Estimate and Validation Cefixime and Azithromycin Simultaneoslyy in Tablt Dosage Froms by RP-HPLC Method
Estimate and Validation Cefixime and Azithromycin Simultaneoslyy in Tablt Dosage Froms by RP-HPLC Method
Estimate and Validation Cefixime and Azithromycin Simultaneoslyy in Tablt Dosage Froms by RP-HPLC Method
MASTER OF PHARMACY
In
PHARMACEUTICAL ANALYSIS AND QUALITY ASSURANCE
By
REDDYBATHULA LEELA
M.Pharm
[Reg. No. 14CD1S0423]
Under The Guidance of
NARASARAOPET
CERTIFICATE
TO ESTIMATE CEFIXIME AND AZITHROMYCIN SIMULTANEOUSLY IN TABLET
DOSAGE FORMS BY RP-HPLC METHOD
NARASARAOPET
original and has not been submitted in part of in full for any degree or diploma
ACKNOWLEDGEMENT
As I inscribe this acknowledgement, all the pleasant memories are flooding my mind.
Right from getting a chance to work under great guides to finishing my work in two years, it
has been dream tenure for me. As I remember the good and the tough times I had, I
remember the people who helped me in various ways in my endeavour and made my
experience a special one. I take this opportunity to thank all of them.
I fondly thank with deepest gratitude to my honourable guide K,Naga Prashanth Asst.
Professor, Narsaraopet Institute of Pharmaceutical sciences who with his deep knowledge,
keen interest of the subject, kind co-operation, untiring patience, constructive criticism,
constant help and encouragement has enabled me to complete my dissertation work. His
principles, discipline, simplicity caring attitude and provision of fearless work environment
will be cherished in all walks of my life.
I First of all, I would like to thank the Super power, the nature, for all that happens in life.
I would like to thank everyone who directly or indirectly helped me in my project work. Last
but not the least I pay my reverence to our institute, Narsaraopet Institute of pharmaceutical
Sciences,I am undeniably proud to be associated with this college.
Leela!!!
4 INTRODUCTION 1
5 LITERATURE REVIEW 25
6 DRUG PROFILE 27
11 BIBILOGRAPHY 79
ABBREVIATIONS
Symbols Description
g Microgram
L Microlitre
AR Analytical reagent
INTRODUCTION
Pharmaceutical Analysis plays a very vital role in the quality assurance and quality
control of bulk drugs and their formulations. Pharmaceutical analysis is a specialized branch
of analytical chemistry which involves separating, identifying and determining the relative
amounts of components in a sample of matter. It is concerned with the chemical
characterization of matter both quantitative and qualitative.
In recent years, several analytical techniques have been evolved.
1. SPECTROPHOTOMETRIC METHODS
2.1.MODES OF CHROMATOGRAPHY
Modes of chromatography are defined essentially according to the nature of the
interactions between the solute and the stationary phase, which may arise from hydrogen
bonding, Vander walls forces, electrostatic forces or hydrophobic forces or basing on the size
of the particles (e.g. Size exclusion chromatography).
Different modes of chromatography are as follows:
A large number of chemically bonded stationary phases based on silica are available
commercially. Silica based stationary phases are still most popular in reversed phase
chromatography however other adsorbents based on polymer (styrene-divinyl benzene
copolymer) are slowly gaining ground.
Simple compounds are better retained by the reversed phase surface, the less water-
soluble (i.e. the more non-polar) they are. The retention decreases in the following order:
aliphatic > induced dipoles (i.e. CCl4) > permanent dipoles (e.g.CHCl3) > weak Lewis bases
(ethers, aldehydes, ketones) > strong Lewis bases (amines) > weak Lewis acids (alcohols,
phenols) > strong Lewis acids (carboxylic acids). Also the retention increases as the number
of carbon atoms increases.
As a general rule the retention increases with increasing contact area between sample
molecule and stationary phase i.e. with increasing number of water molecules, which are
released during the adsorption of a compound. Branched chain compounds are eluted more
rapidly than their corresponding normal isomers.
Chemically bonded octadecyl silane (ODS) an alkaline with 18 carbon atoms it is the
most popular stationary phase used in pharmaceutical industry. Since most pharmaceutical
compounds are polar and water soluble, the majority of HPLC methods used for quality
assurance, decomposition studies, quantitative analysis of both bulk drugs and their
formulations use ODS HPLC columns. The solvent strength in reversed phase
chromatography is reversed from that of adsorption chromatography (silica gel) as stated
earlier. Water interacts strongly with silanol groups, so that, adsorption of sample molecules
become highly restricted and they are rapidly eluted as a result.
Exactly opposite applies in reversed phase system water cannot wet the non-polar
(hydrophobic) alkyl groups such as C18 of ODS phase and therefore does not interact with the
bonded moiety. Hence water is the weakest solvent of all and gives slowest elution rate. The
elution time (retention time) in reversed phase chromatography increases with increasing
amount of water in the mobile phase.
Liquid
GasLiquid Chromatography
Gas
The modern form of column chromatography has been called high performance, high
pressure, and high-resolution and high-speed liquid chromatography.
Recorder Detector Effluent
varies during run. Gradient elution is a means of overcoming the problem of dealing with a
complex mixture of solutes.
Sample introduction systems:
Two means for analyte introduction on the column are injection in to a flowing stream
and a stop flow injection. These techniques can be used with a syringe or an injection valve.
Automatic injector is a microprocessor-controlled version of the manual universal injector.
Usually, up to 100 samples can be loaded in to the auto injector tray. The system parameters
such as flow rates, gradient, run time, volume to be injected, etc. are chosen, stored in
memory and sequentially executed on consecutive injections.
silane) to reduce further the number of silanol groups remaining on the surface (end-capping).
The useful pH range for columns is 2 to 8, since siloxane linkages are cleaved below pH-2
while at pH values above eight silica may dissolve.
In HPLC, generally two types of columns are used, normal phase columns and
reversed phase columns. Using normal phase chromatography, particularly of non-polar and
moderately polar drugs can make excellent separation. It was originally believed that
separation of compounds in mixture takes place slowly by differential adsorption on a
stationary silica phase.
4 .Column-packing materials
The heart of the system is the column. In order to achieve high efficiency of separation, the
column material (micro-particles, 5-10 m size) packed in such a way that highest numbers
of theoretical plates are possible.
Silica (SiO 2 X H2O) is the most widely used substance for the manufacture of
packing materials. It consists of a network of siloxane linkages (Si-O-Si) in a rigid three
dimensional structure containing inter connecting pores. Thus a wide range of commercial
products is available with surface areas ranging from 100 to 800 m 2/g. and particle sizes from
3 to 50 m.
The silanol groups on the surface of silica give it a polar character, which is exploited in
adsorption chromatography using non-polar organic eluents. Silica can be drastically altered
by reaction with organo chloro silanes or organo alkoxy silanes giving Si-O-Si-R linkages
with the surface. The attachment of hydrocarbon change to silica produces a non-polar
surface suitable for reversed phase chromatography where mixtures of water and organic
solvents are used as eluents. The most popular material is octadecyl-silica (ODS-Silica),
which contains C18 chains, but materials with C2, C6, C8 and C22 chains are also available.
During manufacture, such materials may be reacted with a small mono functional silane (e.g.
trimethyl chloro silane) to reduce further the number of silanol groups remaining on the
surface (end-capping). There is a vast range of materials which have intermediate surface
polarities arising from the bonding to silica of other organic compounds which contain
groups such as phenyl, nitro, amino and hydroxyl. Strong ion exchangers are also available in
which sulphonic acid groups or quaternary ammonium groups are bonded to silica. The
useful pH range for columns is 2 to 8, since siloxane linkages are cleaved below pH-2 while
at pH values above eight, silica may dissolve.
In HPLC, generally two types of columns are used, normal phase columns and reversed phase
columns. Using normal phase chromatography, particularly of non-polar and moderately
polar drugs can make excellent separation. It was originally believed that separation of
compounds in mixture takes place slowly by differential adsorption on a stationary silica
phase. However, it now seems that partition plays an important role, with the compounds
interacting with the polar silanol groups on the silica or with bound water molecules.
While normal phase seems the passage of a relatively non-polar mobile phase
over a polar stationary phase, reversed phase chromatography is carried out using a polar
mobile phase such as methanol, acetonitrile, water, buffers etc., over a non-polar stationary
phase. Ranges of stationary phases (C18, C8, -NH2, -CN, -phenyl etc.) are available and very
selective separations can be achieved. The pH of the mobile phase can be adjusted to
suppress the ionization of the drug and thereby increase the retention on the column. For
highly ionized drugs ion-pair chromatography is used.
Phase Description
Silica
Classic normal phase material. Suitable for separating polar non-ionic
Si organic compounds.
TMS, SAS,
Tri methyl silane
RP-2, Dimethyl
C2 Reversed phase material, less retentive than C4, C8, or C18. More retentive than C1.
Butyl: Reversed phase material, useful for ion-pairing chromatography offers less
retention than C8 and C18 phases for non-polar solutes. When bonded to 300 silica, it
is an ideal phase for analyzing large proteins and hydrophobic peptides.
C4
C8 Reversed phase material, similar selectivity to C18 but less retentive. Wide
applicability (e.g. pharmaceuticals, nucleosides, steroids). When bonded to300
silica, it is an ideal phase for peptides, peptide mapping and small hydrophilic
proteins.
C6H5
Phenyl
CN Cyanopropyl, Nitrile
It can be employed as either a reversed phase or normal phase material. It is
slightly polar and exhibits unique selectivity for polar compounds in both RP
and NP modes. It equilibrates very rapidly and suitable for gradient
separations. It has many pharmaceutical applications (e.g. tricyclic
antidepressants).
NH2
Can be employed as reversed phase, normal phase or weak anion exchange material.
Reversed phase: useful for separating carbohydrates. Normal phase: alternative
selectivity to silica, not deactivated by small amounts of water. Ion Exchange: weak
anion exchanger when used with buffers separates anions and organic acids.
Nitro
NO2
Normal phase material. Separates aromatic compounds and compounds with double
bonds.
Diol, Glycerol
OH
Can be employed as either a reversed phase or normal phase material. Reversed
phase: Used for Gel Filtration Chromatography (GFC) of proteins and peptides.
Normal phase: Similar selectivity to silica not deactivated by small amounts of
water.
Ion exchange material. Strong anion exchangers (basic) are useful for separating
nucleosides, nucleotides and organic acids.
Ion exchange material. Strong anion exchangers (basic) are useful for separating
nucleosides, nucleotides and organic acids.
Ion exchange material. Strong cation exchangers (acidic) are useful for separating
organic bases.
CM, Carboxymethyl,
WCX
Weak Acid
Ion exchange material. Weak cation exchangers (acidic) are most useful for
analyzing basic proteins and peptides.
5.Derivatization:
In HPLC derivatization is used to enhance the sensitivity and selectivity of
detection when available detectors are not satisfactory for the underivatized compounds. Both
ultra violet absorbing and fluorescence derivatives have been widely used. Ultra violet
derivatization reagents include N-succinimidyl p-nitro phenyl acetate, phenyl hydrazine and
3, 5-dinitro benzyl chlorides, while fluorescent derivatives can be formed with reagents such
as dansyl chloride, 4-bromo methyl-7-methoxy-coumarin and fluorescamine. Derivative
formation can be carried out before the sample is injected on to the column or by online
chemical reactions between the column out let and the detector.
6. Gradient elution:
Gradient elution or solvent programming is the change of solvent composition
during a separation in which the solvent strength increases from the beginning to the end of
the separation. It is well suited to the analysis of samples of unknown complexity since good
resolution is automatically provided for a wide range of sample polarities. There are two
types of gradient systems: Low-pressure gradient mixtures and high- pressure gradient
mixtures. In the former the solvents are mixed at atmosphere pressure and then pumped to the
column, where as in the later, solvents are pumped in to a mixing chamber at high pressure
before going in to the column.
Performance calculations:
Calculating the following values (which can be include in a custom report) used
to access overall system performance.
Relative retention, Theoretical plates, Capacity factor
Resolution, Peak asymmetry, Plates per meter
The parameters used to calculate these system performance values for the separation of
two chromatographic components.
Theoretical plates:
n = 16 (t / W) 2
Capacity factor:
K' = (t2 / ta) - 1
Rsolution:
R = 2 (t2 - t1) / (W2 + W1)
Peak asymmetry:
T = W0.05 / 2f
Plates per meter:
N=n/L
HETP: L/n
ta = Retention time of an inert peak not retained by the column, measured from point
of injection.
n = Theoretical plates.
t = Retention time of the component.
W = Width of the base of the component peak using tangent method.
K' = Capacity factor.
R = Resolution between a peak of interest (p2) and the peak preceding it (p1)
W2 = Width of the base of component peak 2.
W1 = Width of the base of component peak 1.
T = Peak asymmetry, or tailing factor.
W0.05 = Distance from the leading edge to the tailing edge of the peak,
Measured at a point 5 % of the peak height from the baseline.
f = Distance from the peak maximum to the leading edge of the peak.
L = length of column, in meters
HPLC troubleshooting 3
Abnormal pressure
Air trapped in pump head Degas mobile phase, bleed air from pump and prime pump
You should find out, what caused high back pressure - column or system? We recommend
following procedure:
Remove column from the system and turn on pump. If high back pressure still appears, then
the blockage is in the system:
Fluctuating pressure
See section High back pressure.
Pressure dropping to zero
See sections No pressure reading, no flow and No pressure reading, flow is normal
Pressure dropping, but not to zero
See section Low pressure
Pressure cycling
valve(s)
Pump seal failure Replace pump seal
Insufficient Degas solvent and/or change degassing methods (e.g. use vacuum
degassing degasser)
Leak in system Locate leak and correct it
Using gradient Pressure cycling is normal due to viscosity change
elution
Leaks
Leaks are usually stopped by tightening or replacing a fitting. Be aware, however, that
overtightened metal compression fittings can leak and plasrtic fingertight fittings can
wear out. If a fitting leak does not stop when the fitting is tightened a little, take the
fitting apart and inspect for damage (e.g. distored ferrule, or particles on the sealing
surface). If the fitting or ferrule is damaged, replace it with new one.
Leaky fittings
Injector leaks
Column leaks
*Note: When the particle size of stationary phase is 3 to 4 m, use frit 0.5 m. When particle
size of stationary phase is 5 to 20 m, use frit 2 m.
Detector leaks
Many issues in the LC system appear as changes in the chromatogram. Some of these can be
solved by changes in the instrument, however, other problems require modification of the
assay procedure. Setting the proper column type, pre-column or guard column, tubings,
detector cell and mobile phase are keys to good chromatography.
Peak tailing
Peak fronting
Split peaks
The peak distortion can be caused by sample overload. Reduce the sample size.
The distortion of early eluting peaks can be caused by wrong injection solvent. Reduce the
injection volume, or use weaker injection solvent.
Extra-column Replumb the system (shorter, narrower tubing), use smaller volume
effects detector cell
Secondary retention Add triethylamine (basic compounds) or add acetic acid (acidic
effects, normal-phase compounds) or add water (poly-functional compounds). Only
mode for normal-phase methods which use water-miscible solvents.
You can also try a different LC method.
Secondary retention Add triethylamine (basic samples)
effects, ion-pair
chromatography
Inadequate Use 50 to 100 mM buffer concentration, use buffer with pKa equal to
buffering pH of mobile phase
Extra peaks
METHOD OPTIMISATION: 12
Selection of stationary phase / column: Selection of the column is the first and the most
important step in method development.
Some of the important parameters considered while selecting chromatographic columns
are:
chromatographic separations since a polar mobile phase will give rise to low solute retention
in normal phase and high solute retention in reverse phase LC. The selectivity will be
particularly altered if the buffer pH is close to the pKa of the analytes; the solvent strength is
a measure of its ability to pull analyte from the column. It is generally controlled by the
concentration of the solvent with the highest strength.
The following are the parameters, which shall be taken into consideration while
selecting and optimizing the mobile phase.
Buffer,
pH of the buffer
Mobile phase composition.
Selection of detector:
The detector was chosen depending upon some characteristic property of the analyte
like UV absorbance, fluorescence, conductance, oxidation, reduction etc. characteristics that
are to be fulfilled by a detector to be used in HPLC determination are,
High sensitivity, facilitating trace analysis
Negligible baseline noise. To facilitate lower detection
Low dead volume
Non destructive to sample
Inexpensive to purchase and operate
For the greatest sensitivity max should be used. Higher wavelengths give greater
selectivity.
3. METHOD VALIDATION 17
Method validation can be defined as (ICH) estabilishing documented evidence which
provides a high degree of assurance that specific activity will consistenty produce a desired
result or product meeting its predetermined specifications and quality characteristics.
Method validation is an integral part of the method development; it is the
process of demonstrating that analytical procedures are suitable for their intended use
and that they support the identity, quality, purity, an and drug products. Simply, method
validation is the process of proving that and potency of the drug substances analytical method
is acceptable for its intended purpose.
For chromatographic methods used in analytical applications there is more
consistency in validation practice with key analytical parameters
(a) Recovery (b) Response function (c) Sensitivity (d) Presicion (e) Accuracy (f) limits
of detection (g) Limit of quantitation (h) Ruggedness (i) Robustness (j) stability (k)
system suitability
(a)Recovery:
The absolute recovery of analytical method is measured as the response of a processed
spiked matrix standard expressed as a percentage of the response of pure standard which has
not been subjected to sample pre treatment and indicates whether the method provides a
response for the entire amount of analyte that is present in the sample.
b) Sensitivity:
The method is said to be sensitive if small changes in concentration cause large
changes in response function. The sensitivity of an analytical method is determined from the
slope of the calibration line. The limits of quantification (LOQ) or working dynamic range of
bio analytical method are defined as the highest and lowest concentrations, which can
determined with acceptable accuracy. It is suggested that, this be set at 15% for both the
upper and lower limit of quantitation respectively. Any sample concentration that falls outside
the calibration range cannot be interpolated from the calibration line and extrapolation of the
calibration curve is discouraged. If the concentration is over range, the sample should be
diluted in drug-free matrix and re-assayed.
c) Precision:
The purpose of carrying out a determination is to obtain a valid estimate of a true
value. When one considers the criteria according to which an analytical procedure is selected,
precision and accuracy are usually the first time to come to mind. Precision and accuracy
together determine the error of an individual determination. They are among the most
important criteria for judging analytical procedures by their results.
Precision refers to the reproducibility of measurement within a set, that is, to the scatter of
dispersion of a set about its central value. The term set is defined as referring to a number
(n) of independent replicate measurements of some property. One of the most common
statistical terms employed is the standard deviation of a population of observation. Standard
deviation is the square root of the sum of squares of deviations of individual results for the
mean, divided by one less than the number of results in the set. The standard deviation S, is
given by
1 n
xi x
2
S= n 1 i1
Standard deviation has the same units as the property being measured.
The square of standard deviation is called variance (S 2). Relative standard deviation is
the standard deviation expressed as a fraction of the mean, i.e., S/x. It is sometimes multiplied
by 100 and expressed as a percent relative standard deviation. It becomes a more reliable
expression of precision.
% Relative standard deviation = S x 100 / x
d) Accuracy:
Accuracy normally refers to the difference between the mean x****, of the set of
results and the true or correct value for the quantity measured. According to IUPAC accuracy
relates to the difference between results (or mean) and the true value. For analytical methods,
there are two possible ways of determining the accuracy, absolute method and comparative
method.
Accuracy is best reported as percentage bias, which is calculated from the expression
(measured value - true value)
%Bias = X 100
true value
The accuracy of analytical method is then determined at each concentration by assessing the
agreement between the measured and nominal concentrations of the analytes in the spiked
drug free matrix sampler.
h) Robustness
The concept of robustness of an analytical procedure has been defined by the ICH as a
measure of its capacity to remain unaffected by small but deliberate variations in method
parameters. The robustness of a method is the ability to remain unaffected by
small changes in parameters such as pH of the mobile phase, temperature, %organic solvent
strength and buffer concentration etc. to determine the robustness of the method experimental
conditions were purposely altered and chromatographic characters were evaluated.
i) System suitability
System suitability experiments can be defined as tests to ensure that the method can generate
results of acceptable accuracy and precision. The requirements for system suitability are
usually developed after method development and validation have been completed. (or) The
USP (2000) defines parameters that can be used to determine system suitability prior to
analysis.
The criteria selected will be based on the actual performance of the method as
determined during its validation. For example, if sample retention times form part of the
system suitability criteria, their variation (SD) during validation can be determined system
suitability might then require that retention times fall within a 3 SD range during routine
performance of the method.
LITERATURE REVIEW40
A simple and sensitive reversed phase High Performance Liquid Chromatographic method
has been developed and validated for the simultaneous analysis of the Cefiximetrihydrate
(CEF) and Linezolid (LIN) in tablet dosage form. The separation was carried out using
mobile phase consisting of buffer and methanol with pH 2.5 in the ratio of 70:30, v/v. The
column used was ACE 5 C18, (150 mm x 4.6 mm i.d., 5 m) with flow rate 1.2 ml/min using
PDA detection at 250 nm. The method was linear over a concentration range of 23.33 40
g/ml and 70 120 g/ml for CEF and LIN, respectively. The retention time of CEF and LIN
were found to be 3.30 min and 8.07 min, respectively. Results of analysis were validated
statistically and by recovery studies. The mean recovery was 101.9 0.82 and 102.6 1.15
for CEF and LIN, respectively. The limit of detection (LOD) and the limit of quantification
(LOQ) for CEF and LIN were found to be 0.78 and 2.37 g/ml and 2.42 and 7.34 g/ml,
respectively. The results of the study showed that the proposed RPHPLC method is simple,
rapid, precise and accurate which is useful for the routine determination of CEF and LIN in
its pharmaceutical dosage form.
The objective of the current study was to develop a simple, accurate, precise and rapid RP-
HPLC method with subsequently validate as per ICH guidelines for the determination of
Azithromycin (AZI) and Cefpodoxime Proxetil (CEF) using mobile phase [A mixture of
acetonitrile and Phosphate buffer- pH-7.0 in the ratio of 65:35 was considered to be the
optimal composition] as the solvent. The proposed method involves the measurement of
Retention time at selected analytical wavelength. 270.0 nm was selected as the analytical
wavelength. The retention time of AZI and CEF was found to be 7.318 and 3.521
respectively. The linearity of the proposed method was investigated in the range of 10-50
g/ml (r = 0.9999) for AZI and 8-40 g/ml (r = 0.9998) for CEF respectively. The method
was statistically validated for its linearity, accuracy and precision. Both inter-day and intra-
day variation was found to be showing less % RSD (Relative Standard Deviation) value
indicating high grade of precision of the method
DRUG PROFILE
CEFIXIME8, 9
structure :
IUPAC : 6R,7R)-7-{[2-(2-Amino-1,3-thiazol-4-yl)-2-
(carboxymethoxyimino)acetyl]amino}-3-ethenyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-
2-carboxylic acid
Molecular formula : C16H15N5O7S2
Molecular weight : 453.452 g/mol
Description : Cefixime, an antibiotic, is a third-generation cephalosporin like ceftriaxone and
cefotaxime. Cefixime is highly stable in the presence of beta-lactamase enzymes. As a result,
many organisms resistant to penicillins and some cephalosporins due to the presence of beta-
lactamases, may be susceptible to cefixime. The antibacterial effect of cefixime results from
inhibition of mucopeptide synthesis in the bacterial cell wall.
Categories :
Anti-Bacterial Agents
Cephalosporins
Antibacterials for Systemic Use
Mechanism of action : ike all beta-lactam antibiotics, cefixime binds to specific penicillin-
binding proteins (PBPs) located inside the bacterial cell wall, causing the inhibition of the
third and last stage of bacterial cell wall synthesis. Cell lysis is then mediated by bacterial cell
wall autolytic enzymes such as autolysins; it is possible that cefixime interferes with an
autolysin inhibitor.
Pharmcodynamics : Cefixime, an antibiotic, is a third-generation cephalosporin like
ceftriaxone and cefotaxime. Cefixime is highly stable in the presence of beta-lactamase
enzymes. As a result, many organisms resistant to penicillins and some cephalosporins due to
the presence of beta-lactamases, may be susceptible to cefixime. The antibacterial effect of
cefixime results from inhibition of mucopeptide synthesis in the bacterial cell wall.
Solubility : It is soluble in water and methanol.
Boiling point : ~405.2 C at 760 mmHg (Predicted)
Melting point : 234-236 C
USES : t is primarily used in the treatment of urinary tract infections, although it may be used
against any susceptible aerobic bacterial species It may also be used to treat and prevent
Pneumocystis jiroveci pneumonia. It is generally not recommended for the treatment of
anaerobic infections such as Clostridium difficile colitis (the leading cause of antibiotic-
induced diarrhea).Resistance to trimethoprim is increasing, but it is still a first line antibiotic
in many countries.
AZITHROMYCIN
Structure :
hexopyranosyl]oxy}-1-oxa-6-azacyclopentadec-13-yl 2,6-dideoxy-3-C-methyl-3-O-methyl-
-L-ribo-hexopyranoside
Molecular formula : C38H72N2O12
Molecular weight : 748.984 gmol1
Categories :
Anti-Bacterial Agents
Macrolides
Antibiotics
Ophthalmologicals
Sensory Organs
Antibacterials for Systemic Use
Antiinfectives for Systemic Use
Antiinfectives
Macrolides, Lincosamides and Streptogramins
Cytochrome P-450 CYP1A2 Inhibitors
Cytochrome P-450 CYP1A2 Inducers
CYP2A6 Inhibitors
CYP2A6 Inhibitors (strong)
CYP2A6 Inhibitors (moderate)
CYP2A6 Inducers
CYP2A6 Inducers (strong)
CYP3A4 Inhibitors
Combined Inhibitors of CYP3A4 and P-glycoprotein
first-line therapy .Uncomplicated skin and skin structure infections due to S. aureus, S.
pyogenes, or S. agalactiaeUrethritis and cervicitis due to C. trachomatis or N.
gonorrhoeaeGenital ulcer disease (chancroid) in men due to H. ducreyiIn combination with
ceftriaxone, azithromycin is part of the United States Centers for Disease Control-
recommended regimen for the treatment of gonorrhea. Azithromycin is active as
monotherapy in most cases, but the combination with ceftriaxone is recommended based on
the relatively low barrier to resistance development in gonococci.
PLAN OF WORK
The experimental work has been planned as follows:
Review of the literature for Cefixime and Azithromycinregarding their physical and chemical
properties, various analytical methods that were conducted for Cefixime and
Azithromycinforms the basis for development of new analytical RP-HPLC method
Cefiximeand Ketorolaccombination.
For the mobile phase, the first variable to be decided is whether an organic or aqueous eluent
should be used. With the RP-HPLC analysis, either an aqueous eluent or a very polar organic
solvent such as methanol or acetonitrile should be fixed. If the K values are too large with an
aqueous solvent, organic solvent should be tried. If the K value are too low with organic
solvent the separation should be attempted using a mixture of two solvents with various
properties.
Observation: Two peaks are merged and not separated completely. The trial 1 chromatogram
result was shown in Fig:1.
Trail: 2
Mobile Phase: Degassed Acetonitrile and Water in the ratio of 90:10 V/V.
Parameters Method
METHOD VALIDATION
5.3.1SYSTEM SUITABILITY:
A Standard solution was prepared by using Cefiximeand Azithromycin working
standards as per test method and was injected Five times into the HPLC system.
The system suitability parameters were evaluated from standard chromatograms by
calculating the % RSD from five replicate injections for Cefixime and Azithromycin ,
retention times and peak areas.
ACCEPTANCE CRITERIA:
1. The % RSD for the retention times of principal peak from 5 replicate injections of each
Standard solution should be not more than 2.0 %
2. The % RSD for the peak area responses of principal peak from 5 replicate injections of
each standard Solution should be not more than 2.0%.
3. The number of theoretical plates (N) for the Cefiximeand Azithromycin peaks is NLT 3000.
4. The Tailing factor (T) for the Cefiximeand Azithromycin
peaks is NMT 2.0
OBSERVATION:
The %RSD for retention times and peak areas were found to be within the limit.
Refer table: 1 As shown in fig 6 10.
5.3.2 SPECIFICITY:
Cefiximeand Azithromycin :
Solutions of standard and sample were prepared as per the test method are injected into
chromatographic system.
ACCEPTENCE CRITERIA:
Chromatograms of standard and sample should be identical with near Retention time.
OBSERVATION:
The chromatograms of Standard and Sample were same identical with same retention
time. As shown in fig: 12 and fig: 13
5.3.3 PRECISION:
5.3.3.1Repeatability:
a System precision: Standard solution prepared as per test method and injected five
times.
b Method precision: Prepared six sample preparations individually using single as per
test method and injected each solution.
ACCEPTANCE CRITERIA: The % relative standard deviation of individual Cefiximeand
Azithromycin , from the six units should be not more than 2.0%.
The individual assays of Cefiximeand Azithromycin should be not less than 98% and
not more than 102.0%.
OBSERVATION:
Test results are showing that the test method is precise. Refer tables 2 and 3 for
system precision and for method precision.
5.3.3.2Intermediate precision (analyst to analyst variability):
A study was conducted by two analysts as per test method
ACCEPTENCE CRITERIA:
The individual assays of Cefiximeand Azithromycin should be not less than 98% and not
more than 102% and %RSD of assays should be NMT2.0% by both analysts.
OBSERVATION:
Individual %assays and %RSD of Assay are within limit and passes the intermediate
precision, Refer table: 4
5.3.4 ACCURACY (RECOVERY):
A study of Accuracy was conducted. Drug Assay was performed in triplicate as per
test method with equivalent amount of Cefiximeand Azithromycin into each volumetric flask
for each spike level to get the concentration of Cefiximeand Azithromycin equivalent to
50%, 100%, and 150% of the labeled amount as per the test method. The average % recovery
of Cefiximeand Azithromycin were calculated.
ACCEPTANCE CRITERIA:
The mean % recovery of the Cefiximeand Azithromycin at each spike level should be
not less than 98.0% and not more than 102.0% for both the drugs separately.
OBSERVATION:
Amount found
%Recovery = ---------------------- 100
Amount added
The recovery results indicating that the test method has an acceptable level of
accuracy. Refer table: 5
OBSERVATION:
The linear fit of the system was illustrated graphically. The results are presented in table6.
ACCEPTANCE CRITERIA:
The %RSD of Cefiximeand Azithromycin tablets should be NMT2.0%. The %assay of
Cefiximeand Azithromycin should be between 98.0% and 102.0% for individual drugs.
OBSERVATION:
The results obtained by comparing with both two types were within limit. Refer tables: 3
&9
5.3.7 ROBUSTNESS:
a) Effect of variation of flow rate:
A study was conducted to determine the effect of variation in flow rate. Standard
solution prepared as per the test method was injected into the HPLC system using flow rates,
1.0ml/min and 1.2ml/min. The system suitability parameters were evaluated and found to be
within the limits for 1.0ml/min and 1.2ml/min flow.
Cefiximeand Azithromycin and was resolved from all other peaks and the retention times
were comparable with those obtained for mobile phase having flow rates 1.0ml/min.
ACCEPTANCE CRITERIA:
The Tailing Factor of Cefiximeand Azithromycin standards should be NMT 2.0 for
Variation in Flow.
OBSERVATION:
The tailing factor for Cefiximeand Azithromycin was found to be within the limits. As
shown in table 10.
LOD= 3.3
S
= standard deviation of the response
S = slope of the calibration curve of the analyte.
LOQ = 10
S
= standard deviation of the response
S = slope of the calibration curve of the analyte.
0.14
3.045
0.12
0.10
0.08
AU
4.933
0.06
0.04
0.02
0.00
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50
Minutes
Inference :
1 Cefixime 3.045
2 Azithromycin 4.933
0.025
2.79 3
0.020
3.18 8
0.015
AU
0.010
0.005
0.000
time(min)
1 Cefixime 2.793
2 Azithromycin 3.188
0.040
3.538
0.035
0.030
2.844
0.025
AU
0.020
0.015
0.010
0.005
0.000
1 Cefixime 2.844
2 Azithromycin 3.538
0.080
Cefixime - 2.186
0.070
0.060
Azithromycin - 3.963
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
Azithromycin
2 Azithromycin 3.963
Fig5:Chromatogram of sample
0.080
Cefixime - 2.183
0.070
0.060
Azithromycin - 3.960
0.050
0.040
AU
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
1 Cefixime 2.183
2 3.960
Azithromycin
0.080
Cefixime - 2.183
0.070
0.060
Azithromycin - 3.960
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
0.080
Cefixime - 2.178
0.070
0.060
Azithromycin - 3.955
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
0.080
Cefixime - 2.179
0.070
0.060
Azithromycin - 3.957
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
0.080
Cefixime - 2.179
0.070
0.060
Azithromycin - 3.956
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
0.080
Cefixime - 2.177
0.070
0.060
Azithromycin - 3.954
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
6.3.2: SPECIFICITY:
0.0015
0.0010
0.0005
0.0000
-0.0005
AU
-0.0010
-0.0015
-0.0020
-0.0025
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
0.080
Cefixime - 2.177
0.070
0.060
Azithromycin - 3.954
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
Inference: Got a peak for sample at an Rt of 2.177min for Cefiximeand 3.954min for
Azithromycin
6.3.2: PRECISION:
6.3.2.1Repeatability:
Peak Areas of
Injection
Cefixime %Assay
Concentration
1 2978965 100.51
40ppm
2 2970867 100.24
3 2973742 100.34
4 2978761 100.51
5 2978642 100.50
Peak Areas of
Injection
Azithromycin %Assay
1 429853 100.34
2 429741 100.31
Concentration
40ppm
3 429784 100.32
4 429403 100.23
5 429746 100.31
0.080
Cefixime - 2.186
0.070
0.060
0.040
AU
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
0.080
0.070
A z ith ro m y c in - 3 .9 6 0
0.060
0.050
0.040
AU
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
0.080
Cefix im e - 2.183
0.070
0.060
A z ithrom y c in - 3.960
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
0.080
Cefixime - 2.178
0.070
0.060
Azithromycin - 3.955
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
0.080
Cefixime - 2.179
0.070
0.060
Azithromycin - 3.957
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
(b)Method precision:
Peak Areas of
Injection
Cefixime %Assay
1 2970873 100.24
4 2978462 100.50
5 2976542 100.43
6 2978642 100.50
Peak Areas of
Injection %Assay
Azithromycin
1 429827 100.33
4 429786 100.32
5 429375 100.23
6 429274 100.20
Cefix im e - 2.179
0.080
0.070
0.060
A z ithrom y c in - 3.957
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
0.080
Cefixime - 2.179
0.070
0.060
Azithromycin - 3.956
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
0.080
Cefixime - 2.176
0.070
0.060
Azithromycin - 3.954
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
0.080
Cefixime - 2.177
0.070
0.060
Azithromycin - 3.955
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
0.080
Cefixime - 2.175
0.070
0.060
Azithromycin - 3.953
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
Table4:
Peak Areas of
Injection
Cefixime %Assay
1 2970478 100.23
Concentrati
2 2978492 100.50
on
3 2970874 100.24
40ppm
4 2977892 100.48
5 2970845 100.24
6 2978632 100.50
Peak Areas of
Injection
Azithromycin %Assay
1 429874 100.34
Concentrati
2 429654 100.29
on
3 429658 100.29
40ppm
4 429631 100.29
5 429874 100.34
6 429631 100.34
0.080
Cefixime - 2.174
0.070
0.060
Azithromycin - 3.952
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
0.080
Cefixime - 2.173
0.070
0.060
Azithromycin - 3.953
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
0.080
0.070
0.060
Azithromycin - 3.952
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
TABLE-5:
50% 1486721
20 19.89 99.47 MEAN 99.5433
Injection 1
50%
20 1489764 19.94 99.68
Injection 2
50%
20 1486795 19.90 99.48 %RSD 0.119006
Injection 3
100 % 100.463
40 2978864 40.20 100.51 MEAN
Injection 1 3
100 %
40 2976794 40.18 100.44
Injection 2
100% 0.04022
40 2976874 40.18 100.44 %RSD
Injection 3 8
150%
60 4465274 60.44 100.73 MEAN 100.783
Injection 1
150%
60 4467892 60.47 100.79
Injection 2
150% 0.04994
60 4469874 60.50 100.83 %RSD
Injection 3 1
Concentratio
Amount Amount
n Area % Statistical Analysis
added found
% of spiked Recovery of % Recovery
(ppm) (ppm)
level
50% 214836
20 19.92 99.62 MEAN 99.593
Injection 1
50% 214975
20 19.94 99.68
Injection 2
50% 214558 0.10305
20 19.90 99.48 %RSD
Injection 3 1
100 % 429754
40 40.13 98.92 MEAN 99.66
Injection 1
100 % 429634
40 40.11 99.75
Injection 2
100% 429754 0.70174
40 40.13 100.31 %RSD
Injection 3 3
150% 644876 100.346
60 60.35 99.96 MEAN
Injection 1 7
150% 644968
60 60.36 100.59
Injection 2
150% 644308 0.00734
60 60.29 100.49 %RSD
Injection 3 4
0.035
0.030
0.025
Azithromycin - 3.968
AU
0.020
0.015
0.010
0.005
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
0.040
Cefixime - 2.190
0.035
0.030
0.025
Azithromycin - 3.968
AU
0.020
0.015
0.010
0.005
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
0.080
0.070
0.060
Azithromycin - 3.952
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
Cefixime - 2.173
0.080
0.070
0.060
Azithromycin - 3.952
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
0.12
Cefixime - 2.171
0.10
0.08
Azithromycin - 3.946
0.06
AU
0.04
0.02
0.00
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
0.12
Cefixime - 2.170
0.10
0.08
Azithromycin - 3.949
AU
0.06
0.04
0.02
0.00
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
6.3.5 LINEARITY:
TABLE6:
50 3720677
60 4464812
70 5208948
80 5823083
7000000
6000000
f(x) = 73468.67x + 25087.91
5000000 R = 1
4000000
3000000 Linear ()
2000000
1000000
0
0 10 20 30 40 50 60 70 80 90
50 537344
60 644813
70 752282
80 844751
900000
600000
500000
400000 Linear ()
300000
200000
100000
0
0 10 20 30 40 50 60 70 80 90
0.040
Cefixime - 2.199
0.035
0.030
0.025
Azithromycin - 3.968
AU
0.020
0.015
0.010
0.005
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
0.060
Cefixime - 2.189
0.050
Azithromycin - 3.966
0.040
AU
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
0.070
0.060
0.040
AU
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
Cefixime - 2.171
0.10
0.08
Azithromycin - 3.948
0.06
AU
0.04
0.02
0.00
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
0.12
Cefixime - 2.171
0.10
0.06
AU
0.04
0.02
0.00
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
0.12
Cefixime - 2.170
0.10
0.08
Azithromycin - 3.949
AU
0.06
0.04
0.02
0.00
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
0.16
Cefixime - 2.170
0.14
0.12
0.10
Azithromycin - 3.948
0.08
AU
0.06
0.04
0.02
0.00
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
6.3.6 Ruggedness:
TABLE: 7
System-2
Assay % of
S.NO: Peak area Cefixime
1
2975974 100.41
2 2978542 100.50
3 2978792 100.51
4 2970875 100.23
5 2978453 100.50
6 2971542 100.26
System-2
Assay % of
S.NO: Peak area Azithromycin
1 429871 100.34
2 429637 100.29
3 429654 100.34
4 429875 100.34
5 429875 100.34
6 428754 99.08
0.16
Cefixime - 2.170
0.14
0.12
0.10
Azithromycin - 3.948
0.08
AU
0.06
0.04
0.02
0.00
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
0.080
Cefixime - 2.183
0.070
0.060
Azithromycin - 3.960
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
0.080
Cefixime - 2.178
0.070
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
0.070
0.060
Azithromycin - 3.957
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
0.080
Cefixime - 2.179
0.070
0.060
Azithromycin - 3.956
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
6.3.7 Robustness:
Fig42-44Chromatograms of robustness
0.080
0.070
Cefixime - 2.397
0.060
0.050
Azithromycin - 4.385
0.040
AU
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
0.080
Cefixime - 2.397
0.070
0.060
Azithromycin - 4.386
0.050
0.040
AU
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
0.080
Cefixime - 2.179
0.070
0.060
Azithromycin - 3.956
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
0.080
Cefixime - 2.177
0.070
0.060
Azithromycin - 3.954
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
0.20
0.15
AU
Azithromycin - 3.598
0.10
0.05
0.00
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
Cefixime - 1.982
0.20
0.15
AU
Azithromycin - 3.599
0.10
0.05
0.00
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
Cefixime:
From the linearity plot the LOD and LOQ are calculated:
LOD = 3.3
S
3.32124.413
= ---------------------- = 0.34
20193
LOQ = 10
S
102124.413
= -------------------- = 1.05 20193
Azithromycin;
LOD = 3.3
S
3.32431.578
= ---------------------- = 0.25
31282
LOQ = 10
S
102431.578
= ---------------------- = 0.77
31282
The analytical method was developed by studying different parameters. First of all, maximum
absorbance was found to be at 237nm Cefixime for and 275nm for Azithromycin.Common
wavelength will be 275nm and the peaks purity was excellent. Injection volume was selected
to be 20l which gave a good peak area. The column used for study was Inertsil C 18, ODS
chosen good peak shape. Ambient temperature was found to be suitable for the nature of drug
solution. The flow rate was fixed at 1.0ml/min because of good peak area, satisfactory
retention time and good resolution. Different ratios of mobile phase were studied, mobile
phase with ratio of 55:45 Methanol : Buffer was fixed due to good symmetrical peaks and for
good resolution. So this mobile phase was used for the proposed study.
The present recovery was found to be 98.0-101.50 was linear and precise over the same
range. Both system and method precision was found to be accurate and well within range.
Detection limit was found to be 0.25 Cefixime and 0.34 for Azithromycin. Linearity study
was, correlation coefficient and curve fitting was found to be. The analytical method was
found linearity over the range of 20-80ppm of the target concentration for both the drugs. The
analytical passed both robustness and ruggedness tests. On both cases, relative standard
deviation was well satisfactory.
BIBILOGRAPHY
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