in the manufacture of dialysis solutions without a
further appropriate procedure for the removal of bacterial endotoxins, not more than 0.25 I.U. of
endotoxin per millilitre.
LABELLING
solutions.
PURIFIED WATER IN CONTAINERS
Purified water in containers is purified water in bulk that has been filled and stored in conditions
designed to assure the required microbiological quality. It is free from any added substances.
CHARACTERS
43-6
TESTS
It complies with the tests prescribed in the sub-monograph on Purified Water in Bulk and with the
following additional tests.
Acidity or alkalinity To 10 ml, freshly boiled and cooled in a borosilicate glass flask, add 0.05 ml of
methyl red solution R. The solution is not coloured red.
To 10 ml add 0.1 ml of bromothymol blue solution R1. The solution is not coloured blue.
Oxidisable substances To 100 ml add 10 ml of dilute sulphuric acid R and 0.1 ml of 0.02M potassium
permanganate and boil for 5 min. The solution remains faintly pink.
Chlorides To 10 ml add 1 ml of dilute nitric acid R and 0.2 ml of silver nitrate solution R2. The solution shows no change in appearance for at least 15 min.
Sulphates To 10 ml add 0.1 ml of dilute hydrochloric acid R and 0.1 ml of barium chloride solution R1.
The solution shows no change in appearance for at least 1 h.
Ammonium To 20 ml add 1 ml of alkaline potassium tetraiodomercurate solution R. After 5 min,
examine the solution down the vertical axis of the tube. The solution is not more intensely coloured
than a standard prepared at the same time by adding 1 ml of alkaline potassium tetraiodomercurate
solution R to a mixture of 4 ml of ammonium standard solution (1 ppm NH4) R and 16 ml of
ammonium-free water R (0.2 ppm).
Calcium and magnesium To 100 ml add 2 ml of ammonium chloride buffer solution pH 10.0 R,
50 mg of mordant black 11 triturate R and 0.5 ml of 0.01M sodium edetate. A pure blue colour is
produced.
Residue on evaporation Evaporate 100 ml on a water-bath and dry in an oven at 100C to 105C.
The residue weighs not more than 1 mg (0.001 per cent).
Microbial contamination Total viable aerobic count (2.6.12) not more than 102 microorganisms per millilitre, determined by membrane filtration, using agar medium B.
Bacterial endotoxins (2.6.14). If intended for use in the manufacture of dialysis solutions without a
further appropriate procedure for the removal of bacterial endotoxins, not more than 0.25 I.U. of
endotoxin per millilitre.
LABELLING
solutions.
__________________________________________________________________________________________________________ Ph Eur
43-7
Water for Injections

H 2O
18.02
7732-18-5
Water for Injections complies with the requirements of the 3rd edition of the European Pharmacopoeia [0169].
If Water for Injections in bulk is distributed in individual containers, the label states that the contents
have not been sterilised.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Water for injections is water for the preparation of medicines for parenteral administration when
water is used as vehicle (water for injections in bulk) and for dissolving or diluting substances or
preparations for parenteral administration before use (sterilised water for injections).
WATER FOR INJECTIONS IN BULK
PRODUCTION
Water for injections in bulk is obtained from water that complies with the regulation on water
intended for human consumption laid down by the competent authority or from purified water by
distillation in an apparatus of which the parts in contact with the water are of neutral glass, quartz or
suitable metal and which is fitted with an effective device to prevent the entrainment of droplets. The
correct maintenance of the apparatus is essential. The first portion of the distillate obtained when the
apparatus begins to function is discarded and the distillate is collected.
During production and subsequent storage, appropriate measures are taken to ensure that the total
viable aerobic count is adequately controlled and monitored. Appropriate alert and action limits
should be set so as to detect adverse trends. Under normal conditions, an appropriate action limit is a
total viable aerobic count (2.6.12) of 10 micro-organisms per 100 millilitre when determined by
membrane filtration using agar medium B, and using at least 200 ml of water for injections in bulk.
For aseptic processing, stricter alert and action limits may need to be applied.
In addition, conductivity (2.2.38) (at 20C not more than 1.1 S.cm-1) and total organic carbon
(2.2.44) (not more than 0.5 mg/l) are controlled.
In order to ensure the appropriate quality of the water, validated procedures and in-processmonitoring of the electrical conductivity and regular microbial monitoring are applied.
Water for injections in bulk is stored and distributed in conditions designed to prevent growth of
micro-organisms and to avoid any other contamination.
CHARACTERS
TESTS
It complies with the tests prescribed in the sub-monograph on Purified Water in Bulk (8) and with the
addition of the following test.
Bacterial endotoxins (2.6.14). Not more than 0.25 I.U. of endotoxin per millilitre.
STERILISED WATER FOR INJECTIONS
Sterilised water for injections is water for injections in bulk that has been distributed into suitable
containers, closed and sterilised by heat in conditions which ensure that the product still complies
with the test for bacterial endotoxins. Sterilised water for injections is free from any added
substances.
Examined in suitable conditions of visibility, it is clear and colourless.
Each container contains a sufficient quantity of water for injections to permit the nominal volume
to be withdrawn.
TESTS
It complies with the tests prescribed in the sub-monograph on Purified Water in Containers (8) and
those modified as shown below for acidity and alkalinity, for oxidisable substances, for chlorides (if
the nominal volume of the container is 100 ml or less) and for residue on evaporation. It also
complies with the tests for particulate contamination, sterility and bacterial endotoxins.
Acidity or alkalinity To 20 ml add 0.05 ml of phenol red solution R. If the solution is yellow it
becomes red on the addition of 0.1 ml of 0.01M sodium hydroxide; if red, it becomes yellow on the
addition of 0.15 ml of 0.01M hydrochloric acid.
Conductivity (2.2.38). For containers with a nominal volume of 10 ml or less, the conductivity is
43-8
not more than 25 S.cm-1; if the nominal volume is greater than 10 ml, the conductivity is not more
than 5 S.cm-1.
Oxidisable substances Boil 100 ml with 10 ml of dilute sulphuric acid R. Add 0.2 ml of 0.02M
potassium permanganate and boil for 5 min. The solution remains faintly pink.
Chlorides (2.4.4). For containers with a nominal volume of 100 ml or less, 15 ml complies with the
limit test for chlorides (0.5 ppm). Prepare the standard using a mixture of 1.5 ml of chloride standard
solution (5 ppm Cl) R and 13.5 ml of water R. Examine the solutions down the vertical axes of the
tubes.
Residue on evaporation Evaporate 100 ml to dryness on a water-bath and dry in an oven at 100C
to 105C. For containers with a nominal volume of 10 ml or less, the residue weighs not more than
4 mg (0.004 per cent). For containers with a nominal volume greater than 10 ml, the residue weighs
not more than 3 mg (0.003 per cent).
Particulate contamination: sub-visible particles (2.9.19) It complies with test A or test B, as
appropriate.
Sterility (2.6.1). It complies with the test for sterility.
Bacterial endotoxins (2.6.14). Not more than 0.25 I.U. of endotoxin per millilitre.
__________________________________________________________________________________________________________ Ph Eur
43-9
Wheat Starch
Wheat Starch complies with the requirements of the 3rd edition of the European Pharmacopoeia [0359].
or used.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Wheat starch is obtained from the caryopsis of Triticum aestivum L. (T. vulgare Vill.).
CHARACTERS
A very fine, white powder which creaks when pressed between the fingers; practically insoluble in
cold water and in alcohol. Wheat starch does not contain starch grains of any other origin. It may
contain a minute quantity, if any, of fragments of the tissue of the original plant.
IDENTIFICATION
A. Examined under a microscope using equal volumes of glycerol R and water R, it presents large and
small granules, and, very rarely, intermediate sizes. The large granules, 10 m to 45 m in diameter,
are discoid or, more rarely, reniform when seen face-on. The central hilum and striations are invisible
or barely visible and the granules sometimes show cracks on the edges. Seen in profile, the granules
are elliptical and fusiform and the hilum appears as a slit along the main axis. The small granules,
rounded or polyhedral, are 2 m to 10 m in diameter. Between crossed nicol prisms, the granules
show a distinct black cross intersecting at the hilum.
B. Suspend 1 g in 50 ml of water R, boil for 1 min and cool. A thin, cloudy mucilage is formed.
C. To 1 ml of the mucilage obtained in identification test B, add 0.05 ml of iodine solution R1. A
dark-blue colour is produced which disappears on heating and reappears on cooling.
TESTS
Iron (2.4.9). Shake 1.5 g with 15 ml of dilute hydrochloric acid R. Filter. The filtrate complies with the
glycerol R and water R, not more than traces of cell walls and of cytoplasmic residues are present.
Total protein Not more than 0.3 per cent of total protein (corresponding to 0.048 per cent N2,
conversion factor: 5.7), determined on 6.0 g by sulphuric acid digestion (2.5.9) modified as follows:
wash any adhering particles from the neck into the flask with 25 ml of sulphuric acid R; continue the
heating until a clear solution is obtained; add 45 ml of strong sodium hydroxide solution R.
Microbial contamination. Not more than 103 bacteria and not more than 102 fungi per gram,
determined by plate-count (2.6.12). It complies with the test for E. coli (2.6.13).
at 130C for 90 min.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
43-10
Refined Wheat-germ Oil

Refined Wheat-germ Oil complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Refined wheat-germ oil is the fatty oil obtained from the germ of the grain of Triticum aestivum L. by
cold expression or other suitable mechanical means. It is then refined. A suitable antioxidant may be
added.
CHARACTERS
(40C to 60C).
IDENTIFICATION
A. Carry out the identification of fatty oils by thin-layer chromatography (2.3.2). The chromatogram
obtained is comparable with the type chromatogram for wheat-germ oil.
B. It complies with the test for composition of fatty acids (see Tests).
TESTS
Acid value (2.5.1). Not more than 0.5, determined on 10.0 g. If intended for use in the manufacture
of parenteral dosage forms, not more than 0.3.
(2.4.22). The fatty-acid fraction of the oil has the following composition:
brassicasterol.
0.1 per cent, determined on 5.00 g by the micro-determination of water. Use a mixture of equal
volumes of methanol R and methylene chloride R as solvent.
STORAGE
LABELLING
The label states:
forms,
__________________________________________________________________________________________________________ Ph Eur
43-11
Virgin Wheat-germ Oil

Virgin Wheat-germ Oil complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Virgin wheat-germ oil is the fatty oil obtained from the germ of the grain of Triticum aestivum L. by
cold expression or other suitable mechanical means.
CHARACTERS
A clear, light yellow or golden-yellow liquid, practically insoluble in water and in alcohol, miscible
with light petroleum (40C to 60C).
IDENTIFICATION
A. Carry out the identification of fatty oils by thin-layer chromatography (2.3.2). The chromatogram
obtained is comparable with the typical chromatogram for wheat-germ oil.
B. It complies with test for composition of fatty acids (see Tests).
TESTS
Composition of fatty acids Carry out the test for foreign fatty oils in fatty oils by gas chromatography (2.4.22, Method C). The fatty-acid fraction of the oil has the following composition:
brassicasterol.
Water (2.5.32). Not more than 0.1 per cent, determined on 5.00 g by the micro-determination of
water. Use a mixture of equal volumes of methanol R and methylene chloride R as solvent.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
43-12
Willow Bark
1/01
Willow Bark complies with the requirements of the 3rd edition of the European Pharmacopoeia [1583]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Willow bark consists of the whole or fragmented dried bark of young branches or whole dried pieces
of current year twigs of various species of genus Salix including S. purpurea L., S. daphnoides Vill. and
S. fragilis L. The drug contains not less than 1.5 per cent of total salicylic derivatives, expressed as
salicin (C13H18O7; 286.3), calculated with reference to the dried drug.
CHARACTERS
Willow bark is markedly bitter.
IDENTIFICATION
A. The bark is 1 mm to 2 mm thick and occurs in flexible, elongated, quilled or curved pieces. The
outer surface is smooth or slightly wrinkled longitudinally and greenish-yellow to brownish-grey. The
inner surface is smooth or finely striated longitudinally and white, pale yellow or reddish-brown,
depending on the species. The fracture is short in the outer part and coarsely fibrous in the inner
region. The diameter of current year twigs is not more than 10 mm. The wood is white or pale
yellow.
B. Reduce to a powder (355). The powder is pale yellow, greenish-yellow or light brown. Examine
under a microscope using chloral hydrate solution R. The powder shows: bundles of narrow fibres, up
to about 600 m long, with very thick walls and surrounded by a crystal sheath containing prism
crystals of calcium oxalate; parenchyma of the cortex with thick, pitted and deeply beaded walls, and
containing large cluster crystals of calcium oxalate; uniseriate medullary rays; thickened and suberised
cork cells. Groups of brownish collenchyma from the bud may be present. Twigs show, additionally,
fragments of lignified fibres and vessels from the xylem.
Test solution (a). To 1.0 g of the powdered drug (500) add 2 ml of methanol R; heat in a water-bath
at about 50C, with frequent shaking, for 10 min. Cool and filter.
Test solution (b). To 5.0 ml of test solution (a) add 1.0 ml of a 50 g/l solution of anhydrous sodium
carbonate R and heat in a water-bath at about 60C for 10 min. Cool and filter if necessary.
Reference solution. Dissolve 2.0 mg of salicin R in 1.0 ml of methanol R.
Apply to the plate as bands 20 l of each. Develop over a path of 15 cm using a mixture of 8 volumes
of water R, 15 volumes of methanol R and 77 volumes of ethyl acetate R. Allow the plate to dry in air.
Spray with a mixture of 5 volumes of sulphuric acid R and 95 volumes of methanol R. Heat at 100C
to 105C for 5 min and examine in daylight. The chromatogram obtained with the reference solution
shows in the middle third a reddish-violet zone due to salicin. In the chromatogram obtained with
test solution (a), the zone due to salicin appears with only slight to moderate intensity. In the
chromatogram obtained with test solution (b) the zone due to salicin is clearly more intense and there
are, above the zone due to salicin, one (salicortin or 2-O-acetylsalicortin, or possibly two tremulacin)
faint reddish-violet zones Other blue, yellow or brown zones can occur in both chromatograms.
TESTS
Foreign matter (2.8.2). Not more than 3 per cent of twigs with a diameter greater than 10 mm, and
not more than 2 per cent of other foreign matter.
Loss on drying (2.2.32). Not more than 11 per cent, determined on 1.000 g of the powdered drug
(355), by drying in an oven at 100C to 105C for 2 h.
Total ash (2.4.16). Not more than 10 per cent.
ASSAY
Examine by liquid chromatography (2.2.29), using resorcinol R as the internal standard.
Internal standard solution. Dissolve 50 mg of resorcinol R in 10 ml of methanol R.
Test solution. To 0.5 g of the powdered drug (355) add 50 ml of methanol R and heat under a reflux
condenser for 30 min. Cool and filter. Take up the residue with 50 ml of methanol R. Proceed as
above. Combine the filtrates and evaporate under reduced pressure. Take up the residue with 5.0 ml
of methanol R, add 5.0 ml of 0.1M sodium hydroxide and heat in a water-bath at about 60C under a
43-13
reflux condenser, with frequent shaking for about 1 h. After cooling, add 0.5 ml of 1M hydrochloric
acid. Dilute the solution to 20.0 ml with a mixture of 50 volumes of methanol R and 50 volumes of
water R. Add 1.0 ml of the internal standard solution to 10.0 ml of this solution. Filter through a
membrane filter.
Reference solution (a). Dissolve 18.5 mg of salicin R in 10.0 ml of a mixture of 20 volumes of water R
and 80 volumes of methanol R and add 1.0 ml of the internal standard solution.
Reference solution (b). Dissolve 1.0 mg of picein R in 1.0 ml of reference solution (a).
a stainless steel column, 0.10 m long and 3 mm or 4 mm in internal diameter, packed with
as mobile phase at a flow rate of 1.0 ml/min a mixture of 1.8 volumes of tetrahydrofuran R and
98.2 volumes of water R, containing 0.5 per cent V/V of phosphoric acid R,
a loop injector.
Inject 10 l of reference solution (b). The assay is not valid unless the resolution between the peaks
corresponding to salicin and picein and between the peaks corresponding to picein and resorcinol is
at least 1.5.
Inject five times 10 l of reference solution (a).
Inject three times 10 l of the test solution. Continue the chromatography for four times the
retention time of the peak corresponding to salicin.
Calculate the percentage content of total salicylic derivatives, expressed as salicin, from the
expression:
S1 S 4 m 2 p 2
S2 S3 m1
S1 = area of the peak due to salicin in the chromatogram obtained with the test solution,
S2 = area of the peak due to resorcinol in the chromatogram obtained with the test solution,
S3 = area of the peak due to salicin in the chromatogram obtained with reference solution (a),
S4 = area of the peak due to resorcinol in the chromatogram obtained with reference solution
(a), with the test solution,
m1 = mass of the drug in the test solution, in milligrams,
m2 = mass of salicin in reference solution (a), in milligrams,
p = percentage content of salicin in the reference substance
STORAGE
The following chromatogram is given for information.
300
drug sample #60

mAU
UV VIS 1
WVL:270 nm
250
2
200
1
150
100
50
0
-50
0.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
min
20.0
1. salicin
2. resorcinol
Fig. 1583-1 Chromatogram of the salicylic derivatives from willow bark with resorcinol as the
internal standard
__________________________________________________________________________________________________________ Ph Eur
43-14
Wool Alcohols
Wool Wax Alcohols
Wool Alcohols complies with the requirements of the 3rd edition of the European Pharmacopoeia [0593].
Preparation
Wool Alcohols Ointment
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Wool alcohols is a mixture of sterols and higher aliphatic alcohols from wool fat. It contains not less
than 30.0 per cent of cholesterol. It may contain not more than 200 parts per million of
butylhydroxytoluene.
CHARACTERS
A pale-yellow to brownish-yellow, brittle mass becoming plastic when warm, practically insoluble in
water, soluble in methylene chloride, in ether and in boiling ethanol, slightly soluble in alcohol
(90 per cent V/V).
IDENTIFICATION
Dissolve 50 mg in 5 ml of methylene chloride R and add 1 ml of acetic anhydride R and 0.1 ml of
sulphuric acid R. Within a few seconds, a green colour develops.
TESTS
Appearance of solution To 1.0 g add 10 ml of light petroleum R1 and shake while warming in a
water-bath. The substance dissolves completely. After cooling, the solution is clear (2.2.1).
Alkalinity Dissolve 2.0 g in 25 ml of hot alcohol (90 per cent V/V) R and add 0.5 ml of
phenolphthalein solution R1. No red colour develops.
Melting point (2.2.15). Not lower than 58C. Melt the substance to be examined by heating in a
water-bath at a temperature which exceeds the expected melting point by not more than 10C;
introduce the substance to be examined into the capillary tubes and allow to stand at 15C to 17C
for not less than 16 h.
Acid value (2.5.1). Not more than 2. If necessary, heat in a water-bath under a reflux condenser to
dissolve the substance to be examined.
Hydroxyl value (2.5.3, Method A). 120 to 180.
Peroxide value (2.5.5). Not more than 15. Take from the substance to be examined wedge-shaped
pieces whose base consists of part of the surface. Melt the pieces before carrying out the determination.
Saponification value (2.5.6). Not more than 12, determined on 2.00 g. Heat under reflux for 4 h.
Butylhydroxytoluene Not more than 200 ppm, determined by gas chromatography (2.2.28), using
methyl decanoate R as the internal standard.
Internal standard solution. Dissolve 0.20 g of methyl decanoate R in carbon disulphide R and dilute to
100.0 ml with the same solvent. Dilute 1.0 ml of the solution to 10.0 ml with carbon disulphide R.
Test solution (a). Dissolve 1.0 g of the substance to be examined in carbon disulphide R and dilute to
Test solution (b). Dissolve 1.0 g of the substance to be examined in carbon disulphide R, add 1.0 ml of
the internal standard solution and dilute to 10.0 ml with carbon disulphide R.
Reference solution. Dissolve 0.20 g of butylhydroxytoluene R in carbon disulphide R and dilute to
100.0 ml with the same solvent. Dilute 1.0 ml of this solution to 10.0 ml with carbon disulphide R. To
1.0 ml of this solution add 1.0 ml of the internal standard solution and dilute to 10.0 ml with carbon
disulphide R.
a column 1.5 m long and 4 mm in internal diameter packed with silanised diatomaceous earth for
gas chromatography R impregnated with 10 per cent m/m of polydimethylsiloxane R; the column is
preceded by a column containing silanised glass wool,
the detector at 300C. Inject the chosen volumes of test solutions (a) and (b) and of the reference
solution.
43-15
100C to 105C.
Water-absorption capacity Place 0.6 g of the substance to be examined and 9.4 g of white soft
paraffin R in a mortar and melt on a water-bath. Allow to cool and incorporate 20 ml of water R,
added in portions. Within 24 h no water is released from the almost white, ointment-like emulsion.
ASSAY
Dissolve 0.1000 g in 12 ml of hot alcohol (90 per cent V/V) R. Allow to stand for 18 h, filter through a
sintered-glass filter (16) and wash the filter with two quantities, each of 15 ml, of alcohol (90 per cent
V/V) R. Combine the filtrate and washings, add 20 ml of a freshly prepared 10 g/l solution of
digitonin R in alcohol (90 per cent V/V) R and heat to about 60C. Allow to cool, filter through a
sintered-glass filter (16), wash the residue with 10 ml of alcohol (90 per cent V/V) R and dry to
constant mass at 100C to 105C.
1 g of residue is equivalent to 0.239 g of cholesterol.
STORAGE
Store protected from light in a well-filled container.
LABELLING
The label states, where applicable, the concentration of added butylhydroxytoluene.
__________________________________________________________________________________________________________ Ph Eur
43-16
Wool Fat
Anhydrous Lanolin
1/01
Wool Fat complies with the requirements of the 3rd edition of the European Pharmacopoeia [0134]. These
Preparation
Hydrous Wool Fat
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Wool fat is a purified, anhydrous, waxy substance obtained from the wool of the sheep (Ovis aries). It
may contain not more than 200 parts per million of butylhydroxytoluene.
CHARACTERS
A pale-yellow, unctuous substance with a characteristic odour. When melted, it is a clear or almost
clear, yellow liquid. It is practically insoluble in water, soluble in ether and slightly soluble in boiling
ethanol. A solution in light petroleum is opalescent.
IDENTIFICATION
A. In a test-tube, dissolve 0.5 g in 5 ml of chloroform R and add 1 ml of acetic anhydride R and 0.1 ml
of sulphuric acid R. A green colour develops.
B. Dissolve 50 mg in 5 ml of chloroform R, add 5 ml of sulphuric acid R and shake. A red colour
develops and an intense green fluorescence appears in the lower layer.
TESTS
Water-soluble acid or alkaline substances Melt 5.0 g on a water-bath and shake vigorously for
2 min with 75 ml of water R previously heated to 90C to 95C. Allow to cool and filter through filter
paper previously rinsed with water R. To 60 ml of the filtrate (which may not be clear) add 0.25 ml of
bromothymol blue solution R1. Not more than 0.2 ml of 0.02M hydrochloric acid or 0.15 ml of 0.02M
sodium hydroxide is required to change the colour of the indicator.
Drop point (2.2.17). 38C to 44C. To fill the metal cup, melt the wool fat on a water-bath, cool to
about 50C, pour into the cup and allow to stand at 15C to 20C for 24 h.
Water-absorption capacity Place 10 g in a mortar. Add water R in portions of 0.2 ml to 0.5 ml
from a burette, stirring vigorously after each addition to incorporate the water R. The end-point is
reached when visible droplets remain which cannot be incorporated. Not less than 20 ml of water R is
absorbed.
Saponification value (2.5.6). 90 to 105, determined on 2.00 g. Heat under reflux for 4 h.
Water-soluble oxidisable substances To 10 ml of the filtrate obtained in the test for water-soluble
acid or alkaline substances add 1 ml of dilute sulphuric acid R and 0.1 ml of 0.02M potassium
permanganate. After 10 min, the solution is not completely decolourised.
100.0 ml with the same solvent. Dilute 1.0 ml of the solution to 10.0 ml with carbon disulphide R.
Test solution (a). Dissolve 1.0 g of the substance to be examined in carbon disulphide R and dilute to
Test solution (b). Dissolve 1.0 g of the substance to be examined in carbon disulphide R, add 1.0 ml of
the internal standard solution and dilute to 10.0 ml with carbon disulphide R.
Reference solution. Dissolve 0.2 g of butylhydroxytoluene R in carbon disulphide R and dilute to 100.0 ml
with the same solvent. Dilute 1.0 ml of this solution to 10.0 ml with carbon disulphide R. To 1.0 ml of
this solution add 1.0 ml of the internal standard solution and dilute to 10.0 ml with carbon
disulphide R.
gas chromatography R impregnated with 10 per cent m/m of poly(dimethyl)siloxane R; the column
is preceded by a column containing silanised glass wool,
43-17
Maintain the temperature of the column at 150C, that of the injection port at 180C and that of the
detector at 300C.
Inject the chosen volumes of test solutions (a) and (b) and of the reference solution.
Paraffins The tap and cotton plugs used must be free from grease. Prepare a column of anhydrous
aluminium oxide 0.23 m long and 20 mm in diameter by adding a slurry of anhydrous aluminium
oxide R and light petroleum R1 to a glass tube fitted with a tap and containing light petroleum R1 (before
use, dehydrate the anhydrous aluminium oxide by heating it in an oven at 600C for 3 h). Allow to
settle and reduce the depth of the layer of solvent above the column to about 40 mm. Dissolve 3.0 g
of the substance to be examined in 50 ml of warm light petroleum R1, cool, pass the solution through
the column at a flow rate of 3 ml/min and wash with 250 ml of light petroleum R1. Concentrate the
combined eluate and washings to low bulk by distillation, evaporate to dryness on a water-bath and
heat the residue at 105C for periods of 10 min until two successive weighings do not differ by more
than 1 mg. The residue weighs not more than 30 mg (1.0 per cent).
Pesticide residues Not more than 0.05 ppm for each organochlorine pesticide, 0.5 ppm for each
other pesticide and 1 ppm for the sum of all the pesticides.
All glassware used is thoroughly washed using phosphate-free detergent as follows. The glassware is
immersed in a bath of detergent solution (5 per cent in deionised water) and allowed to soak for 24 h.
The detergent is washed off with copious amounts of acetone and hexane for pesticide analysis. It is
important to keep glassware specifically for pesticide analyses, it must not be mixed up with glassware
used for other applications. The glassware used must be free of chlorinated solvents, plastics and
rubber materials, in particular phthalate plasticisers, oxygenated compounds and nitrogenated
solvents such as acetonitrile. Use hexane, toluene and acetone for pesticide analysis. Use HPLC
grade reagents for ethyl acetate, cyclohexane and water.
The test consists of the isolation of pesticide residues by size-exclusion chromatography followed
by solid phase extraction and identification by gas chromatography coupled with an electron capture
detector or a thermionic detector.
ISOLATION OF THE PESTICIDE RESIDUES. As detector, use a UV-visible spectrophotometer
set at a wavelength of 254 nm to calibrate the chromatographic column for gel permeation.
Calibration is extremely important in gel permeation chromatography to check that the pressure,
solvent flow rate, solvent ratio, temperature and column conditions remain constant. The gel
permeation column is to be calibrated at regular intervals using a standard mixture prepared as
follows: into a 1000 ml volumetric flask, introduce 50.00 g of maize oil R, 2.00 g of di(2-ethylhexyl)
phthalate R, 0.20 g of methoxychlor R, 50.0 mg of perylene R, 50.0 mg of naphthalene R, and 80.0 mg
of sulphur R. Dilute to 1000.0 ml with a mixture of equal volumes of cyclohexane R and ethyl
acetate R.
To calibrate the column, set the mobile phase at a flow rate of 5 ml/min with a mixture of equal
volumes of cyclohexane R and ethyl acetate R. Inject 5 ml of the standard mixture and record the
resulting chromatogram. The retention times for the analytes must not vary by more than 5 per
cent between calibrations. If the retention time shift is greater than 5 per cent, take corrective
action. Excessive retention time shifts may be caused by:
poor laboratory temperature control,
the pump contains air. This can be verified by measuring the flow rate: collect 25 ml of column
eluate in a volumetric flask and record the time (300 5 s),
a leak in the system.
Changes in pressure, in mobile phase flow rate or in column temperature conditions, as well as
column contamination, can affect pesticide retention times and are to be monitored. If the flow rate
or column pressure are outside desired bands the guard column or column is to be replaced.
Test solution. In a volumetric flask, dissolve 1 g of wool fat, accurately weighed, in a mixture of 1
volume of ethyl acetate R and 7 volumes of cyclohexane R. Add 1 ml of an internal standard (2 ppm)
either isodrin R or ditalimphos R and dilute to 20 ml. The internal standard solutions are used to establish
that recoveries of the pesticides from the GPC purification stage, evaporation and solid phase extraction stage
are at acceptable levels. Recovery levels of the internal standard solutions from the wool fat are determined by
comparing the peak areas of the wool fat extracts with peak areas of solutions of the internal standards.
a guard column 0.075 m long and 21.2 mm in internal diameter and a gel permeation column
0.3 m long and 21.2 mm in internal diameter, both packed with styrene-divinylbenzene
copolymer R (5 m),
as mobile phase at a flow rate of 5 ml/min a mixture of 1 volume of ethyl acetate R and 7 volumes
of cyclohexane R,
Inject 5 ml of the test solution. Discard the first 95 ml (19 min) of eluate containing the substance
to be examined. Collect the next 155 ml of eluate (31 min) containing any pesticide residues in an
evaporating vessel.
Place the 155 ml of the eluate collected from the gel permeation chromatography column into an
43-18
evaporating vessel. Place this vessel in an autoevaporator setting the water-bath temperature at 45C
and the nitrogen pressure at 55 kPa. Evaporate the eluate down to 0.5 ml.
To prepare the solid phase extraction cartridges take some magnesium silicate for pesticide residue
analysis R and heat it in a muffle furnace at 700C for 4 h to remove moisture and polychlorinated
biphenyls. After 2 h transfer the magnesium silicate directly to an oven at 100C to 105C, and allow
to stand for 30 min. Transfer the magnesium silicate to a stoppered glass jar and allow to equilibrate
for 48 h. This material may be used for up to 2 weeks. After that period the magnesium silicate is to
be reactivated, by heating at 600C for 2 h in a muffle furnace. Remove the magnesium silicate from
the furnace, cool and store in a stoppered glass jar. The magnesium silicate is deactivated by adding
1 per cent water R. After the water has been added, shake the magnesium silicate intermittently over
15 min just prior to use. The deactivated magnesium silicate is suitable for use for up to 1 week. It is
essential that only deactivated magnesium silicate is used.
Take a 6 ml empty solid phase extraction cartridge and weigh into the cartridge 1 g of the
deactivated magnesium silicate.
At this stage the GPC fraction still contains about 10 per cent of the substance to be examined, so
further clean-up is necessary. A separate isolation procedure is carried out a) for organochlorine and
synthetic pyrethroid pesticides and b) for organophosphorus pesticides. Place a preconditioned solid
phase extraction cartridge containing l g of deactivated magnesium silicate for pesticide residue analysis R
onto a vacuum manifold.
Condition the cartridge by adding 10 ml of toluene R and allowing the solvent to elute through the
cartridge. Place the 0.5 ml of the solvent fraction from the evaporating vessel on the preconditioned
cartridge. Elute the pesticide fractions from the cartridges using 20 ml of either of the two different
solvent systems shown below:
a) for determination of the organochlorine and synthetic pyrethroid pesticides: toluene R. A very
small amount of the substance to be examined is co-eluted,
b) for determination of the organophosphorus pesticides: a mixture of 2 volumes of acetone R and
98 volumes of toluene R. This solvent system is used to elute all the pesticides including the more
polar organophosphorus pesticides. Unfortunately some of the substance to be examined is coeluted with this solvent system, which can interfere with the electron capture detector.
Collect the eluate from the extraction cartridges in 25 ml glass vials. Quantitatively transfer the eluate
to an evaporating vessel, washing the vial with three quantities each of 10 ml of hexane R.
Place the evaporating vessel on the autoevaporator and evaporate the solid phase extraction
fractions down to 0.5 ml. The water-bath temperature is set at 45C and the nitrogen pressure is
55 kPa.
Examine the residues by gas chromatography using electron capture and thermionic detectors as
described below.
Recovery. Calculate the recovery correction factor (Rcf) of the internal standards (ditalimphos R or
isodrin R) added to the test solution using the following expression:
peak area of internal standard extracted from the test solution

100
peak area of an internal standard 1 ppm in solution
5 ml of the 20 ml test solution containing 1 ml of 2 ppm internal standard concentrated to 0.5 ml
is equivalent to 1 ppm of the internal standard in the solution.
If the recovery of the internal standards falls outside the range 70 per cent to 110 per cent the test
is not valid.
Reference solutions. Prepare reference solutions of pesticides using the pesticides standards at a
concentration of 0.5 ppm (see composition of reference solutions A to F in Table 0134.-1).
Commercially available pesticides may be purchased. The individual standards have a concentration
of 10 ppm.
At the same time prepare solutions of pesticides equivalent to the limit of detection of the method
(see recommended compositions in Table 0134.-1). These reference solutions are used to optimise
the electron capture detector and thermionic detector to achieve the detection limits of the method
(reference solutions G and H).
To prepare the reference solutions at the different concentrations use a calibrated pipette and
volumetric flasks. To prepare the internal standard solutions I and J use a four-place analytical
balance, pipette and volumetric flasks.
IDENTIFICATION AND QUANTIFICATION OF THE PESTICIDE RESIDUES. To identify any
pesticide residues compare the chromatograms obtained with chromatograms obtained with reference
solutions A to F.
The identity of the pesticides can be confirmed by spiking samples or overlaying chromatograms
using an integration package on a computer. The interpretation of pesticides in trace residue analyses
is extremely complex. The detectors, particularly the electron capture detector, are prone to
interference, both from the substance to be examined itself, and from solvents, reagents and
apparatus used in the extraction. These peaks can easily be misinterpreted or quoted as a false
43-19
positive. Confirmation of pesticides can be achieved by running samples and standards on different
capillary columns (see chromatographic systems A or B described below). The peaks can be
identified by using the information in Table 0134.-2.
A knowledge of the different responses the pesticides have in the two detectors is useful in
identification of unknown peaks.
Once pesticides have been identified, calculate the content of each pesticide using the following
expression:
Cp =
Pp D Ce
Pe
100
R cf
Cp = concentration of identified pesticide (ppm),

Pp = peak area of the individual pesticide in the test sample obtained,
Ce = concentration of the individual pesticide in the external standard (ppm),
Pe = peak area of the individual pesticide in the external standard,
D = dilution factor,
Rcf = recovery correction factor.
The dilution factor (D) can be defined as follows:
volume of sample obtained after the 2nd evaporation stage
GPC injection volume
sample weight
sample volumetric flask volume
43-20
Table 01341 Composition of the reference solutions
Reference solution A
(0.5 ppm or 0.5 mg/l)
(organochlorine and
synthetic pyrethroid
pesticides)
Reference solution B
(0.5 ppm or 0.5 mg/l)
(organochlorine and synthetic
pyrethroid pesticides)
Cyhalothrin R
Cypermethrin R
Aldrin R
o,p -DDT
R
p,p -DDD
R
p,p -DDD
R
Dieldrin R
R
o,p -DDE
p,p -DDE
R
p,p -DDT
R
Deltamethrin R
Endrin R
Heptachlor R
Heptachlor epoxide R
Hexachlorobenzene R
Lindane R
Tecnazene R
-Endosulphan R
-Endosulphan R
Fenvalerate R
-hexachlorocyclohexane
Methoxychlor R
Permethrin R
Reference solution C
(0.5 ppm or 0.5 mg/l)
(organophosphorus
pesticides)
Reference solution D
(0.5 ppm or 0.5 mg/l)
(organophosphorus
pesticides)
Bromophos-ethyl R
Bromophos R
Carbophenothion R
Chlorpyriphos R
Chlorfenvinphos R
Chlorpyriphos-methyl R
Diazinon R
Coumaphos R
Dichlofenthion R
Phosalone R
Ethion R
Pirimiphos-ethyl R
Fenchlorphos
Tetrachlorvinphos R
Malathion R
Propetamphos R
Reference solution E
(2 ppm or 2.0 mg/l)
(organochlorine pesticide)
Reference solution F
(2 ppm or 2.0 mg/l)
(organochlorine pesticide)
Chlordane R
Toxaphene R
Reference solution G
(electron capture detector
calibration mixture)
Reference solution H
(thermionic detector
calibration mixture)
Aldrin R (0.01 mg/l)
Chlorfenvinphos R (0.05 mg/l)
Cypermethrin R (0.1 mg/l)
Diazinon R (0.05 mg/l)
o,p -DDD
R (0.01 mg/l)
Ethion R (0.05 mg/l)
Deltamethrin R (0.1 mg/l)
Fenchlorphos R (0.05 mg/l)
Endrin R (0.01 mg/l)
Propetamphos R (0.05 mg/l)
-Hexachlorocyclohexane R
(0.01 mg/l)
Reference solution I
(internal standard organophosphorus pesticide)
Reference solution J
(internal standard organochlorine pesticide)
Ditalimphos R
(2 ppm or 2.0 mg/l)
Isodrin R (2 ppm or 2.0 mg/l)
Ditalimphos R
(1 ppm or 1.0 mg/l)
Isodrin R (1 ppm or 1.0 mg/l)
43-21
Table 01342 Elution order of the pesticides in chromatographic
systems A and B
Chromatographic system A Chromatographic system B
Tecnazene
-Hexachlorocyclohexane
Hexachlorobenzene
Tecnazene
Hexachlorobenzene
Lindane
Propetamphos
Diazinon
Dichlofenthion
Chlorpyriphos-methyl
Heptachlor
Fenchlorphos
Diazinon
Lindane
Propetamphos
Heptachlor
Dichlofenthion
Aldrin
Chlorpyriphos-methyl
Fenchlorphos
Aldrin
Malathion
Pyrimiphos-ethyl
Chlorpyriphos-ethyl
Chlorpyriphos-ethyl
Bromophos
Bromophos
Pyrimiphos-ethyl
Malathion
Heptachlor epoxide
Heptachlor epoxide
Chlorfenvinphos (E)
o,p -DDE
Chlorfenvinphos (Z)
Chlorfenvinphos (E)
Bromophos-ethyl
-Endosulphan
o,p -DDE
Chlorfenvinphos (Z)
-Endosulphan
Bromophos-ethyl
Tetrachlorvinphos
p,p -DDE
Dieldrin
Dieldrin
p,p -DDE
Tetrachlorvinphos
o,p -DDT
o,p -DDT
Endrin
Endrin
-Endosulphan
o,p -DDD
o,p -DDD
p,p -DDD
p,p -DDD
-Endosulphan
Ethion
Ethion
Carbophenothion
p,p -DDT
p,p -DDT
Carbophenothion
Methoxychlor
Methoxychlor
Phosalone
Cyhalothrin
Cyhalothrin (2 isomers)
cis-Permethrin
cis-Permethrin
Phosalone
trans-Permethrin
trans-Permethrin
Coumaphos
Cypermethrin (4 isomers)
Cypermethrin (4 isomers)
Coumaphos
Fenvalerate (2 isomers)
Fenvalerate (2 isomers)
Deltamethrin
Deltamethrin
The chromatographic procedure may be carried out using chromatographic system A:

a deactivated silica guard column 4.5 m long and 0.53 mm in internal diameter and a fusedsilica column 60 m long and 0.25 mm internal diameter coated with
poly(dimethyl)(diphenyl)siloxane R (film thickness 0.25 m).
43-22
helium for chromatography R as the carrier gas at a linear velocity of 25 cm/s and at a pressure of
180 kPa,
Column
Time
(min)
Temperature
(C)
01
15
5 30
75
75 175
175 275
30 40 275 285
40 55 285
Injection port
300
Detector
350
Rate (C
per min)
24
4
1
Comment
isothermal
linear gradient
linear gradient
linear gradient
isothermal
either an electron capture or thermionic specific detector.

Confirmation analysis may be carried out using chromatographic system B:
a deactivated silica guard column 4.5 m long and 0.53 mm in internal diameter and a fusedsilica column 60 m long and 0.25 mm in internal diameter coated with
poly(cyanopropyl)(7)(phenyl)(7)(methyl)(86) siloxane R (film thickness 0.25 m),
helium for chromatography R as the carrier gas at a linear velocity of 25 cm/s and a pressure of
180 kPa,
Column
Time
(min)
Temperature
(C)
01
15
5 30
75
75 175
175 275
30 40 275 285
40 55 285
Injection port
300
Detector
350
Rate (C
per min)
24
4
1
Comment
isothermal
linear gradient
linear gradient
linear gradient
isothermal
electron capture or thermionic specific detector.

Inject 2 l of the each solution.
Chlorides Boil 1.0 g with 20 ml of alcohol (90 per cent V/V) R in a round-bottomed flask fitted with a
reflux condenser for 5 min. Cool, add 40 ml of water R and 0.5 ml of nitric acid R and filter. To the
filtrate add 0.15 ml of a 10 g/l solution of silver nitrate R in alcohol (90 per cent V/V) R. Allow to stand
for 5 min protected from light. Any opalescence in the solution is not more intense than that in a
standard prepared at the same time by adding 0.15 ml of a 10 g/l solution of silver nitrate R in alcohol
(90 per cent V/V) R to a mixture of 0.2 ml of 0.02M hydrochloric acid, 20 ml of alcohol (90 per cent
V/V) R, 40 ml of water R and 0.5 ml of nitric acid R (150 ppm).
100C to 105C for 1 h.
Sulphated ash (2.4.14). Not more than 0.15 per cent. Ignite 5.0 g and use the residue to determine
the sulphated ash.
STORAGE
At a temperature not exceeding 25C.
LABELLING
__________________________________________________________________________________________________________ Ph Eur
43-23
Hydrogenated Wool Fat

1/01
Hydrogenated Wool Fat complies with the requirements of the 3rd edition of the European Pharmacopoeia
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Hydrogenated wool fat is a mixture of higher aliphatic alcohols and sterols obtained from the direct,
high-pressure, high-temperature hydrogenation of wool fat (0134) during which the esters and acids
present are reduced to the corresponding alcohols. It may contain not more than 200 ppm of
butylhydroxytoluene.
CHARACTERS
A white or pale yellow, unctuous substance, practically insoluble in water, soluble in boiling alcohol
and in light petroleum.
IDENTIFICATION
B. Examine the chromatograms obtained in the test for fatty alcohols and sterols. The retention time
and size of the principal peaks in the chromatogram obtained with the test solution are approximately
the same as those of the principal peaks in the chromatogram obtained with reference solution (a).
C. Dissolve 50 mg in 5 ml of methylene chloride R and add 1 ml of acetic anhydride R and 0.1 ml of
sulphuric acid R. A green colour is produced.
TESTS
Melting point (2.2.15). 45C to 55C. Allow to stand at 20C for 16 h.
Hydroxyl value (2.5.3, Method A). 140 to 180.
Saponification value (2.5.6). Not more than 8.0. Heat under reflux for 4 h.
Fatty alcohols and sterols Examine by gas chromatography (2.2.28).
Test solution. Dissolve 0.25 g of the substance to be examined in 60 ml of ethanol R and dilute to
Reference solution (a). Dissolve 0.25 g of hydrogenated wool fat CRS in 60 ml of ethanol R and dilute to
Reference solution (b). Dissolve 50 mg of cetyl alcohol CRS and 50 mg of stearyl alcohol CRS in 60 ml of
ethanol R and dilute to 100.0 ml with the same solvent.
a fused-silica column 30 m long and 0.25 mm in internal diameter with a film of
polydimethylsiloxane R or another non-polar phase of 0.25 m thickness,
helium for chromatography R as the carrier gas at a pressure of 100 kPa,
Time
(min)
Column
Temperature
(C)
05
100
5 45 100 300
45 60 300
Injection port
325
Detector
350
Rate (C
per min)
5
Comment
isothermal
linear gradient
isothermal
Inject 1 l of each solution. The chromatogram obtained with the test solution does not differ
significantly from the chromatogram obtained with reference solution (a) (Fig. 09691) and it does
not show enhanced peaks with retention times corresponding to cetyl alcohol and stearyl alcohol
present in the chromatogram obtained with reference solution (b).
2,4-dimethyl-6-tert-butylphenol R as the internal standard.
43-24
Internal standard solution. Dissolve 0.2500 g of 2,4-dimethyl-6-tert-butylphenol R in hexane R and dilute
Stock solution. Dissolve 0.2500 g of butylhydroxytoluene R in hexane R and dilute to 250.0 ml with the
same solvent.
Test solution. Use the solution within 3 h after its preparation. Melt about 100.0 g of the substance to be
examined in a microwave oven. Introduce 10.0 g (m) of the molten sample into a 100 ml volumetric
flask. Whilst still molten, add a sufficient quantity (a few millilitres) of hexane R to take the sample up
into solution and mix well. Add 2.0 ml of the internal standard solution and dilute to 100.0 ml with
hexane R. Place the volumetric flask in an incubator at 10C for 1 h.
Place a solid phase extraction cartridge on a vacuum manifold and place a 30 ml glass vial beneath
the outlet of the cartridge. Condition a 5 g solid phase extraction cartridge by passing through the
cartridge 10 ml of a mixture of equal volumes of hexane R and toluene R. Before the column starts to
dry out add 2.5 ml of the prepared sample and pass through with 20 ml of a mixture of equal
volumes of hexane R and toluene R. Quantitatively transfer the eluent collected to a turbo teat and
evaporate down to 1 ml using an autoevaporator. The concentrated eluent obtained is used as test
solution.
Reference solution. Use the solution within 3 h after its preparation. Melt about 100.0 g of BHT-free
hydrogenated wool fat CRS in a microwave oven. Introduce 10.0 g (m) of the molten sample into a
100 ml volumetric flask. Whilst still molten, add a sufficient quantity (a few millilitres) of hexane R to
take the sample up into solution and mix well. Add 2.0 ml of the internal standard solution and
2.0 ml of stock solution. Dilute to 100.0 ml with hexane R. Place 2.5 ml of this solution on a solid
phase extraction cartridge preconditioned with 10 ml of a mixture of equal volumes of hexane R and
toluene R. Pass 20 ml of the same mixture of solvents through the column to elute the butylhydroxytoluene and the internal standard. Quantitatively transfer the eluent collected to a turbo
teat and evaporate down to 1 ml using an autoevaporator. The concentrated eluent obtained is used
as reference solution.
a column 12 m long and 0.22 mm in internal diameter coated with poly(dimethyl)siloxane R (film
thickness 0.25 m),
Column
Time
(min)
Temperature
(C)
Rate (C
per min)
02
25
57
7 10
10 30
0
100
100 180
180
25
Injection port 0 1
14
Detector
180 250
250
210
25
Comment
isothermal
linear gradient
isothermal
linear gradient
isothermal
210 350
350 0
300
Inject 1 l of the test solution and 1 l of the reference solution. Continue the chromatography of
the test solution for about 30 min. When the chromatograms are recorded in the prescribed conditions, the retention times are: internal standard, about 3.5 min and butylhydroxytoluene, about
4.6 min.
Determine the concentration of butylhydroxytoluene in parts per million in the substance to be
examined from the expression:
200 ABHT
K
10
A
K BHT m
ABHT = area of the peak due to butylhydroxytoluene in the chromatogram obtained with the test
solution,
A = area of the peak due to the internal standard in the chromatogram obtained with the test
solution,
KBHT = area of the peak due to butylhydroxytoluene in the chromatogram obtained with the
reference solution,
K = area of the peak due to the internal standard in the chromatogram obtained with the
reference solution,
m = mass of substance to be examined used to prepare the test solution, in grams.
43-25
100C to 105C for 1 h.
STORAGE
Store in a well-filled container, protected from light.
46.060
43.964
43.370
46.775
47.407
44.736
45.852
42.222
42.728
36.596
37.931
38.446
40.147
41.213
35.473
32.263
33.757
36.835
34.917
30.086
28.780
22.915
17.784
19.955
20.754
2.0e4
26.387
22.323
3.0e4
26.783
23.535
24.252
28.130
39.507
4.0e4
33.480
31.672
24.978
40.695
LABELLING
1.0e4
10
20
30
40
50
Fig. 09691 Chromatogram for the test for fatty alcohols and sterols (reference solution (a)).
__________________________________________________________________________________________________________ Ph Eur
43-26
Hydrous Wool Fat

Lanolin
Hydrous Wool Fat complies with the requirements of the 3rd edition of the European Pharmacopoeia [0135].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Hydrous wool fat is a mixture of 75 per cent m/m of wool fat and 25 per cent m/m of water. It is
obtained by the gradual addition of water to melted wool fat with continuous stirring. It may contain
not more than 150 ppm of butylhydroxytoluene.
CHARACTERS
A pale yellow, unctuous substance.
IDENTIFICATION
A. In a test-tube, dissolve 0.5 g in 5 ml of chloroform R and add 1 ml of acetic anhydride R and 0.1 ml
of sulphuric acid R. A green colour develops.
B. Dissolve 50 mg in 5 ml of chloroform R, add 5 ml of sulphuric acid R and shake. A red colour
develops and an intense green fluorescence appears in the lower layer.
TESTS
Water-soluble acid or alkaline substances Melt 6.7 g on a water-bath and shake vigorously for
2 min with 75 ml of water R previously heated to 90C to 95C. Allow to cool and filter through filter
paper previously rinsed with water R. To 60 ml of the filtrate (which may not be clear) add 0.25 ml of
bromothymol blue solution R1. Not more than 0.2 ml of 0.02M hydrochloric acid or 0.15 ml of 0.02M
Drop point (2.2.17). 38C to 44C, determined on the residue obtained in the test for wool-fat
content. To fill the metal cup, melt the residue on a water-bath, cool to about 50C, pour into the
cup and allow to stand at 15C to 20C for 24 h.
Water-absorption capacity Place 10 g of the residue obtained in the test for wool-fat content in a
mortar. Add water R in portions of 0.2 ml to 0.5 ml from a burette, stirring vigorously after each
addition to incorporate the water R. The end-point is reached when visible droplets remain which
cannot be incorporated. Not less than 20 ml of water R is absorbed.
Saponification value (2.5.6). 67 to 79, determined on 2.00 g. Heat under reflux for 4 h.
Water-soluble oxidisable substances To 10 ml of the filtrate obtained in the test for water-soluble
acid or alkaline substances add 1 ml of dilute sulphuric acid R and 0.1 ml of 0.02M potassium
permanganate. After 10 min, the solution is not completely decolorised.
100.0 ml with the same solvent. Dilute 1.0 ml of this solution to 10.0 ml with carbon disulphide R.
Test solution (a). Dissolve 1.0 g of the residue obtained in the test for wool-fat content in carbon
disulphide R and dilute to 10.0 ml with the same solvent.
Test solution (b). Dissolve 1.0 g of the residue obtained in the test for wool-fat content in carbon
disulphide R, add 1.0 ml of the internal standard solution and dilute to 10.0 ml with carbon
disulphide R.
Reference solution. Dissolve 0.2 g of butylhydroxytoluene R in carbon disulphide R and dilute to 100.0 ml
with the same solvent. Dilute 1.0 ml of this solution to 10.0 ml with carbon disulphide R. To 1.0 ml of
this solution add 1.0 ml of the internal standard solution and dilute to 10.0 ml with carbon
disulphide R.
gas chromatography R impregnated with 10 per cent m/m of polydimethylsiloxane R; the column is
preceded by a column containing silanised glass wool,
43-27
Maintain the temperature of the column at 150C, that of the injection port at 180C and that of the
detector at 300C. Inject the chosen volumes of test solutions (a) and (b) and of the reference solution.
Paraffins The tap and cotton plugs used must be free from grease. Prepare a column of anhydrous
aluminium oxide 230 mm long and 20 mm in diameter by adding a slurry of anhydrous aluminium
oxide R and light petroleum R1 to a glass tube fitted with a tap and containing light petroleum R1. Allow
to settle and reduce the depth of the layer of solvent above the column to about 40 mm. Dissolve
3.0 g of the residue obtained in the test for wool-fat content in 50 ml of warm light petroleum R1, cool,
pass the solution through the column at a rate of 3 ml per minute and wash with 250 ml of light
petroleum R1. Concentrate the combined eluate and washings to low bulk by distillation, evaporate to
dryness on a water-bath and heat the residue at 105C for periods of 10 min until two successive
weighings do not differ by more than 1 mg. The residue weighs not more than 30 mg (1.0 per cent).
Chlorides Boil 1.3 g with 20 ml of alcohol (90 per cent V/V) R under a reflux condenser for 5 min.
Cool, add 40 ml of water R and 0.5 ml of nitric acid R and filter. To the filtrate add 0.15 ml of a
10 g/l solution of silver nitrate R in alcohol (90 per cent V/V) R. Allow to stand for 5 min, protected
from light. Any opalescence in the solution is not more intense than that in a standard prepared at
the same time by adding 0.15 ml of a 10 g/l solution of silver nitrate R in alcohol (90 per cent V/V) R to
a mixture of 0.2 ml of 0.02M hydrochloric acid, 20 ml of alcohol (90 per cent V/V) R, 40 ml of water R
and 0.5 ml of nitric acid R (115 ppm).
Sulphated ash (2.4.14). Not more than 0.1 per cent. Ignite 5.0 g and use the residue to determine
the sulphated ash.
Wool-fat content 72.5 per cent to 77.5 per cent. In a suitable tared dish containing a glass rod, heat
30.0 g to constant mass on a water-bath, stirring continuously. Weigh the residue.
STORAGE
Store in a well-closed container, at a temperature not exceeding 25C.
LABELLING
__________________________________________________________________________________________________________ Ph Eur
43-28
Wormwood
Wormwood complies with the requirements of the 3rd edition of the European Pharmacopoeia [1380]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Wormwood consists of the basal leaves or slightly leafy, flowering tops, or of a mixture of these dried,
whole or cut organs of Artemisia absinthium L. It contains not less than 2 ml/kg of essential oil,
calculated with reference to the dried drug.
CHARACTERS
IDENTIFICATION
A. The leaves are greyish to greenish, densely tomentose on both surfaces. The basal leaves, with
long petioles, have triangular to oval bipinnatisect to tripinnatisect lamina, with rounded to lanceolate
segments. The cauline leaves are less segmented and the apical leaves are lanceolate. The stem of the
flower-bearing region is greenish-grey, tomentose, up to 2.5 mm in diameter and usually with five
flattened longitudinal grooves. The capitula are arranged as loose, axillary panicles, inserted at the
level of the lanceolate to slightly pinnatisect leaves; they are spherical to flattened hemispherical,
2 mm to 4 mm in diameter and consist of a grey, tomentose involucre, the outer bracts linear, inner
layer ovate, blunt at the apices with scarious margins, a receptacle with very long paleae up to 1 mm
or more long, numerous yellow, tubular, hermaphroditic florets about 2 mm long and few yellow, ray
florets.
B. Reduce to a powder (355). The powder is greenish-grey. Examine under a microscope using
chloral hydrate solution R. The powder shows many T-shaped trichomes with a short uniseriate stalk
consisting of one to five small cells, perpendicularly capped by a very long, undulating terminal cell
tapering at the ends; fragments of epidermises with sinuous to wavy walls, anomocytic stomata
(2.8.3) and secretory trichomes each with a short, biseriate, two celled stalk and a biseriate head with
two or four cells; fragments of the tubular and ray florets, some containing small cluster crystals of
calcium oxalate; numerous paleae each composed of a small cell forming a stalk and a very long,
cylindrical and thin-walled terminal cell about 1 mm to 1.5 mm long; spheroidal pollen grains, about
30 m in diameter, with three pores and a finely warty exine; groups of fibres, small vessels with
spiral and annular thickening, larger vessels with bordered pits and parenchyma with moderately
thickened and pitted walls, from the stem.
substance.
Test solution. Place 2 g of the powdered drug (355) in 50 ml of boiling water R and allow to stand for
5 min, shaking the flask several times. After cooling, add 5 ml of a 100 g/l solution of lead acetate R.
Mix and filter. Rinse the flask and the residue on the filter with 20 ml of water R. Shake the filter with
50 ml of methylene chloride R. Separate the organic layer, dry over anhydrous sodium sulphate R filter
and evaporate the filtrate to dryness on a water-bath. Dissolve the residue in 0.5 ml of alcohol R.
Reference solution. Dissolve 2 mg of methyl red R and 2 mg of resorcinol R in 10.0 ml of methanol R.
mixture of 10 volumes of acetone R, 10 volumes of glacial acetic acid R, 30 volumes of toluene R and
50 volumes of methylene chloride R. Allow the plate to dry in air. Spray the plate with acetic anhydridesulphuric acid solution R and examine in daylight. The chromatogram obtained with the test solution
shows the blue zone of artabsin shortly above the red zone of methyl red in the chromatogram
obtained with the reference solution. Examine in daylight while heating at 100C to 105C for 5 min.
The chromatogram obtained with the reference solution shows in the middle third the red zone of
methyl red and below it the light pink zone of resorcinol. The chromatogram obtained with the test
solution shows an intense red to brownish-red zone of absinthin with a similar Rf to that of the zone
due to resorcinol in the chromatogram obtained with the reference solution. Other zones are visible,
but less intense than that of absinthin.
TESTS
Foreign matter (2.8.2). Not more than 5 per cent of stems with a diameter greater than 4 mm and
2 per cent of other foreign matter.
Bitterness value. Not less than 10,000. The bitterness value is determined by comparison with
quinine hydrochloride, the bitterness value of which is set at 200,000; the bitterness value is the
reciprocal of the dilution that still has a bitter taste.
Quinine hydrochloride stock solution. Dissolve 0.100 g of quinine hydrochloride R in water R and dilute to
43-29
100.0 ml with the same solvent. Dilute 1.0 ml of the solution to 100.0 ml with water R.
Wormwood extract. To 1.0 g of powdered drug (710) add 1000 ml of boiling water R. Heat on a
water-bath for 30 min, stirring continuously. Allow to cool and dilute to 1000 ml with water R. Shake
vigorously and filter, discarding the first 20 ml of filtrate.
Prepare a series of dilutions by placing in the first tube 4.2 ml of quinine hydrochloride stock
solution and increasing the volume by 0.2 ml in each subsequent tube to a total of 5.8 ml; dilute the
contents of each tube to 10.0 ml with water R. Determine as follows the dilution with the lowest
concentration that still has a bitter taste. Take 10.0 ml of the weakest solution into the mouth and
pass it from side to side over the back of the tongue for 30 s. If the solution is not found to be bitter,
spit it out and wait for 1 min. Rinse the mouth with water R. After 10 min, use the next dilution in
order of increasing concentration.
Calculate the correction factor (k) from the expression:
5.00/n
n = number of millilitres of quinine hydrochloride stock solution in the dilution of lowest
concentration that is bitter.
Dilute 10/k ml of wormwood extract to 100.0 ml with water R. 10.0 ml of this dilution has a bitter
taste.
ASSAY
Carry out the determination of essential oil in vegetable drugs (2.8.12). Use 50.0 g of the cut drug, a
1000 ml round-bottomed flask and 500 ml of water R as the distillation liquid. Add 0.5 ml of xylene R
in the graduated tube. Distil at a rate of 2 ml/min to 3 ml/min for not less than 3 h.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
43-30
Xanthan Gum
Xanthan Gum complies with the requirements of the 3rd edition of the European Pharmacopoeia [1277].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Xanthan gum is a high-molecular-mass anionic polysaccharide produced by fermentation of
carbohydrate with Xanthomonas campestris. It consists of a principal chain of (14)-linked Dglucose units with trisaccharide side chains, on alternating anhydroglucose units, consisting of a
glucuronic acid unit included between two mannose units. Most of the terminal units contain a
pyruvate moiety and the mannose unit adjacent to the principal chain may be acetylated at C-6.
Xanthan gum has a molecular mass of approximately 1 106. It contains not less than 1.5 per cent
of pyruvoyl groups (C3H3O2; relative mass of the group 71.1), calculated with reference to the dried
substance. Xanthan gum exists as the sodium, potassium or calcium salt.
CHARACTERS
A white or yellowish-white, free-flowing powder, soluble in water giving a highly viscous solution,
practically insoluble in organic solvents.
IDENTIFICATION
A. In a flask, suspend 1 g in 15 ml of 0.1M hydrochloric acid. Close the flask with a fermentation bulb
containing barium hydroxide solution R and heat carefully for 5 min. The barium hydroxide solution
shows a white turbidity.
B. To 300 ml of water R, previously heated to 80C and stirred rapidly with a mechanical stirrer in a
400 ml beaker, add, at the point of maximum agitation, a dry blend of 1.5 g of carob bean gum R and
1.5 g of the substance to be examined. Stir until the mixture forms a solution, and then continue
stirring for 30 min or longer. Do not allow the water temperature to drop below 60C during stirring.
Discontinue stirring and allow the mixture to stand for at least 2 h. A firm rubbery gel forms after the
temperature drops below 40C but no such gel forms in a 1 per cent control solution of the sample
prepared in the same manner but omitting the carob bean gum.
pH (2.2.3). The pH of a 10.0 g/l solution is 6.0 to 8.0.
Viscosity (2.2.10). The viscosity at 24 1C is not less than 600 mPas. Add 3.0 g within 45 s to
90 s into 250 ml of a 12 g/l solution of potassium chloride R in a 500 ml beaker stirring with a lowpitch propeller-type stirrer rotating at 800 r/min. When adding the substance take care that
agglomerates are destroyed. Add an additional quantity of 44 ml of water R, to rinse any adhering
residue from the walls of the beaker. Stir the preparation at 800 r/min for 2 h whilst maintaining the
temperature at 24 1C. Determine the viscosity within 15 min using a rotating viscosimeter set at
60 r/min and equipped with a rotating spindle 12.7 mm in diameter and 1.6 mm high which is
attached to a shaft 3.2 mm in diameter. The distance from the top of the cylinder to the lower tip of
the shaft being 25.4 mm, and the immersion depth being 50.0 mm.
2-Propanol Not more than 750 ppm, determined by gas chromatography (2.2.28) using 2-methyl-2propanol R as the internal standard.
Internal standard solution. Dilute 0.50 g of 2-methyl-2-propanol R to 500 ml with water R.
Test solution. To 200 ml of water R in a 1000 ml round-bottomed flask, add 5.0 g of the substance to
be examined and 1 ml of a 10 g/l emulsion of dimeticone R in liquid paraffin R, stopper the flask and
shake for 1 h. Distil about 90.0 ml, mix the distillate with 4.0 ml of the internal standard solution and
Reference solution. Dilute a suitable quantity of 2-propanol R, accurately weighed, with water R to
obtain a solution having a known concentration of 2-propanol of about 1 mg/ml. To 4.0 ml of this
solution add 4.0 ml of the internal standard solution and dilute to 100.0 ml with water R.
copolymer R,
maintaining the temperature of the column at 165C, and that of the injection port and of the
detector at 200C.
Inject 5 l of the test solution and 5 l of the reference solution. The retention time of 2-methyl-2propanol is about 1.5 relative to that of 2-propanol.
43-31
Other polysaccharides Examine by thin-layer chromatography (2.2.27), using a TLC silica gel
plate R.
Test solution. To 10 mg in a thick-walled centrifuge test tube add 2 ml of a 230 g/l solution of
trifluoroacetic acid R, shake vigorously to dissolve the forming gel, stopper the test tube, and heat the
mixture at 120C for 1 h. Centrifuge the hydrolysate, transfer the clear supernatant liquid carefully
into a 50 ml flask, add 10 ml of water R and evaporate the solution to dryness under reduced
pressure. Take up the residue thus obtained in 10 ml of water R and evaporate to dryness under
reduced pressure. Wash three times with 20 ml of methanol R and evaporate under reduced pressure.
To the resulting clear film which has no odour of acetic acid, add 0.1 ml of water R and 1 ml of
methanol R. Centrifuge to separate the amorphous precipitate. Dilute the supernatant liquid, if
necessary, to 1 ml with methanol R.
Reference solution. Dissolve 10 mg of glucose R and 10 mg of mannose R in 2 ml of water R and dilute
to 10 ml with methanol R.
Apply separately to the plate, as bands, 5 l of each solution. Develop over a path of 15 cm using a
mixture of 10 volumes of a 16 g/l solution of sodium dihydrogen phosphate R, 40 volumes of butanol R
and 50 volumes of acetone R. Spray evenly with a solution of 0.5 g of diphenylamine R in 25 ml of
methanol R to which 0.5 ml of aniline R and 2.5 ml of phosphoric acid R have been added. Heat for
5 min at 120C. Examine in daylight. The test is not valid unless the chromatogram obtained with
the reference solution shows two clearly separated greyish-brown zones due to glucose and mannose
in the middle third. The chromatogram obtained with the test solution shows corresponding zones.
In addition, one weak reddish and two faint bluish-grey bands may be visible just above the starting
line. One or two bluish-grey bands may also be seen in the upper quarter of the chromatogram. No
other bands are visible.
at 100C to 105C for 2.5 h.
Total ash (2.4.16). 6.5 per cent to 16.0 per cent.
fungi per gram, determined by plate count. It complies with the test for Escherichia coli (2.6.13).
ASSAY
Test solution. Dissolve a quantity of the substance to be examined corresponding to 120.0 mg of the
dried substance in water R and dilute to 20.0 ml with the same solvent.
Reference solution. Dissolve 45.0 mg of pyruvic acid R in water R and dilute to 500.0 ml with the same
solvent.
Place 10.0 ml of the test solution in a 50 ml round-bottomed flask, add 20.0 ml of 0.1M hydrochloric
acid and weigh. Boil on a water-bath under a reflux condenser for 3 h. Weigh and adjust to the initial
mass with water R. In a separating funnel mix 2.0 ml of the solution with 1.0 ml of dinitrophenylhydrazine-hydrochloric solution R. Allow to stand for 5 min and add 5.0 ml of ethyl acetate R. Shake
and allow the solids to settle. Collect the upper layer and shake with three quantities, each of 5.0 ml,
of sodium carbonate solution R. Combine the aqueous layers and dilute to 50.0 ml with sodium
carbonate solution R. Mix. Treat 10.0 ml of the reference solution at the same time and in the same
manner as for the test solution.
Immediately measure the absorbance of the two solutions (2.2.25) at 375 nm, using sodium
carbonate solution R as the compensation liquid.
The absorbance of the test solution is not less than that of the reference solution, which corresponds to a content of pyruvic acid of not less than 1.5 per cent.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
43-32
Xylitol
1/01
CH2OH
H
OH
HO
OH
CH2OH
C5H12O5
152.1
87-99-0
Xylitol complies with the requirements of the 3rd edition of the European Pharmacopoeia [1381]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Xylitol contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent of
meso-xylitol, calculated with reference to the anhydrous substance.
CHARACTERS
White crystalline powder or crystals, very soluble in water, sparingly soluble in alcohol.
IDENTIFICATION
obtained with xylitol CRS.
same solvent.
Reference solution (a). Dissolve 25 mg of xylitol CRS in water R and dilute to 5.0 ml with the same
solvent.
Reference solution (b). Dissolve 25 mg of mannitol CRS and 25 mg of xylitol CRS in water R and dilute
principal spot in the chromatogram obtained with the reference solution (a). The test is not valid
TESTS
Appearance of solution Dissolve 2.5 g in water R and dilute to 50.0 ml with the same solvent. The
solution is not more opalescent than reference suspension IV (2.2.1) and not more intensely coloured
than reference solution BY7 (Method II, 2.2.2).
Reducing sugars Dissolve 5.0 g in 6 ml of water R with the aid of gentle heat. Cool and add 20 ml of
cupri-citric solution R and a few glass beads. Heat so that boiling begins after 4 min and maintain
43-33
Related substances Examine by gas chromatography (2.2.28) as described under Assay. The sum
of the percentage contents of the related substances in the chromatogram obtained with the test
solution is not greater than 2.0 per cent.
Lead (2.4.10). It complies with the limit test for lead in sugars (0.5 ppm). Dissolve the substance to
be examined in 150.0 ml of the prescribed mixture of solvents.
of water.
xylitol and not more than 2.5 I.U. of endotoxin per gram for parenteral dosage forms having a
concentration of 100 g/l or more of xylitol.
ASSAY
Examine by gas chromatography (2.2.28), using erythritol as the internal standard.
Internal standard solution. Dissolve 5 mg of erythritol R in water R and dilute to 25.0 ml with the same
solvent.
the same solvent.
Reference solution. Dissolve 25.0 mg each of L-arabinitol CRS, galactitol CRS, mannitol CRS and sorbitol
CRS in water R and dilute to 100.0 ml with the same solvent. To 10.0 ml of the solution add about
490 mg of xylitol CRS, accurately weighed, to obtain the reference solution containing a known
concentration of about 49 mg of xylitol CRS per millilitre.
a stainless steel column 2 m long and 2 mm in internal diameter packed with acid washed
silanised diatomaceous earth for gas chromatography R (150 m to 180 m) and impregnated with
3 per cent m/m of poly[(cyanopropyl)(methyl)][(phenyl)(methyl)]siloxane R,
nitrogen R as the carrier gas at a flow rate of 30 ml/min,
of 6C per minute to 200C and maintaining the temperature of the injection port and of the
detector at 250C.
Pipet 1.0 ml of the reference solution and 1.0 ml of the test solution into separate 100 ml roundbottomed flasks. To each flask, add 1.0 ml of the internal standard solution and evaporate the
respective mixtures to dryness in a water-bath at 60C with the aid of a rotary evaporator. Dissolve
each dry residue in 1 ml of anhydrous pyridine R and add 1 ml of acetic anhydride R to each flask and
boil each solution under reflux for 1 h to complete acetylation.
Inject 1 l each of the solutions obtained from the reference solution and the test solution.
When the chromatograms are recorded in the prescribed conditions, the relative retentions calculated with reference to xylitol are: erythritol about 0.3, arabinitol about 0.7, mannitol about 1.8,
galactitol about 2.0 and sorbitol about 2.2.
Calculate the percentage content of C5H12O5, taking into account the declared content of xylitol
CRS, and the percentage content of each related substance in the substance to be examined by the
formula:
100
ms R u
mu R s
ms = mass of the respective component in 1 ml of the reference solution, in milligrams,

mu = mass of the substance to be examined in 1 ml of the test solution, in milligrams,
Rs = ratio of the surface areas of the derivatised component peak to the derivatised erythritol
peak in the chromatogram obtained with the reference solution,
Ru = ratio of the surface areas of the derivatised component peak to the derivatised erythritol
peak in the chromatogram obtained with the test solution.
LABELLING
The label states:
forms.
43-34
IMPURITIES
OH
H
OH
HO
HO
H
OH
A. L-arabinitol,
OH
H
OH
HO
HO
OH
OH
B. meso-galactitol,
C. mannitol,
D. sorbitol.
__________________________________________________________________________________________________________ Ph Eur
43-35
Xylometazoline Hydrochloride
Me
H
N
,HCl
N
Me
C 16H24N 2,HCl
But
280.8
1218-35-5
Xylometazoline Hydrochloride complies with the requirements of the 3rd edition of the European Pharmacopoeia [1162]. These requirements are reproduced after the heading Definition below.
Preparation
Xylometazoline Nasal Drops
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Xylometazoline hydrochloride contains not less than 99.0 per cent and not more than the equivalent
of 101.0 per cent of 2-(4-1,1-dimethylethyl-2,6-dimethylbenzyl)-2-imidazoline hydrochloride, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, freely soluble in water, in alcohol and in methanol,
IDENTIFICATION
obtained with xylometazoline hydrochloride CRS.
C. Dissolve about 0.5 mg in 1 ml of methanol R and add 0.5 ml of a freshly prepared 50 g/l solution
10 min and add 1 ml of an 80 g/l solution of sodium bicarbonate R. A violet colour develops.
D. Dissolve 0.2 g in 1 ml of water R, add 2.5 ml of alcohol R and 2 ml of 1M sodium hydroxide. Mix
thoroughly and examine in ultraviolet light at 365 nm. The solution shows no fluorescence or at most
the same fluorescence as a blank solution prepared in the same manner. The identification is not
valid unless a solution prepared in the same manner using naphazoline hydrochloride CRS instead of
the substance to be examined shows a distinct bluish fluorescence.
TESTS
Acidity or alkalinity Dissolve 0.25 g in water R and dilute to 25 ml with the same solvent. Add
0.1 ml of methyl red solution R and 0.1 ml of 0.01M hydrochloric acid. The solution is red. Not more
coating substance.
Reference solution (a). Dissolve 20 mg of xylometazoline hydrochloride CRS in methanol R and dilute to
Reference solution (b). Dissolve 4 mg of xylometazoline impurity A CRS in methanol R and dilute to
43-36
volumes of concentrated ammonia R and 100 volumes of methanol R. Allow the plate to dry. At the
bottom of a chromatography tank place an evaporating dish containing a mixture of 1 volume of
water R, 1 volume of hydrochloric acid R1 and 2 volumes of a 15 g/l solution of potassium
permanganate R. Close the tank and allow to stand for 15 min. Place the dried plate in the tank and
re-close the tank. Leave the plate in contact with the chlorine vapour for 5 min. Withdraw the plate
and place it in a current of cold air until the excess of chlorine is removed and an area of the coating
below the points of application does not give a blue colour with a drop of potassium iodide and starch
solution R. Spray with potassium iodide and starch solution R. Any spot in the chromatogram obtained
with test solution (a) corresponding to xylometazoline impurity A, is not more intense than the
principle spot in the chromatogram obtained with reference solution (b) (0.2 per cent). Any spot in
the chromatogram obtained with test solution (a), apart from the principal spot and the spot corresponding to xylometazoline impurity A is not more intense than the principle spot in the chromatogram obtained with reference solution (c) (0.2 per cent).
100C to 105C.
ASSAY
STORAGE
IMPURITIES
H2N
Me
H
N
O
Me
Me
A. N-(2-aminoethyl)-2-(4-1,1-dimethylethyl-2,6-dimethyl-phenyl)acetamide.
__________________________________________________________________________________________________________ Ph Eur
43-37
Xylose
O
OH
HO
C 5H10O5
and epimer at C*
OH
OH
150.1
58-86-6
Xylose complies with the requirements of the 3rd edition of the European Pharmacopoeia [1278]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Xylose is (+)-D-xylopyranose.
CHARACTERS
A white, crystalline powder or colourless needles, freely soluble in water, soluble in hot alcohol.
IDENTIFICATION
obtained with xylose CRS. Examine the substances prepared as discs.
B. Examine by thin-layer chromatography (2.2.27), using a suitable silica gel as the coating
substance.
Test solution. Dissolve 10 mg of the substance to be examined in a mixture of 2 volumes of water R
Reference solution (a). Dissolve 10 mg of xylose CRS in a mixture of 2 volumes of water R and 3
Reference solution (b). Dissolve 10 mg of fructose R, 10 mg of glucose R and 10 mg of xylose R in a
mixture of 2 volumes of water R and 3 volumes of methanol R and dilute to 20 ml with the same
volumes of anhydrous acetic acid R and 50 volumes of ethylene chloride R. The solvents should be
measured accurately since a slight excess of water produces cloudiness. Dry the plate in a current of
warm air. Spray uniformly with a 5 g/l solution of thymol R in a mixture of 5 ml of sulphuric acid R
and 95 ml of alcohol R. Heat in an oven at 130C for 10 min. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the
C. Dissolve 0.1 g in 10 ml of water R. Add 3 ml of cupri-tartaric solution R1 and heat. An orange to
red precipitate is formed.
TESTS
Acidity or alkalinity To 50 ml of solution S add 0.3 ml of phenolphthalein solution R1. The solution
is colourless. Not more than 0.2 ml of 0.1M sodium hydroxide is required to change the colour of the
indicator to pink.
Specific optical rotation (2.2.7). Dissolve 10.0 g in 80 ml of water R, add 1 ml of dilute
ammonia R2 and dilute to 100.0 ml with water R. Allow to stand for 30 min. The specific optical
rotation is +18.5 to +19.5, calculated with reference to the dried substance.
100C to 105C at a pressure not exceeding 0.7 kPa.
43-38
STORAGE
__________________________________________________________________________________________________________ Ph Eur
43-39
Yarrow
Yarrow complies with the requirements of the 3rd edition of the European Pharmacopoeia [1382]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Yarrow consists of the whole or cut, dried flowering tops of Achillea millefolium L. It contains not less
than 2 ml/kg of essential oil and not less than 0.02 per cent of proazulenes, expressed as chamazulene
(C14H16; Mr 184.3), both calculated with reference to the dried drug.
CHARACTERS
IDENTIFICATION
A. The leaves are green or greyish-green, faintly pubescent on the upper surface and more pubescent
on the lower surface, two to three pinnately divided with linear lobes and a finely pointed whitish tip.
The capitula are arranged in a corymb at the end of the stem. Each capitulum, 3 mm to 5 mm in
diameter, consists of the receptacle, usually four or five ligulate ray-florets and three to twenty tubular
disk-florets. The involucre consists of three rows of imbricate lanceolate, pubescent green bracts
arranged with a brownish or whitish, membranous margin. The receptacle is slightly convex and, in
the axillae of paleae, bears a ligulate ray floret with a three-lobed, whitish or reddish ligule and
tubular disk-florets with a radial, five-lobed, yellowish or light brownish corolla. The pubescent
green, partly brown or violet stems are longitudinally furrowed, up to 3 mm thick with a lightcoloured medulla.
B. Reduce to a powder (355). The powder is green or greyish-green. Examine under a microscope,
using chloral hydrate solution R. The powder shows fragments of the stems, leaves and bracts bearing
very rare glandular trichomes with a short stalk and a head formed of two rows of three to five cells
enclosed in a bladder-like membrane and uniseriate covering trichomes consisting of four to six small,
more or less isodiametric cells at the base and a thick-walled, often somewhat tortuous terminal cell,
about 400 m to greater than 1000 m long; fragments of the ligulate corolla with papillary epidermal
cells; small-celled parenchyma from the corolla tubes containing cluster crystals of calcium oxalate;
groups of lignified and pitted cells from the bracts; spherical pollen grains, about 30 m in diameter,
with three germinal pores and spiny exine; groups of sclerenchymatous fibres and small vessels with
spiral or annular thickening, from the stem.
C. Add 2.5 ml of dimethylaminobenzaldehyde solution R8 to 0.1 ml of solution S (see Tests) and heat
on a water-bath for 2 min. Allow to cool. Add 5 ml of light petroleum R and shake the mixture
vigorously. The aqueous layer shows a blue or greenish-blue colour.
substance.
Test solution. Use the solution prepared in identification test C.
Reference solution. Dissolve 10 mg of cineole R and 10 mg of guaiazulene R in 20 ml of toluene R.
Spray the plate with anisaldehyde solution R. Examine in daylight while heating at 100C to 105C for
5 min to 10 min. The chromatogram obtained with the reference solution shows in the upper part a
red zone (guaiazulene) and in the middle part a blue or greyish-blue zone (cineole). The chromatogram obtained with the test solution shows: a violet zone a little above the zone due to guaiazulene in
the chromatogram obtained with the reference solution; below this zone a reddish-violet zone; below
which, one or two not clearly separated greyish-violet to greyish zones (which changes to greenishgrey after a few hours) and a reddish-violet zone a little above the zone due to cineole in the chromatogram obtained with the reference solution. Further faint zones are present.
TESTS
Solution S To 2.0 g of the powdered drug (710) add 25 ml of ethyl acetate R, shake for 5 min and
filter. Evaporate to dryness on a water-bath and dissolve the residue in 0.5 ml of toluene R.
Foreign matter (2.8.2). Not more than 5 per cent of stems, with a diameter greater than 3 mm and
not more than 2 per cent of other foreign matter.
Loss on drying (2.2.32). Not more than 12.0 per cent determined on 0.500 g of powdered drug
43-40
ASSAY
Essential oil Carry out the determination of essential oil in vegetable drugs (2.8.12). Use 20.0 g of
cut drug, a 1000 ml round-bottomed flask and 500 ml of a mixture of 1 volume of water R and 9
volumes of ethylene glycol R as the distillation liquid. Add 0.2 ml of xylene R in the graduated tube.
Distil at a rate of 2 ml/min to 3 ml/min for 2 h.
Stop cooling at the end of distillation and continue distilling until the blue, steam-volatile
components have reached the lower end of the cooler. Immediately start cooling again, taking care to
avoid warming the separation space. Stop the distillation after 5 min. Replace the 1000 ml roundbottomed flask by a 250 ml round-bottomed flask containing a mixture of 0.4 ml of xylene R and
50 ml of water R. Distil for 15 min. After 10 min read the total volume. To determine the blank
value, use 0.2 ml of xylene R in the graduated tube and distil a mixture of 0.4 ml of xylene R and
50 ml of water R for 15 min.
Proazulenes To ensure that as little water as possible is transferred, transfer the blue essential oilxylene mixture obtained in the assay of essential oil to a 50 ml volumetric flask with the aid of small
portions of xylene R, rinsing the graduated tube of the apparatus with xylene R and dilute to 50.0 ml
with the same solvent. Measure the absorbance (2.2.25) at 608 nm using xylene R as the compensation liquid.
Calculate the percentage content of proazulenes, expressed as chamazulene from the expression:
A 2.1
m
i.e. taking the specific absorbance of chamazulene to be 23.8.
m = mass of the drug to be examined in grams.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
43-41
Zidovudine
O
Me
HN
O
HOCH2
N
O
N3
C10H13N5O4
267.2
30516-87-1
Zidovudine complies with the requirements of the 3rd edition of the European Pharmacopoeia [1059]. These
Action and use Antiviral.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Zidovudine contains not less than 97.0 per cent and not more than the equivalent of 102.0 per cent
of 1-(3-azido-2,3-dideoxy--D-ribofuranosyl)-5-methylpyrimidine-2,4(1H,3H)-dione, calculated with
CHARACTERS
A white or brownish powder, sparingly soluble in water, soluble in ethanol. It melts at about 124C.
IDENTIFICATION
Examine by infrared absorption spectrophotometry (2.2.24) comparing with the spectrum obtained
with zidovudine CRS. If the spectra obtained in the solid state show differences, dissolve the
substance to be examined and the reference substance separately in the minimum quantity of water R
and evaporate to dryness in a desiccator, under high vacuum over diphosphorus pentoxide R. Record
new spectra using the residues.
TESTS
Appearance of solution Dissolve 0.5 g in 50 ml of water R, heating if necessary. The solution is not
more intensely coloured than reference solution BY5 (Method II, 2.2.2).
solvent. The specific optical rotation, determined at 25C is +60.5 to +63.0, calculated with reference to the dried substance.
Related substances
A. Examine by thin-layer chromatography (2.2.27), using a plate coated with a suitable silica gel
containing a fluorescent indicator having an optimal intensity at 254 nm.
the same solvent.
Reference solution (a). Dissolve 20 mg each of thymine R, zidovudine impurity A CRS and
triphenylmethanol R in methanol R, add 1.0 ml of the test solution and dilute to 100 ml with
methanol R.
Reference solution (b). Dilute 5.0 ml of reference solution (a) to 10 ml with methanol R.
examine in ultraviolet light at 254 nm. In the chromatogram obtained with the test solution: any spot
corresponding to zidovudine impurity A is not more intense than the corresponding spot in the
chromatogram obtained with reference solution (b) (0.5 per cent), and any spot apart from the
principal spot and any spot corresponding to zidovudine impurity A and thymine (which is limited by
liquid chromatography) is not more intense than the spot corresponding to zidovudine in the chromatogram obtained with reference solution (b) (0.5 per cent). Spray the plate with a 10 g/l solution of
vanillin R in sulphuric acid R. In the chromatogram obtained with the test solution, any spot corres-
43-42
ponding to triphenylmethanol is not more intense than the corresponding spot in the chromatogram
obtained with reference solution (a) shows four clearly separated spots, corresponding to thymine,
zidovudine impurity A, zidovudine and triphenylmethanol, in order of increasing Rf value.
B. Examine by liquid chromatography as described in the assay. Inject separately 10 l each of test
solution (a), reference solution (b), reference solution (d) and reference solution (e). Continue the
chromatography for 1.5 times the retention time of the principal peak in the chromatogram obtained
with test solution (a). When the chromatograms are recorded in the prescribed conditions, the
substances are eluted in the following sequence: thymine, zidovudine and zidovudine impurity B. In
the chromatogram obtained with test solution (a): the area of any peak corresponding to thymine is
not greater than the area of the peak in the chromatogram obtained with reference solution (b) (2 per
cent); the area of any peak corresponding to zidovudine impurity B is not greater than the area of the
corresponding peak in the chromatogram obtained with reference solution (d) (1 per cent); the area
of any other peak apart from the principal peak is not greater than the area of the peak in the chromatogram obtained with reference solution (e) (0.5 per cent). The total surface area of all the peaks
apart from the principal peak obtained in the chromatogram with test solution (a) is not greater than
six times the area of the peak obtained with reference solution (e) (3.0 per cent). Disregard any peak
with an area less than 10 per cent of the area of the peak in the chromatogram obtained with reference solution (e).
100C to 105C.
ASSAY
Reference solution (a). Dissolve 10.0 mg of zidovudine CRS in the mobile phase and dilute to 50.0 ml
Reference solution (b). Dissolve 10.0 mg of thymine R in methanol R and dilute to 50.0 ml with the
same solvent. Dilute 5.0 ml of the solution to 50.0 ml with the mobile phase.
Reference solution (c). Dissolve 5 mg of zidovudine impurity B CRS in 25.0 ml of reference solution (a)
Reference solution (d). Dilute 5.0 ml of reference solution (c) to 50.0 ml with the mobile phase.
Reference solution (e). Dilute 0.25 ml of test solution (a) to 50.0 ml with the mobile phase.
as mobile phase at a flow rate of 1.2 ml per minute a mixture of 20 volumes of methanol R and
80 volumes of water R,
Inject 10 l of reference solution (c). Adjust the sensitivity of the detector so that the height of the
principal peaks in the chromatogram obtained is not less that 70 per cent of the full scale of the
recorder. The test is not valid unless the resolution between the peaks due to zidovudine and
zidovudine impurity B is at least 1.0. Inject separately 10 l of test solution (b) and 10 l of reference
solution (a). Adjust the sensitivity of the detector so that the height of the peaks in the chromatograms obtained is not less than 50 per cent of the full scale of the recorder.
Calculate the content of C10H13N5O4 from the areas of the peaks and the declared content of
zidovudine CRS.
STORAGE
43-43
IMPURITIES
O
Me
HN
O
HOCH2
O
A. 1-[(2R,5S)-5-hydroxymethyl-2,5-dihydro-2-furyl]-5-methylpyrimidine-2,4(1H,3H)-dione,
O
Me
HN
O
HOCH2
O
Cl
B. 1-(3-chloro-2,3-dideoxy--D-ribofuranosyl)-5-methylpyrimidine-2,4(1H,3H)-dione,
O
Me
HN
O
C. thymine,
Ph 3 COH
D. triphenylmethanol.
__________________________________________________________________________________________________________ Ph Eur
43-44
Zinc Acetate
C4H6O4Zn,2H2O
219.5
5970-45-6
Zinc Acetate complies with the requirements of the 3rd edition of the European Pharmacopoeia for Zinc
Acetate Dihydrate [1482]. These requirements are reproduced after the heading Definition below.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Zinc acetate dihydrate contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of C4H6O4Zn,2H2O.
CHARACTERS
A white crystalline powder or leaflets, freely soluble in water, soluble in alcohol.
IDENTIFICATION
A. It gives reaction (a) of acetate (2.3.1).
B. It gives the reaction of zinc (2.3.1).
TESTS
Reducing substances Boil for 5 min a mixture of 10 ml of solution S, 90 ml of water R, 5 ml of
dilute sulphuric acid R and 1.5 ml of a 0.3 g/l solution of potassium permanganate R. The pink colour of
the solution remains.
chlorides (50 ppm).
Aluminium Not more than 5 ppm of Al, determined by atomic absorption spectrometry (Method I,
2.2.23).
Test solution. Dissolve 2.50 g of the substance to be examined in 20 ml of a 200 g/l solution of
cadmium- and lead-free nitric acid R and dilute to 25.0 ml with the same acidic solution.
Reference solutions. Prepare the reference solutions using aluminium standard solution (200 ppm Al) R,
diluted with a 200 g/l solution of cadmium- and lead-free nitric acid R.
Measure the absorbance at 309.3 nm, using an aluminium hollow-cathode lamp as a source of
radiation and an air-acetylene flame.
Cadmium Not more than 2 ppm of Cd, determined by atomic absorption spectrometry (Method I,
2.2.23).
Test solution. Use the solution described in the test for aluminium.
Reference solutions. Prepare the reference solutions using cadmium standard solution (0.1 per cent Cd) R,
Measure the absorbance at 228.8 nm, using a cadmium hollow-cathode lamp as a source of radiation
Copper Not more than 50 ppm of Cu, determined by atomic absorption spectrometry (Method I,
2.2.23).
Test solution. Use the solution described in the test for iron.
Reference solutions. Prepare the reference solutions using copper standard solution (10 ppm Cu) R,
Measure the absorbance at 324.8 nm, using a copper hollow-cathode lamp as source of radiation and
Iron Not more than 50 ppm of Fe, determined by atomic absorption spectrometry (Method I,
2.2.23).
cadmium- and lead-free nitric acid R and dilute to 25.0 ml with the same acid solution.
43-45
Reference solutions. Prepare the reference solutions using iron standard solution (20 ppm Fe) R, diluted
with a 200 g/l solution of cadmium- and lead-free nitric acid R.
Measure the absorbance at 248.3 nm, using an iron hollow-cathode lamp as a source of radiation and
Lead Not more than 10 ppm of Pb, determined by atomic absorption spectrometry (Method I,
2.2.23).
cadmium- and lead-free nitric acid R and dilute to 25.0 ml with the same acid solution.
Reference solutions. Prepare the reference solutions using lead standard solution (0.1 per cent of Pb) R,
diluting with a 200 g/l solution of cadmium- and lead-free nitric acid R.
Measure the absorbance at 283.3 nm, using a lead hollow-cathode lamp as a source of radiation and
ASSAY
Dissolve 0.200 g in 5 ml of dilute acetic acid R. Carry out the complexometric titration of zinc
(2.5.11).
1 ml of 0.1M sodium edetate is equivalent to 21.95 mg of C4H6O4Zn,2H2O.
STORAGE
Store in a well-closed non metallic container.
__________________________________________________________________________________________________________ Ph Eur
43-46
Zinc Acexamate
O
H
N
Me
Zn
O
O
C16H28N2O6Zn
409.8
70020-71-2
Zinc Acexamate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1279].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Zinc acexamate contains not less than 97.5 per cent and not more than the equivalent of 101.0 per
cent of zinc 6-(acetylamino)hexanoate, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, soluble in water, practically insoluble in acetone and in
alcohol. It dissolves in dilute nitric acid.
IDENTIFICATION
obtained with zinc acexamate CRS. Examine the substances prepared as discs.
B. 5 ml of solution S (see Tests) gives the reaction of zinc (2.3.1).
TESTS
Appearance of solution Solution S is not more opalescent than reference suspension IV (2.2.1) and
6-Aminohexanoic acid Examine by thin-layer chromatography (2.2.27), using a suitable silica gel
as the coating substance.
same solvent.
Reference solution. Dissolve 15 mg of 6-aminohexanoic acid R in water R and dilute to 10 ml with the
Apply to the plate 5 l of the test solution and 5 l of the reference solution. Allow the plate to dry in
air. Develop over a path of 15 cm using a mixture of 2 volumes of ammonia R, 30 volumes of water R
and 68 volumes of alcohol R. Dry the plate in a current of warm air. Spray with ninhydrin solution R
and heat at 100C to 105C for 15 min. In the chromatogram obtained with the test solution, any
spot corresponding to 6-aminohexanoic acid is not more intense than the spot in the chromatogram
Test solution (a). Dissolve 0.50 g of the substance to be examined in water R and dilute to 100.0 ml
Test solution (b). To 20.0 ml of test solution (a), add 20 ml of the mobile phase, 0.4 ml of a 100 g/l
solution of phosphoric acid R and dilute to 50.0 ml with the mobile phase.
Reference solution (a). Dissolve 40 mg of N-acetyl--caprolactam R in water R and dilute to 100.0 ml
Reference solution (b). Dilute 5.0 ml of reference solution (a) to 100.0 ml with water R.
Reference solution (c). Dissolve 20 mg of zinc acexamate impurity A CRS in water R and dilute to
Reference solution (d). Dissolve 40 mg of caprolactam R in water R and dilute to 100.0 ml with the
Reference solution (e). To 20.0 ml of test solution (a), add 5.0 ml of reference solution (b), 5.0 ml of
reference solution (c), 5.0 ml of reference solution (d) and 0.4 ml of a 100 g/l solution of phosphoric
acid R and dilute to 50.0 ml with the mobile phase.
43-47
Reference solution (f). To 5.0 ml of reference solution (c), add 5.0 ml of reference solution (b), 5.0 ml
of reference solution (d) and 0.4 ml of a 100 g/l solution of phosphoric acid R and dilute to 50.0 ml
as mobile phase at a flow rate of 1.2 ml per minute a mixture of 0.2 volumes of phosphoric acid R,
8 volumes of acetonitrile R and 92 volumes of water R, adjusted to pH 4.5 with dilute
ammonia R1,
When the chromatograms are recorded in the prescribed conditions, the elution order is: zinc
acexamate, -caprolactam, zinc acexamate impurity A and N-acetyl--caprolactam. Inject 20 l of
reference solution (e). Continue the chromatography for eight times the retention time of zinc
acexamate. Adjust the sensitivity of the system so that the height of the peak corresponding to zinc
acexamate impurity A is at least 50 per cent of the full scale of the recorder. The test is not valid
unless the resolution between the first peak (zinc acexamate) and the second peak (-caprolactam) is
at least 3.0. If necessary adjust the pH of the mobile phase to 4.7 with dilute ammonia R1.
Inject 20 l of test solution (b) and 20 l of reference solution (f). In the chromatogram obtained
with test solution (b): the area of any peak corresponding to N-acetyl--caprolactam is not greater
than that of the corresponding peak in the chromatogram obtained with reference solution (f)
(0.1 per cent); the area of any peak corresponding to zinc acexamate impurity A is not greater than
that of the corresponding peak in the chromatogram obtained with reference solution (f) (2 per cent);
the area of any peak corresponding to -caprolactam is not greater than that of the corresponding
peak in the chromatogram obtained with reference solution (f) (0.1 per cent); the sum of the areas of
all the peaks, apart from the principal peak and the peak due to zinc acexamate impurity A is not
greater than five times the area of the peak due to N-acetyl--caprolactam in the chromatogram
obtained with reference solution (f) (0.5 per cent). Disregard any peak with an area less than 0.5
times that of the peak due to N-acetyl--caprolactam in the chromatogram obtained with reference
solution (f).
Cadmium Not more than 2 ppm of Cd, determined by atomic absorption spectrometry (Method I,
2.2.23).
Reference solutions. Prepare the reference solutions using cadmium standard solution (0.1 per cent of
Cd) R, diluted with a 200 g/l solution of cadmium- and lead-free nitric acid R.
Measure the absorbance at 228.8 nm, using a cadmium hollow-cathode lamp as a source of radiation
Iron Not more than 50 ppm of Fe, determined by atomic absorption spectrometry (Method I,
2.2.23).
Reference solutions. Prepare the reference solutions using iron standard solution (20 ppm Fe) R, diluted
with a 200 g/l solution of cadmium- and lead-free nitric acid R.
Measure the absorbance at 248.3 nm, using an iron hollow-cathode lamp as a source of radiation and
Lead Not more than 10 ppm of Pb, determined by atomic absorption spectrometry (Method I,
2.2.23).
Test solution. Dissolve 5.00 g of the substance to be examined in 20 ml of 200 g/l solution of
Reference solutions. Prepare the reference solutions using lead standard solution (0.1 per cent of Pb) R,
diluting with a 200 g/l solution of cadmium- and lead-free nitric acid R.
Measure the absorbance at 283.3 nm, using a lead hollow-cathode lamp as a source of radiation and
100C to 105C.
ASSAY
(2.5.11).
1 ml of 0.1M sodium edetate is equivalent to 40.98 mg of C16H28N2O6Zn.
43-48
STORAGE
Store in a well-closed, non-metallic container.
IMPURITIES
O
Me
H
N
N
H
COOH
A. 6-[[6-(acetylamino)hexanoyl]amino]hexanoic acid,
H2N
COOH
B. 6-aminohexanoic acid (6-aminocaproic acid),

O
O
Me
C. 1-acetylhexahydro-2H-azepin-2-one (N-acetyl--caprolactam),
O
HN
D. hexahydro-2H-azepin-2-one (-caprolactam).
__________________________________________________________________________________________________________ Ph Eur
43-49
Zinc Chloride
ZnCl2
136.3
7646-85-7
Zinc Chloride complies with the requirements of the 3rd edition of the European Pharmacopoeia [0110].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Zinc chloride contains not less than 95.0 per cent and not more than the equivalent of 100.5 per cent
of ZnCl2.
CHARACTERS
A white, crystalline powder or cast in white sticks, deliquescent, very soluble in water, freely soluble
in alcohol and in glycerol.
IDENTIFICATION
A. Dissolve 0.5 g in dilute nitric acid R and dilute to 10 ml with the same acid. The solution gives
reaction (a) of chlorides (2.3.1).
B. 5 ml of solution S (see Tests) gives the reaction of zinc (2.3.1).
TESTS
Solution S To 2.0 g add 38 ml of carbon dioxide-free water R prepared from distilled water R and add
dilute hydrochloric acid R dropwise until dissolution is complete. Dilute to 40 ml with carbon dioxidefree water R prepared from distilled water R.
pH (2.2.3). Dissolve 1.0 g in 9 ml of carbon dioxide-free water R, ignoring any slight turbidity. The pH
of the solution is 4.6 to 5.5.
Oxychlorides Dissolve 1.5 g in 1.5 ml of carbon dioxide-free water R. The solution is not more opalescent than reference suspension II (2.2.1). Add 7.5 ml of alcohol R. The solution may become cloudy
within 10 min. Any cloudiness disappears on the addition of 0.2 ml of dilute hydrochloric acid R.
test for sulphates (200 ppm). Prepare the standard using a mixture of 5 ml of sulphate standard solution (10 ppm SO4) R and 10 ml of distilled water R.
Aluminium, calcium, heavy metals, iron, magnesium To 8 ml of solution S add 2 ml of concentrated ammonia R and shake. The solution is clear (2.2.1) and colourless (Method II, 2.2.2). Add 1 ml
of disodium hydrogen phosphate solution R. The solution remains clear for at least 5 min. Add 0.2 ml of
sodium sulphide solution R. A white precipitate is formed and the supernatant liquid remains colourless.
Ammonium (2.4.1). 0.5 ml of solution S diluted to 15 ml with water R complies with the limit test
for ammonium (400 ppm).
ASSAY
(2.5.11).
1 ml of 0.1M sodium edetate is equivalent to 13.63 mg of ZnCl2.
STORAGE
Store in a non-metallic, well-closed container.
__________________________________________________________________________________________________________ Ph Eur
43-50
Zinc Oxide
ZnO
81.4
1341-13-2
Zinc Oxide complies with the requirements of the 3rd edition of the European Pharmacopoeia [0252]. These
Action and use Mild astringent.
Preparations
Zinc Cream
Coal Tar and Zinc Ointment
Zinc Ointment
Zinc and Castor Oil Ointment
Compound Zinc Paste
Zinc and Salicylic Acid Paste
Zinc and Coal Tar Paste
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Zinc oxide contains not less than 99.0 per cent and not more than the equivalent of 100.5 per cent of
ZnO, calculated with reference to the ignited substance.
CHARACTERS
A soft, white or faintly yellowish-white, amorphous powder, free from gritty particles, practically
insoluble in water and in alcohol. It dissolves in dilute mineral acids.
IDENTIFICATION
A. It becomes yellow when strongly heated; the yellow colour disappears on cooling.
B. Dissolve 0.1 g in 1.5 ml of dilute hydrochloric acid R and dilute to 5 ml with water R. The solution
gives the reaction of zinc (2.3.1).
TESTS
Alkalinity Shake 1.0 g with 10 ml of boiling water R. Add 0.1 ml of phenolphthalein solution R and
filter. If the filtrate is red, not more than 0.3 ml of 0.1M hydrochloric acid is required to change the
Carbonates and substances insoluble in acids Dissolve 1.0 g in 15 ml of dilute hydrochloric acid R.
It dissolves without effervescence and the solution is not more opalescent than reference suspension
II (2.2.1) and is colourless (Method II, 2.2.2).
Cadmium Not more than 10 ppm of Cd, determined by atomic absorption spectrophotometry
(Method II, 2.2.23).
Test solution. Dissolve 2.0 g of the substance to be examined in 14 ml of a mixture of equal volumes
of water R and cadmium- and lead-free nitric acid R, boil for 1 min, cool and dilute to 100.0 ml with
water R.
Reference solutions. Prepare the reference solutions using cadmium standard solution (0.1 per cent Cd) R
and diluting with a 3.5 per cent V/V solution of cadmium- and lead-free nitric acid R.
Measure the absorbance at 228.8 nm using a cadmium hollow-cathode lamp as source of radiation
and an air-acetylene or an air-propane flame.
Iron (2.4.9). Dissolve 50 mg in 1 ml of dilute hydrochloric acid R and dilute to 10 ml with water R.
The solution complies with the limit test for iron (200 ppm). Use in this test 0.5 ml of thioglycollic
acid R.
Lead Not more than 50 ppm of Pb, determined by atomic absorption spectrophotometry (Method II,
2.2.23).
Test solution. Dissolve 5.0 g in 24 ml of a mixture of equal volumes of water R and cadmium- and leadfree nitric acid R, boil for 1 min, cool and dilute to 100.0 ml with water R.
Reference solutions. Prepare the reference solutions using lead standard solution (0.1 per cent Pb) R and
diluting with a 3.5 per cent V/V solution of cadmium- and lead-free nitric acid R.
Measure the absorbance at 283.3 nm using a lead hollow-cathode lamp as source of radiation and an
air-acetylene flame. Depending on the apparatus, the line at 217.0 nm may be used.
43-51
ASSAY
(2.5.11).
1 ml of 0.1M sodium edetate is equivalent to 8.14 mg of ZnO.
__________________________________________________________________________________________________________ Ph Eur
43-52
Zinc Stearate
(C17H35CO2)2Zn
632
557-05-1
Zinc Stearate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0306].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Zinc stearate [(C17H35COO)2Zn; Mr 632] may contain varying proportions of zinc palmitate
[(C15H31COO)2Zn; Mr 576.2] and zinc oleate [(C17H33COO)2Zn; Mr 628]. It contains not less than
10.0 per cent and not more than 12.0 per cent of Zn.
CHARACTERS
A light, white, amorphous powder, free from gritty particles, practically insoluble in water, in ethanol
and in ether.
IDENTIFICATION
A. The residue obtained in the preparation of solution S (see Tests) has a freezing point (2.2.18) not
lower than 53C.
B. Neutralise 5 ml of solution S to red litmus paper R with strong sodium hydroxide solution R. The
solution gives the reaction of zinc (2.3.1).
TESTS
Solution S To 5.0 g add 50 ml of ether R and 40 ml of a 7.5 per cent V/V solution of cadmium- and
lead-free nitric acid R in distilled water R. Heat under a reflux condenser until dissolution is complete.
Allow to cool. In a separating funnel, separate the aqueous layer and shake the ether layer with two
quantities, each of 4 ml, of distilled water R. Combine the aqueous layers, wash with 15 ml of ether R
and heat on a water-bath until ether is completely eliminated. Allow to cool and dilute to 50.0 ml
with distilled water R (solution S). Evaporate the ether layer to dryness and dry the residue at 105C.
Appearance of solution Solution S is not more intensely coloured than reference solution Y6
(Method II, 2.2.2).
Appearance of solution of fatty acids Dissolve 0.5 g of the residue obtained in the preparation of
solution S in 10 ml of chloroform R. The solution is clear (2.2.1) and not more intensely coloured than
Acidity or alkalinity Shake 1.0 g with 5 ml of alcohol R and add 20 ml of carbon dioxide-free water R
and 0.1 ml of phenol red solution R. Not more than 0.3 ml of 0.1M hydrochloric acid or 0.1 ml of 0.1M
Acid value of the fatty acids (2.5.1). 195 to 210, determined on 0.20 g of the residue obtained in
the preparation of solution S, dissolved in 25 ml of the prescribed mixture of solvents.
Sulphates (2.4.13). Dilute 1 ml of solution S to 50 ml with distilled water R. 12.5 ml of the solution
diluted to 15 ml with distilled water R complies with the limit test for sulphates (0.6 per cent).
Cadmium Not more than 5 ppm of Cd, determined by atomic absorption spectrophotometry
(Method II, 2.2.23).
Test solution. Dilute 20.0 ml of solution S to 50.0 ml with a 3.5 per cent V/V solution of cadmiumand lead-free nitric acid R.
Reference solutions. Prepare the reference solutions using cadmium standard solution (0.1 per cent Cd) R
and diluting with a 3.5 per cent V/V solution of cadmium- and lead-free nitric acid R.
Measure the absorbance at 228.8 nm using a cadmium hollow-cathode lamp as source of radiation
and an air-acetylene or an air-propane flame.
Lead Not more than 25 ppm of Pb, determined by atomic absorption spectrophotometry (Method II,
2.2.23).
Test solution. Use solution S.
Reference solutions. Prepare the reference solutions using lead standard solution (0.1 per cent Pb) R and
diluting with a 3.5 per cent V/V solution of cadmium- and lead-free nitric acid R.
Measure the absorbance at 283.3 nm using a lead hollow-cathode lamp as source of radiation and an
air-acetylene flame. Depending on the apparatus the line at 217.0 nm may be used.
43-53
ASSAY
To 1.000 g add 50 ml of dilute acetic acid R and boil for at least 10 min or until the layer of fatty acids
is clear, adding more water R as necessary to maintain the original volume. Cool and filter. Wash the
filter and the flask with water R until the washings are no longer acid to blue litmus paper R. Combine
the filtrate and washings. Carry out the complexometric titration of zinc (2.5.11).
1 ml of 0.1M sodium edetate is equivalent to 6.54 mg of Zn.
__________________________________________________________________________________________________________ Ph Eur
43-54
Zinc Sulphate
ZnSO4,7H2O
287.5
7446-20-0
Zinc Sulphate complies with the requirements of the 3rd edition of the European Pharmacopoeia [0111].
Preparations
Zinc Sulphate Eye Drops
Zinc Sulphate Lotion
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Zinc sulphate contains not less than 99.0 per cent and not more than the equivalent of 104.0 per cent
of ZnSO4,7H20.
CHARACTERS
A white, crystalline powder or colourless, transparent crystals, efflorescent, very soluble in water,
practically insoluble in alcohol.
IDENTIFICATION
A. Solution S (see Tests) gives the reactions of sulphates (2.3.1).
B. Solution S gives the reaction of zinc (2.3.1).
TESTS
(100 ppm). Use in this test 0.5 ml of thioglycollic acid R.
ASSAY
(2.5.11).
1 ml of 0.1M sodium edetate is equivalent to 28.75 mg of ZnSO4,7H2O.
STORAGE
Store in a non-metallic, well-closed container.
__________________________________________________________________________________________________________ Ph Eur
43-55
Zinc Undecenoate
H
COO
H 2C
C 22H38O4Zn
Zn 2+
2
431.9
557-08-4
Zinc Undecenoate complies with the requirements of the 3rd edition of the European Pharmacopoeia for Zinc
Undecylenate [0539]. These requirements are reproduced after the heading Definition below.
Action and use Used topically in treatment of fungal infections.
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Zinc undecylenate contains not less than 98.0 per cent and not more than the equivalent of 102.0 per
cent of zinc di(undec-10-enoate), calculated with reference to the dried substance.
CHARACTERS
A white or almost white, fine powder, practically insoluble in water, in alcohol and in ether.
It melts at 116C to 121C but may leave a slight solid residue.
IDENTIFICATION
A. To 2.5 g add 10 ml of water R and 10 ml of dilute sulphuric acid R. Shake with two quantities, each
of 10 ml, of ether R. Reserve the aqueous layer for identification test C. Wash the combined ether
layers with water R and evaporate to dryness. To the residue add 2 ml of freshly distilled aniline R and
boil under a reflux condenser for 10 min. Allow to cool and add 30 ml of ether R. Shake with three
quantities, each of 20 ml, of dilute hydrochloric acid R and then with 20 ml of water R. Evaporate the
organic layer to dryness on a water-bath. The residue, after recrystallisation twice from alcohol (70 per
cent V/V) R and drying in vacuo for 3 h, melts (2.2.14) at 66C to 68C.
B. Dissolve 0.1 g in a mixture of 2 ml of dilute sulphuric acid R and 5 ml of glacial acetic acid R. Add
dropwise 0.25 ml of potassium permanganate solution R. The colour of the potassium permanganate is
discharged.
C. A mixture of 1 ml of the aqueous layer obtained in identification test A and 4 ml of water R gives
the reaction of zinc (2.3.1).
TESTS
Alkalinity Mix 1.0 g with 5 ml of alcohol R and 0.5 ml of phenol red solution R. Add 50 ml of carbon
dioxide-free water R and examine immediately. No reddish colour appears.
Alkali and alkaline-earth metals To 1.0 g add 25 ml of water R and 5 ml of hydrochloric acid R and
heat to boiling. Filter whilst hot. Wash the filter and the residue with 25 ml of hot water R. Combine
the filtrate and washings and add concentrated ammonia R until alkaline. Add 7.5 ml of thioacetamide
solution R and heat on a water-bath for 30 min. Filter and wash the precipitate with two quantities,
each of 10 ml, of water R. Combine the filtrate and washings, evaporate to dryness on a water-bath
and ignite. The residue weighs not more than 20 mg (2 per cent).
Sulphates (2.4.13). To 0.1 g add a mixture of 2 ml of dilute hydrochloric acid R and 10 ml of distilled
water R and heat to boiling. Cool, filter and dilute to 15 ml with distilled water R. The solution
complies with the limit test for sulphates (500 ppm). Prepare the standard using 5 ml of sulphate
standard solution (10 ppm SO4) R and 10 ml of distilled water R.
100C to 105C.
Degree of unsaturation Dissolve 0.100 g in a mixture of 5 ml of dilute hydrochloric acid R and 30 ml
of glacial acetic acid R. Using 0.05 ml of indigo carmine solution R1, added towards the end of the
titration as indicator, titrate with 0.0167M bromide-bromate until the colour changes from blue to
yellow. 9.1 ml to 9.4 ml of 0.0167M bromide-bromate is required. Carry out a blank titration.
ASSAY
To 0.350 g add 25 ml of dilute acetic acid R and heat to boiling. Carry out the complexometric titration of zinc (2.5.11).
1 ml of 0.1M sodium edetate is equivalent to 43.19 mg of C22H38O4Zn.
STORAGE
__________________________________________________________________________________________________________ Ph Eur
43-56
Zolpidem Tartrate
Me
N
H OH
, HOOC
N
Me
COOH
H OH
(C19H21N3O)2,C4H6O6
NMe2
764.9
99294-93-6
Zolpidem Tartrate complies with the requirements of the 3rd edition of the European Pharmacopoeia [1280].
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Zolpidem tartrate contains not less than 98.5 per cent and not more than the equivalent of 101.0 per
cent of bis[N,N-dimethyl-2-[6-methyl-2-(4-methylphenyl)imidazo[1,2-a]pyridin-3-yl]acetamide]
(2R,3R)-2,3-dihydroxybutanedioate, calculated with reference to the anhydrous substance.
CHARACTERS
A white or almost white, crystalline powder, hygroscopic, slightly soluble in water, sparingly soluble
in methanol, practically insoluble in methylene chloride.
IDENTIFICATION
First identification: A, C
A. Dissolve 0.10 g in 10 ml of 0.1M hydrochloric acid. Add 10 ml of water R. Add dropwise with
stirring 1 ml of dilute ammonia R2. Filter and collect the resulting precipitate. Wash the precipitate
with water R and then dry at 100C to 105C for 2 h (test precipitate). Carry out the same operation
using zolpidem tartrate CRS (reference precipitate). Examine the precipitates by infrared absorption
spectrophotometry (2.2.24) comparing the spectra obtained. Examine the precipitates as discs.
Test solution. Dissolve 50 mg of the substance to be examined in 5 ml of methanol R, add 0.1 ml of
diethylamine R and dilute to 10 ml with methanol R.
Reference solution (a). Dissolve 50 mg of zolpidem tartrate CRS in 5 ml of methanol R, add 0.1 ml of
diethylamine R and dilute to 10 ml with methanol R.
Reference solution (b). Dissolve 50 mg of flunitrazepam CRS in 5 ml of methylene chloride R and dilute
to 10 ml with the same solvent. Mix 1 ml of the solution with 1 ml of reference solution (a).
10 volumes of diethylamine R, 45 volumes of cyclohexane R and 45 volumes of ethyl acetate R. Allow
the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained
C. Dissolve about 0.1 g in 1 ml of methanol R heating gently. 0.1 ml of the solution gives reaction (b)
of tartrates (2.3.1).
TESTS
Appearance of solution Prepare the solutions protected from light and carry out the test as rapidly as
possible. Triturate 0.25 g with 0.125 g of tartaric acid R. Dissolve the mixture in 10 ml of water R and
dilute to 25 ml with the same solvent. The solution is clear (2.2.1) and not more intensely coloured
than reference solution Y6 or BY6 (Method II, 2.2.2).
Reference solution (a). Dissolve 5 mg of zolpidem impurity A CRS in the mobile phase and dilute to
50 ml with the mobile phase.
Reference solution (b). Dissolve 5 mg of the substance to be examined in the mobile phase and dilute
43-57
to 50 ml with the mobile phase. To 10 ml of the solution, add 10 ml of reference solution (a).
Reference solution (c). Dilute 2.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute
as mobile phase at a flow rate of 1.5 ml/min a mixture of 18 volumes of acetonitrile R, 23
volumes of methanol R and 59 volumes of a 5.6 g/l solution of phosphoric acid R adjusted to pH
5.5 with triethylamine R,
peak due to zolpidem impurity A is at least 50 per cent of the full scale of the recorder. The test is not
valid unless the resolution between the peaks due to zolpidem impurity A and zolpidem tartrate is at
least 2.0.
Inject 20 l of the test solution and 20 l of reference solution (c). In the chromatogram obtained
with the test solution, the sum of the areas of any peaks, apart from the principal peak, is not greater
than the area of the principal peak in the chromatogram obtained with reference solution (c) (0.2 per
cent). Disregard any peak with an area less than 0.1 times that of the principal peak in the chromatogram obtained with reference solution (c) and any peak (with a relative retention time of 0.16 to the
zolpidem peak) corresponding to tartaric acid.
of water.
ASSAY
Titrate with 0.1M perchloric acid, determining the end-point potentiometrically (2.2.20). Carry out a
blank titration.
STORAGE
IMPURITIES
Me
Me
N
N
O
NMe2
A. N,N-dimethyl-2-[7-methyl-2-(4-methylphenyl)imidazo[1,2-a]pyridin-3-yl]acetamide.
__________________________________________________________________________________________________________ Ph Eur
43-58
Zopiclone
Cl
O
N
N
N
N
O
H
N
MeN
and enantiomer
C17H17ClN6O3
388.8
43200-80-2
Zopiclone complies with the requirements of the 3rd edition of the European Pharmacopoeia [1060]. These
Ph Eur ___________________________________________________________________________________________________________
DEFINITION
Zopiclone contains not less than 98.5 per cent and not more than the equivalent of 100.5 per cent of
(5RS)-6-(5-chloropyridin-2-yl)-7-oxo-6,7-dihydro-5H-pyrrolo[3,4-b]pyrazin-5-yl 4methylpiperazine-1-carboxylate, calculated with reference to the solvent-free substance.
CHARACTERS
A white or slightly yellowish powder, practically insoluble in water, freely soluble in methylene
chloride, sparingly soluble in acetone, practically insoluble in alcohol. It dissolves in dilute mineral
acids.
IDENTIFICATION
Second identification: A, C
A. Dissolve 50.0 mg in a 3.5 g/l solution of hydrochloric acid R and dilute to 100.0 ml with the same
solvent. Dilute 2.0 ml of this solution to 100.0 ml with a 3.5 g/l solution of hydrochloric acid R. Examined between 220 nm and 350 nm (2.2.25), the solution shows an absorption maximum at 303 nm.
obtained with zopiclone CRS. Examine the substances prepared as discs.
Reference solution. Dissolve 10 mg of zopiclone CRS in methylene chloride R and dilute to 10 ml with the
same solvent.
2 volumes of triethylamine R, 50 volumes of acetone R and 50 volumes of ethyl acetate R. Allow the
obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution.
TESTS
Solution S Dissolve 1.0 g in dimethylformamide R and dilute to 20.0 ml with the same solvent.
not more intensely coloured than intensity 5 of the range of reference solutions of the most appropriate colour (Method II, 2.2.2).
Optical rotation (2.2.7). Dilute 10.0 ml of solution S to 50.0 ml with dimethylformamide R. The
angle of optical rotation is 0.05 to + 0.05.
43-59
before use.
Reference solution (c). Dissolve 10.0 mg of zopiclone oxide CRS in the mobile phase and dilute to
25.0 ml with the mobile phase. To 10.0 ml of this solution, add 1.0 ml of the test solution and dilute
62 volumes of a solution containing 8.1 g/l of sodium lauryl sulphate R and 1.6 g/l of sodium
dihydrogen phosphate R adjusted to pH 3.5 with a 10 per cent V/V solution of phosphoric acid R,
Inject 20 l of reference solution (c). Adjust the sensitivity of the system so that the height of each
of the two principal peaks in the chromatogram obtained is at least 30 per cent of the full scale of the
recorder. When the chromatograms are recorded in the prescribed conditions, the retention time of
zopiclone is 27 min to 31 min. If necessary, adjust the concentration of acetonitrile in the mobile
phase (increasing the concentration decreases the retention times and decreasing the concentration
increases the retention times). The test is not valid unless, in the chromatogram obtained with reference solution (c), the resolution between the peaks due to zopiclone oxide and zopiclone is at least
3.0. If the prescribed resolution is not obtained, adjust the mobile phase to pH 4.0 with a 10 per cent
V/V solution of phosphoric acid R.
Continue the chromatography for 1.5 times the retention time of zopiclone. In the chromatogram
the area of the principal peak in the chromatogram obtained with reference solution (a) (0.3 per cent)
and not more than two such peaks have an area greater than the area of the principal peak in the
2-Propanol Not more than 0.7 per cent m/m of 2-propanol, determined by gas chromatography
(2.2.28) using ethanol R1 as the internal standard.
Internal standard solution. Dilute 5 ml of ethanol R1 to 100 ml with ethylene chloride R. Dilute 1 ml of
this solution to 10 ml with ethylene chloride R.
Test solution. Dissolve 0.25 g of the substance to be examined in ethylene chloride R, add 0.5 ml of the
internal standard solution and dilute to 5.0 ml with ethylene chloride R.
Reference solution. Dilute 4.5 ml of 2-propanol R to 100.0 ml with ethylene chloride R. To 1.0 ml of this
solution, add 10.0 ml of the internal standard solution and dilute to 100.0 ml with ethylene chloride R.
a fused-silica column 10 m long and about 0.53 mm in internal diameter with a 20 m coating
of styrene-divinylbenzene copolymer R,
Column
Injection port
Detector
Time
(min)
Temp.
(C)
Rate
(C/min)
Comment
05
510
50
5070
1014
1420.5
70
70200
20
20.527.5
200
isothermal
linear
gradient
isothermal
linear
gradient
isothermal
150
250
43-60
Inject 1 l of the test solution and 1 l of the reference solution. Calculate the percentage content
m/m of 2-propanol taking its density to be 0.785 g/ml at 20C.
ASSAY
Dissolve 0.300 g in a mixture of 10 ml of acetic acid R and 40 ml of acetic anhydride R. Titrate with
STORAGE
IMPURITIES
Cl
N
R=
O
H N
N
N
O
N
O N
OR
and enantiomer
Me
A. (5RS)-6-(5-chloropyridin-2-yl)-7-oxo-6,7-dihydro-5H-pyrrolol[3,4-b]pyrazin-5-yl 4methylpiperazine-1-carboxylate 4-oxide (zopiclone oxide)
HO
and enantiomer
B. (7RS)-6-(5-chloropyridin-2-yl)-7-hydroxy-6,7-dihydro-5 H-pyrrolo[3,4-b]pyrazin-5-one,
H
C. 6-(5-chloropyridin-2-yl)-6,7-dihydro-5H-pyrrolo[3,4-b]pyrazin-5-one.
__________________________________________________________________________________________________________ Ph Eur
43-61
Zuclopenthixol Acetate
OAc
N
H
N
Cl
S
C24H27ClN2O2S
443.0
85721-05-7
Definition Zuclopenthixol Acetate is (Z)-2-{4-[3-(2-chlorothioxanthene-9-ylidene)propyl]piperazin1-yl}ethyl acetate. It contains not less than 98.0% and not more than 102.0% of C24H27ClN2O2S,
Characteristics A yellowish, viscous oil.
Very slightly soluble in water; very soluble in dichloromethane, in ethanol (96%) and in ether.
Identification
A. The light absorption of a 0.0015% w/v solution in ethanol (96%), Appendix II B, in the range 210
to 350 nm exhibits two maxima at 230 and 268 nm. The absorbances at the maxima are about 1.18
and 0.51 respectively.
zuclopenthixol acetate (RS 363).
from light, using a silica gel F254 precoated plate (Merck silica gel 60 F254 plates are suitable), a
mixture of 10 volumes of diethylamine, 40 volumes of dichloromethane and 50 volumes of cyclohexane
as the mobile phase but using an unlined tank. Apply separately to the plate 4 l of each of the
following solutions. Solutions (1) to (3) contain (1) 0.250% w/v, (2) 0.00025% w/v and (3)
0.000125% w/v respectively of the substance being examined in dichloromethane. Solution (4)
contains 0.00050% w/v of 2-chlorothioxanthone BPCRS in dichloromethane. Solution (5) contains
0.000625% w/v of zuclopenthixol hydrochloride BPCRS in dichloromethane containing a few drops of
diethylamine. After removal of the plate, allow it to dry in air, spray with a mixture of equal volumes
of sulphuric acid and absolute ethanol, heat at 110 for 5 minutes and examine under ultraviolet light
(365 nm) immediately. In the chromatogram obtained with solution (1) any spot corresponding to
2-chlorothioxanthone is not more intense than the spot in the chromatogram obtained with solution
(4) (0.2%) and any spot corresponding to zuclopenthixol is not more intense than the spot in the
chromatogram obtained with solution (5) (0.25%). Any other secondary spot in the chromatogram
obtained with solution (1) is not more intense than the spot in the chromatogram obtained with
solution (2) (0.1%) and not more than three such spots are more intense than the spot in the chromatogram obtained with solution (3) (0.05%).
trans-Isomer Carry out the method for liquid chromatography, Appendix III D, protected from light,
using three solutions in dichloromethane containing (1) 0.040% w/v of the substance being examined,
(2) 0.00046% w/v of trans-clopenthixol acetate dihydrochloride BPCRS (equivalent to 0.00040% w/v of
trans-clopenthixol acetate) and (3) 0.020% w/v of the substance being examined and 0.023% w/v of
trans-clopenthixol acetate dihydrochloride BPCRS.
(25 cm 4.6 mm) packed with stationary phase A (5 m) (Spherisorb S 5W is suitable), (b) a mixture
of 0.03 volume of 13.5M ammonia, 45 volumes of dichloromethane, 45 volumes of heptane and 50
volumes of acetonitrile as the mobile phase with a flow rate of 2 ml per minute and (c) a detection
Inject 15 l of each solution. The test is not valid unless the resolution factor between the principal
peaks in the chromatogram obtained with solution (3) is at least 2.6.
In the chromatogram obtained with solution (1) the area of any peak corresponding to transclopenthixol acetate is not greater than the area of the peak in the chromatogram obtained with
solution (2) (1%).
Assay Dissolve 0.2 g in 50 ml of anhydrous acetic acid and carry out Method I for non-aqueous titra-
43-62
tion, Appendix VIII A, determining the end point potentiometrically. Each ml of 0.1M perchloric acid
VS is equivalent to 22.15 mg of C24H27ClN2O2S.
Storage Zuclopenthixol Acetate should be kept in a well-closed container, protected from light and
Preparation
Zuclopenthixol Acetate Injection
IMPURITIES
CH3[CH2] 8COO
N
H
Cl
S
A. trans-clopenthixol acetate(trans-isomer)
O
Cl
S
B. 2-chlorothioxanthone
N
H
Cl
S
C. zuclopenthixol
OH
43-63
Zuclopenthixol Decanoate
N
H
OCO[CH2] 8CH3
N
Cl
S
C32H43ClN2O2S
555.3
64053-00-5
Definition Zuclopenthixol Decanoate is (Z)-2-{4-[3-(2-chlorothioxanthene-9ylidene)propyl]piperazin-1-yl}ethyl decanoate. It contains not less than 98.0% and not more than
102.0% of C32H43ClN2O2S, calculated with reference to the dried substance.
Characteristics A yellow, viscous oil.
Very slightly soluble in water; very soluble in dichloromethane, in ethanol (96%) and in ether.
Identification
and 0.36 respectively.
zuclopenthixol decanoate (RS 364). Examine as a mull in liquid paraffin.
mixture of 3 volumes of diethylamine, 10 volumes of propan-1-ol and 90 volumes of cyclohexane as the
mobile phase but using an unlined tank. Apply separately to the plate 4 l of each of the following
solutions. Solutions (1) to (3) contain (1) 0.250% w/v, (2) 0.00025% w/v and (3) 0.000125% w/v
respectively of the substance being examined in dichloromethane. Solution (4) contains 0.00050% w/v
of 2-chlorothioxanthone BPCRS in dichloromethane. Solution (5) contains 0.000625% w/v of
zuclopenthixol hydrochloride BPCRS in dichloromethane containing a few drops of diethylamine. After
removal of the plate, allow it to dry in air, spray with a mixture of equal volumes of sulphuric acid and
absolute ethanol, heat at 110 for 5 minutes and examine under ultraviolet light (365 nm) immediately.
In the chromatogram obtained with solution (1) any spot corresponding to 2-chlorothioxanthone is
not more intense than the spot in the chromatogram obtained with solution (4) (0.2%) and any spot
corresponding to zuclopenthixol is not more intense than the spot in the chromatogram obtained
with solution (5) (0.25%). Any other secondary spots in the chromatogram obtained with solution (1)
are not more intense than the spot in the chromatogram obtained with solution (2) (0.1%) and not
more than three such spots are more intense than the spot in the chromatogram obtained with solution (3) (0.05% each).
using the following solutions in ethanol (96%). Solution (1) contains 0.040% w/v of the substance
being examined. Solution (2) contains 0.00057% w/v of trans-clopenthixol decanoate dihydrochloride
BPCRS (equivalent to 0.00050% w/v of trans-clopenthixol decanoate). Solution (3) contains 0.020%
w/v of the substance being examined and 0.028% w/v of trans-clopenthixol decanoate dihydrochloride
BPCRS.
(15 cm 4.6 mm) packed with stationary phase C (5 m) (Waters Symmetry C18 is suitable), (b) as
the mobile phase with a flow rate of 1 ml per minute a mixture of 25 volumes of 20 mM dioctyl sodium
sulphosuccinate and 75 volumes of ethanol (96%), the mixture containing 0.1% v/v of orthophosphoric
acid and (c) a detection wavelength of 230 nm, maintaining the column temperature at 40.
Inject 20 l of each solution. The test is not valid unless the resolution factor between the principal
peaks in the chromatogram obtained with solution (3) is at least 1.8.
In the chromatogram obtained with solution (1) the area of any peak corresponding to transclopenthixol decanoate is not greater than the area of the peak in the chromatogram obtained with
solution (2) (1.25%).
Loss on drying When dried at 60 at a pressure not exceeding 0.7 kPa, loses not more than 0.5% of
its weight. Use 1 g.
43-64
Assay Dissolve 0.2 g in 50 ml of anhydrous acetic acid and carry out Method I for non-aqueous titration, Appendix VIII A, determining the end point potentiometrically. Each ml of 0.1M perchloric acid
VS is equivalent to 27.76 mg of C32H43ClN2O2S.
Storage Zuclopenthixol Decanoate should be kept in a well-closed container, protected from light
and stored at a temperature not exceeding 20.
Preparation
Zuclopenthixol Decanoate Injection
IMPURITIES
CH3[CH2]8COO
N
H
Cl
S
A. trans-clopenthixol decanoate(trans-isomer)
O
Cl
S
B. 2-chlorothioxanthone
N
H
Cl
S
C. zuclopenthixol
OH
43-65
Zuclopenthixol Hydrochloride
OH
N
H
N
Cl
,2HCl
S
C22H25ClN2OS,2HCl
473.9
633-59-0
Definition Zuclopenthixol Hydrochloride is (Z)-2-{4-[3-(2-chlorothioxanthene-9-ylidene)propyl]piperazin-l-yl}ethanol dihydrochloride. It contains not less than 98.0% and not more than 101.0% of
C22H25ClN2OS,2HCl, calculated with reference to the anhydrous substance.
Characteristics An off-white, granular powder.
Very soluble in water; sparingly soluble in ethanol (96%); slightly soluble in chloroform; very slightly
soluble in ether.
Identification
and 0.42, respectively.
zuclopenthixol hydrochloride (RS 365).
mixture of 30 volumes of dichloromethane and 70 volumes of toluene as the mobile phase but using an
unlined tank and allowing the mobile phase to ascend 10 cm above the line of application. Apply
separately to the plate 4 l of each of the following solutions. Solutions (1) to (3) contain (1) 2.5%
w/v, (2) 0.0025% w/v and (3) 0.00125% w/v respectively of the substance being examined in dichloromethane containing a few drops of diethylamine. Solution (4) contains 0.0025% w/v of 2-chlorothioxanthone BPCRS in dichloromethane. Solution (5) contains 0.0025% w/v of 9-allyl-2-chlorothioxanthen9-ol BPCRS in dichloromethane. After removal of the plate, allow it to dry in air, spray with a mixture
of equal volumes of sulphuric acid and absolute ethanol, heat at 110 for 5 minutes and examine under
ultraviolet light (365 nm) immediately. In the chromatogram obtained with solution (1) any spots
corresponding to 2-chlorothioxanthone and to 9-allyl-2-chlorothioxanthenol are not more intense
than the spots in the chromatograms obtained with solutions (4) and (5) respectively (0.1% each),
any other secondary spot is not more intense than the spot in the chromatogram obtained with solution
(2) (0.1%) and not more than four such spots are more intense than the spot in the chromatogram
obtained with solution (3) (0.05%).
Free amine Carry out the method for thin-layer chromatography, Appendix III A, protected from
light, using a silica gel F254 precoated plate (Merck silica gel 60 F254 plates are suitable) and a
mixture of 2 volumes of water, 10 volumes of 13.5M ammonia, 20 volumes of butan-1-ol and 65
volumes of acetone as the mobile phase. Apply separately to the plate 4 l of each of the following
solutions. Solution (1) is a 0.25% w/v solution of the substance being examined in dichloromethane;
add a few drops of diethylamine to aid dissolution. For solution (2), dilute 1 volume of solution (1) to
400 volumes with dichloromethane. After removal of the plate, allow it to dry in air, spray with a
mixture of equal volumes of sulphuric acid and absolute ethanol, heat at 110 for 5 minutes and examine under ultraviolet light (365 nm) immediately. In the chromatogram obtained with solution (1) any
secondary spot of the same colour and at an Rf value lower than that of the principal spot is not more
intense than the spot in the chromatogram obtained solution (2) (0.25%).
using the following solutions. For solution (1) dissolve 10 mg of the substance being examined in
5 ml of water, add 25 ml of the mobile phase, shake thoroughly for 1 minute and dilute to 50 ml with
the mobile phase; use the upper layer. For solution (2) prepare a 0.004% w/v solution of transclopenthixol hydrochloride BPCRS in water, shake 5 ml of this solution with 25 ml of the mobile phase
for 1 minute and dilute to 50 ml with the mobile phase; use the upper layer. For solution (3) dissolve
43-66
10 mg of the substance being examined in 5 ml of a 0.004% w/v solution of trans-clopenthixol hydrochloride BPCRS, add 25 ml of the mobile phase, shake thoroughly for 1 minute, dilute to 50 ml with
the mobile phase and use the upper layer.
(25 cm 4.6 mm) packed with stationary phase A (5 m) (Spherisorb S 5W is suitable), (b) a mixture
of 0.2 volume of water, 0.3 volume of 13.5M ammonia, 15 volumes of propan-2-ol and 85 volumes of
n-heptane as the mobile phase with a flow rate of 1.5 ml per minute and (c) and a detection wavelength of 254 nm.
The test is not valid unless the chromatogram obtained with solution (3) shows a similar resolution
to that in the reference chromatogram supplied with trans-clopenthixol hydrochloride BPCRS.
In the chromatogram obtained with solution (1) the area of any peak corresponding to transclopenthixol hydrochloride is not greater than 1.5 times the area of the peak in the chromatogram
obtained with solution (2) (3%).
Water Not more than 2.5%, determined on 0.5 g, Appendix IX C.
Assay Dissolve 0.5 g in 50 ml of anhydrous acetic acid and carry out Method I for non-aqueous titration, Appendix VIII A, using crystal violet solution as indicator. Each ml of 0.1M perchloric acid VS is
equivalent to 23.70 mg of C22H27Cl3N2OS.
Storage Zuclopenthixol Hydrochloride should be kept in a well-closed container and protected from
light.
Preparation
Zuclopenthixol Tablets
IMPURITIES
2+
A. 2-chloro-9-(1-hydroxy-3-{(4-(2-hydroxyethyl)]piperazin-1-yl}propyl)thioxanthen-9-ol,
HO
N
N
H
Cl
S
B. trans-clopenthixol,
O
Cl
S
C. 2-chlorothioxanthone,
CH2
HO
Cl
S
D. 9-allyl-2-chlorothioxanthen-9-ol,
CH2
Cl
S
E. 9-allylidene-2-chlorothioxanthene,
43-67
NH
H
N
Cl
S
F. 2-chloro-9-(3-piperazin-1-ylpropylidene)thioxanthene.

British Pharmacopea - 02 PDF

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24-1

C. (S)-2-[(3S,8aS)-3-(4-aminobutyl)-1,4-dioxo-1,2,3,4,6,7,8,8a-octahydropyrrolo[1,2a]piperazin-2-yl]-4-phenylbutanoic acid ((S,S,S)-diketopiperazine),

D. (S)-2-[(3S,8aR)-3-(4-aminobutyl)-1,4-dioxo-1,2,3,4,6,7,8,8a-octahydropyrrolo[1,2a]piperazin-2-yl]-4-phenylbutanoic acid ((R,S,S)-diketopiperazine),

E. N-[N-[(R)-1-carboxy-3-phenylpropyl)-L-lysyl]-L-proline (lisinopril (R,S,S)-isomer),

F. N-[N-[(S)-1-carboxy-3-cyclohexylpropyl)-L-lysyl]-L-proline (cyclohexyl analogue).

Definition Loprazolam Mesilate is (Z)-6-(2-chlorophenyl)-2,4-dihydro-2-(4-methylpiperazin-1ylmethylene)-8-nitroimidazo[1,2-a][1,4]benzodiazepin-1-one methanesulphonate monohydrate. It

as detector a spectrophotometer set at 238 nm.

A. (1S,7S,8S,8aR)-8-[2-[(2R,4R)-4-hydroxy-6-oxotetrahydro-2H-pyran-2-yl]ethyl]-7-methyl1,2,3,7,8,8a-hexahydronaphthalen-1-yl (2S)-2-methylbutanoate (mevastatin),

B. (3R,5R)-7-[(1S,2S,6R,8S,8aR)-2,6-dimethyl-8-[[(2S)-2-methylbutanoyl]oxy]1,2,6,7,8,8a-hexahydronaphthalen-1-yl]-3,5-dihydroxyheptanoic acid (hydroxyacid

C. (1S,3R,7S,8S,8aR)-3,7-dimethyl-8-[2-[(2R)-6-oxo-3,6-dihydro-2H-pyran-2-yl]ethyl]1,2,3,7,8,8a-hexahydronaphthalen-1-yl (2S)-2-methylbutanoate (dehydrolovastatin),

Freezing point (C)

Macrogol Cetostearyl Ether

Ethylene oxide units

Iodine value (2.5.4) Not more than 2.0.

Macrogol 6 Glycerol Caprylocaprate

Macrogol Glycerol Cocoates

Iodine value (2.5.4). Not more than 5.0.

Macrogol Lauryl Ether

Iodine value (2.5.4). Not more than 2.0.

Macrogol Oleyl Ether

*This broad range is needed since two different grades of oleyl

Macrogol Stearyl Ether

Iodine value (2.5.4). Not more than 2.0.

Heavy Magnesium Carbonate

Light Magnesium Carbonate

Partially Hydrated Magnesium Chloride

Magnesium Chloride Hexahydrate

Heavy Magnesium Oxide

Light Magnesium Oxide

Time (min) Temperature

Magnesium Sulphate Heptahydrate

Dried Magnesium Sulphate

Refined Maize Oil

C. ethyl and methyl (2RS)-2-(dimethoxyphosphinothioyl)butanedioate (methyl analogue).

V0 = total volume of glucose standard solution, in millilitres,

Manganese Sulphate Monohydrate

A. 6-hydroxy-6-methyl-3,20-dioxopregn-4-en-17-yl acetate (6-hydroxymedroxyprogesterone

D. 6-methyl-3,20-dioxopregn-4-en-17-yl acetate (6-epimedroxyprogesterone acetate),

E. 6-methylene-3,20-dioxopregn-4-en-17-yl acetate (6-methylenehydroxyprogesterone acetate),

F. 6-methyl-3,20-dioxo-5-pregnan-17-yl acetate (4,5-dihydromedroxyprogesterone acetate),

Definition Megestrol Acetate is 6-methyl-3,20-dioxopregna-4,6-dien-17-yl acetate. It contains

Definition Meglumine is 1-deoxy-1-methylamino-D-glucitol. It contains not less than 99.0% and

A. ethyl 4-hydroxy-2-methyl-2H-1,2-benzothiazine-3-carboxylate 1,1-dioxide

D. methyl 4-hydroxy-2-methyl-2H-1,2-benzothiazine-3-carboxylate 1,1-dioxide,

Definition Melphalan is 4-bis(2-chloroethyl)amino-L-phenylalanine. It contains not less than

Menadiol Sodium Phosphate

Definition Menadiol Sodium Phosphate is tetrasodium 2-methylnaphthalene-1,4-diyl

Definition Metaraminol Tartrate is (1R,2S)-2-amino-1-(3-hydroxyphenyl)propan-1-ol hydrogen

Methacrylic AcidEthyl Acrylate Copolymer (1:1)

Methacrylic AcidEthyl Acrylate Copolymer (1:1) Dispersion 30 per

Methacrylic AcidMethyl Methacrylate Copolymer (1:1)

Methacrylic AcidMethyl Methacrylate Copolymer (1:2)

Definition Methanol is methyl alcohol.

B. R1 = NH2, R2 = H: (S)-2-[4-[[(2,4-diaminopteridin-6-yl)methyl]amino]benzoylamino]pentanedioic acid (4-aminofolic acid, aminopterine),

D. R = OH: 4-[[(2-amino-4-hydroxypteridin-6-yl)methyl]methylamino]benzoic acid

Definition Methoxamine Hydrochloride is all-rac-2-amino-1-(2,5-dimethoxyphenyl)propan-1-ol

Industrial Methylated Spirit

Industrial Methylated Spirit (Ketone-free)