DMF For Acyclovir: Index

DMF FOR ACYCLOVIR
INDEX
3.2.S.1 General Information ...................................................................................................................................... 2

3.2.S.1.1 Nomenclature ..................................................................................................................................... 1
3.2.S.1.2 Structure ............................................................................................................................................. 2
3.2.S.1.3 General Properties .............................................................................................................................. 2
3.2.S.2 Manufacture................................................................................................................................................... 1
3.2.S.2.1 Manufacturer ...................................................................................................................................... 1
3.2.S.2.2 Description of manufacturing process and process control ................................................................ 2
3.2.S.3 Characterisation ............................................................................................................................................. 9
3.2.S.3.1 Elucidation of structure and other characteristics ............................................................................... 9
3.2.S.3.2 Impurities.......................................................................................................................................... 24
3.2.S.4 Control of Drug Substance ....................................................................................................................... 26
3.2.S.4.1 Specification ..................................................................................................................................... 26
3.2.S.4.2 Analytical Procedures ....................................................................................................................... 26
3.2.S.4.3 Validation of Analytical Procedures ................................................................................................. 33
3.2.S.5 Reference standards or materials ................................................................................................................110
3.2.S.6 Container closure systems ......................................................................................................................... 120
3.2.S.6.1 Primary packaging .......................................................................................................................... 120
3.2.S.6.2 Secondary Packaging...................................................................................................................... 141
3.2.S.6.3 Justification of selecting the packaging materials for direct contact with API ............................... 146
3.2.S.6.4 Label ............................................................................................................................................... 146
3.2.S.7 Stability ..................................................................................................................................................... 148
3.2.S.7.1 Stability summary and conclusions ................................................................................................ 148
3.2.S.7.2 Post-approval stability protocol and stability commitment ............................................................ 149
3.2.S.7.3 Stability data ................................................................................................................................... 149
DMF for Acyclovir Hubei Yitai Pharmaceutical Co., Ltd.
3.2.S.1 General Information

3.2.S.1.1 Nomenclature
International Non-proprietary Name: Acyclovir
Chemical Name: 2-amino-9-[(2-hydroxyethoxy)methyl]-1,9-dihydro-6H-purin-6-one
CAS Registry Number: 59277-89-3
3.2.S.1.2 Structure
Chemical Structure:
O
N
HN
OH
N N
H2N
O
Molecular Formula: C8H11N5O3
Molecular Weight: 225.20
3.2.S.1.3 General Properties

Appearance: White to off-white, crystalline powder.
Solubility: Slightly soluble in water, very slightly soluble in ethanol (96 per cent), practically
insoluble in heptane. It dissolves in dilute solutions of mineral acids and alkali hydroxides.
Acyclovir is soluble when the PH of aqueous solution is 14, sparingly soluble when the PH of
aqueous solution is 1 and 13, slightly soluble when the PH of aqueous solution is between 2and 12.
Melting Point:Melts at temperatures higher than 250℃, with decomposition.
pH: Not applicable
Optical Rotation: No optical rotation.
Crystal Form: Form V (2/3 hydrate)
pKa(25℃):2.52, 9.35
Particle size distribution: D10≤15μm, D50≤45μm, D90≤90μm
Isomerism:
There is one isomerism likely to be found in Acyclovir, it is the impurity C in EP. The name is
7-isomer acyclovir, chemical name is 2-amino-7-[(2-hydroxyethoxy)methyl]-1,7-dihydro-
6H-purin-6-one, the chemical structure is:

Stoichiometric ration between the API under the form it is presented and its
pharmacodinamically active compound: not applicable
Refractive index: NA
Partition coefficient: 0.33(water: n-butanol=1:1)
UV maxima absorption: A254±2nm=0.3~0.6, 10μg/ml in water.
Hygroscopicity: very slightly hygroscopicity
Solubility in physiological pH (37℃): pH1.2: 13.2mg/ml; pH4.0: 3.0mg/ml; pH6.8: 2.6mg/ml;
water: 2.3mg/ml.
Polymorphism: According to the reference documents, Acyclovir show polymorphism. It has 6
kinds of crystal forms, that are anhydrous forms(Form I, Form II, Form III and Form IV), Form
V(2/3 hydrate), Form VI(2 hydrate). The crystal form of Acyclovir manufactured in our company is
Form V (2/3 hydrate).

3.2.S.2 Manufacture
3.2.S.2.1 Manufacturer
●Manufacturer
Name: Hubei Yitai Pharmaceutical Co., Ltd

Address: Fengchengyuan, Suburban district of Tianmen city, Hubei province, China
GPS location: N 30.64°, E113.19 °
Post Code: 431700
Contact person: Xiaoyu Wu
Tel: 0728-5331701
Fax: 0728-5335936
E-mail: [email protected]
Website: www.hbyitai.com.cn
FEI No.: 3004844161
DUNS No.: 531149169
Responsibility: Hubei Yitai Pharmaceutical Co., Ltd has full responsibility for the manufacturing
and testing of Acyclovir.
● DMF Holder
Name: Hubei Yitai Pharmaceutical Co., Ltd

Address: Fengchengyuan, Suburban district of Tianmen city, Hubei province, China
GPS location: N 30.64°, E113.19 °
Post Code: 431700
Contact person: Xiaoyu Wu
Tel: 0728-5331701
Fax: 0728-5335936
E-mail: [email protected]
Website: www.hbyitai.com.cn
FEI No.: 3004844161
DUNS No.: 531149169
Responsibility: Hubei Yitai Pharmaceutical Co., Ltd has full responsibility for the manufacturing
and testing of Acyclovir.
1
3.2.S.2.2 Description of manufacturing process and process control

3.2.S.2.2.1 Description of manufacturing process and process control
Manufacturing process of Acyclovir:1,3-Dioxolane(intermediate A) is synthesized from glycol and
paraformaldehyde by using sulfuric acid as catalyst. Intermediate B is synthesized from
intermediate A and acetic anhydride by using p-toluene sulfonic acid as catalyst. Intermediate I is
synthesized from diacetylguanine as starting material and intermediate B using p-toluene sulfonic
acid as catalyst by consendsation. After hydrolyzed by sodium hydroxide, then obtain the
intermediate II. Neutralized the intermediate II by acetic acid, after decolorization by active carbon,
after crystallizing, washing with ethanol, filtration, drying, sieving, mixing and packaging, then
obtain the final product Acyclovir.
3.2.S.2.2.2 Flow-chart of Production
3.2.S.2.2.2.1 Reaction equation
Cyclization reaction
O
OH H2 SO4
HO + (HCHO)n + H2 O
O
Glycol Paraformadehyde 1,3-Dioxolane(Intermediate A)
M.F.:C2 H 6 O 2 M.W.:62.1 M.F.:C3 H 6 O 2 M.W.:74.1
Acylation Reaction
O
O O O
O p-toluene sulfonic acid
+ O
O O +
O OH
O
O
1,3-Dioxolane(Intermediate A) Acetical anhydride 2-Ox-1,4-diacetoxybutane(Intermediate B) Acetical acid
M.F.:C3 H6 O2 M.W.:74.1 M.F.:C4 H6 O3 M.W.:102.1 M.F.:C7 H8 O5 M.W.:176.2 M.F.:C2 H4 O2 M.W.:60.0
Condensation reaction
O
O
O O O O
N
N p-toluene sulfonic acid O HN
O HN
+ O O
O O
+ O
N CH3
N N H3C NH N
NH O O
O
Diacetylguanine(SM) 2-Ox-1,4-diacetoxybutane(Intermediate B) Diacetyl Acyclovir(Intermediate I)
M.F.:C9 H 9 N 5 O 3 M.W.:235.2 M.F.:C7 H 8 O 5 M.W.:176.2 M.F.:C12H 15N 5O 5 M.W.:309.3
Hydrolysis reaction
2
O ONa
C2 H5 OH N
O HN
N O N
O + NaOH
OH + 2CH 3 COONa
CH3 N N
H3C NH N N H2N
O O
Diacetyl Acyclovir(Intermediate I) Sodium Hydroxide Acyclovir Sodium(Intermediate II)

M.F.:C12H 15N 5O 5 M.W.:309.3 M.F.:NaOH M.W.:40.0 M.F.:C8H 10N 5NaO 3 M.W.:247.2
Neutralization reaction
ONa OH
N O N
N N
OH + OH + CH3COONa
N OH
H2N N N N
H2N
O O
Acyclovir Sodium(Intermediate II) Acetical acid Acyclovir

M.F.:C8H 10N 5NaO 3 M.W.:247.2 M.F.:C2 H 4 O 2 M.W.:60.0 M.F.:C8H 11N 5O 3 M.W.:225.2
3.2.S.2.2.2.2 Flow-chart
Preparation of side chain:
3
Glycol
Graphical
description T02201
Pumping to ethylene
Materiel glycol metering tank
Intermediate
Equipment number PFA R02201

Mark in the upper right Stirring and
corner Concentrated heating
Process step sulfuric acid
(Distillation
Process
residue)
parameters Refluxing 30~40 mins
Standard
The key operation Atmospheric

distillation* 90℃~130℃
marked As “*”, the Apply mechanically for 6
key parameters use times ,then to sewage treatment
bold font format T02205
Crude
Dioxolane
R02202
Cooling, ≤20℃,divided into three
NaOH
stirring* times
Stirring for 30~40 mins,

Static layering static standing for 1~1.5h
The under layer to sewage
treatment
Stirring and static Stirring for 30~40 mins, static

NaOH
stratification standing for 1~1.5h
The under layer to sewage
treatment
Stirring and static Jog for 3 times, static standing

NaOH
stratification over 4h
The next batch apply
mechanically the under layer
T02203
Acyclovir
intermediates A Weight yield 90~120%
(Dioxolane)
Unfinished, next page
Remark：PFA is the abbreviation of Paraformaldehyde
4
Continued from previous page Acetic

anhydride
Graphical T02204
description Pumping to acetic
anhydride metering
Materiel tank
Intermediate
Equipment number R02204
Mark in the upper right P - toluene Stirring, heating and
corner 45~55℃
sulfonic acid adding dropwise*
Process step
Process
parameters Keep warming* 58~62℃，4~4.5h
Standard
Sodium acetate Cooling and

The key operation anhydrous neutralization
≤30℃，pH4~5
marked As “*”,
the key parameters
use bold font format
Stirring 20~25℃，2~3h
S02201
Centrifugal
filtration Filter cake to environmental
treatment
Side chain T02213
crude
R02205
Preliminary
≤90℃ fraction to incineration
distillation*
treatment
Residual liquid to incineration
treatment
R02206
R02207
Rectification* Reflux ratio 3:1
≤115~125℃ fraction
50~90℃fraction、residual liquid to
incineration treatment
R02208
Rectification T02222 T02223
T02224 T02225
Acyclovir side Weight yield
chain 118.4~180.0%
5
Condensation
Toluene
Graphical
description T02301~
T02306
Materiel Pumping into
Intermediate toluene
metering tank
Equipment number Diacetyl R02303~
Mark in the upper guanine R02308
right corner
Adding
Process steps P - toluene
Process parameters sulfonic
acid
Heating and
Standards refluxing to No obvious drops of
separate water
Side-chain
The critical water
operations are
marked as "*", T02307~
T02312
the critical Drawing into
parameters are Adding 103~112℃
side-chain
marked as bold dropwise*
metering tank
Toluene
Refluxing 30~40min
T02301
~T02306
Pumping into
Refluxing and 960±20L,
toluene
distillation 16~16.5h
metering tank
Distill toluene for
recovery
R02301
~R02302
Cooling 20~28℃
S02301
Methanol Cooling and ~S02306
Recycling mother
Centrifugal
liquior
filtration
T02316~
T02317
Drawing into
methanol Washing 40~60L/ centrifuge
metering tank
Methanol washing solution
is recycled separately for
incineration
Crude Diacetyl
Weight 509~560kg
acyclovir
6
Hydrolysis reaction
Graphical
description
Process
Materiel ethanol
Intermediate
R02404~
Equipment number R02406
Pumping into Process ethanol
In the upper right corner
the reactor concentration 82~90%
Process step
Process parameters
Sodium
Cooling feed
hydroxide
Standard
Crude Heat
The key
Diacetyl preservation 20~38℃
operation
acyclovir feed
marked "*", the
key parameters
for bold font Heat
format preservation 1~1.5h
stirring
Temperature 1.5~2h，
rise to
Reflux 30~40min
reflux
S02401~
S02407
Cooling and 22~30℃
filtering*
Filtrate to recycle
Acyclovir sodium Weight 437~727kg
7
Neutralization, refining, drying and packaging:
Graphical
description R02501
Acyclovir sodium R02502
Materiel Feeding,Stirrin
Intermediate Purified water
g and
Equipment number acetic dissolving
In the upper acid
right corner T02508
Pump into
Stirring
Acetic acid pH7~8
Process step neutralization *
metering tank
Process parameters
Activated 95~100℃,
Decolorization
Standard carbon 20~30min
Ethanol F02501
The key F02502
operation F02503 Reflux &
Reflux 10~15min
marked "*", the Filter filtration
key parameters
for bold font T02504
format Ethanol
metering tank
R02503 R02505
Process step
或R02504 R02506
Regional
Crystallization 0~5℃
S02501
S02502
Centrifugation S02503
Purified Purified water

25~30min
water washing *
Ethanol
30±5L/Centrifuge
washing
Spin-dry 30~40min
The mother liquor
wastewater treatment
D02501
D02502
Drying Vacuum degree≤-0.09MPa
50~95℃,4~6h
M02501
Smashing
M02502
Sieving
60 or 80 mesh
M02503
Total mixing* 45~60min
D-class Inner packing
Outer packing
Acyclovir
8
3.2.S.3 Characterisation
3.2.S.3.1 Elucidation of structure and other characteristics
3.2.S.3.1.1 Elucidation of structure
The sources of test sample and reference substances are shown in table 3.3-1. Chemical HPLC
chromatographic purity of test sample is shown in Fig.3.2.S.3-1~Fig.3.2.S.3-2.
Table 3.3-1 The sources of test sample and reference substances
Sample Source Purity Test method Batch number Code
Reference substances USP 100% HPLC K0L616 AC-D
Test sample Self-made 99.85% HPLC 02171109 AC-Y
99.91% HPLC 02190501 4
Test sample Self-made 99.87% HPLC 02190502 2
99.91% HPLC 02190503 1
Test organization: Wuhan University Test Center.
Source of test sample and reference substance
Test Sample: Produced by Hubei Yitai Pharmaceutical Co. Ltd. as directed in 3.2.S.2.2
Batch number: 02171109
Reference Substance:USP
Batch number: K0L616
Purity of the test sample:
Chemical purity of test sample 02171109: 99.85%, HPLC chromatogram is shown in Figure
3.2.S.3-1.
A. Elemental analysi
1. Test instrument: VarioEL Ⅲ (Germany)
2. Test method: The content of carbon, hydrogen and nitrogen is determined by automatic element
analyzer.
3. Test results：
Original test report of the Acyclovir (AC-Y) and RS of Acyclovir (AC-D) see Fig. 3.2.S.3-2, and
the test results see in table 3.2.S.3-2.
Table 3.2.S.3-2 Results of elemental analysis for Structure Confirmation
Content C% H% N%
Theoretical value 40.47 5.200 29.51
40.34 5.255 29.38
Sample Determined value
40.27 5.263 29.32
(AC-Y)
Average value 40.305 5.259 29.35
USP Determined value 40.41 5.263 29.42
9
reference 40.37 5.271 29.33

(AC-D) Average value 40.39 5.267 29.38
4. Analysis: Seen from the test result of elemental analysis, the absolute difference between the
determined values of percentage composition of C, H and N with the theoretical value is within
0.5%.
B. Infrared absorption spectrum (IR)
1. Instrument：Nicolet 5700 FT-IR，resolution: 4cm-1, calibrated by polystyrene film.
2. Test condition: KBr pellet.
3. Sample: Acyclovir AC-Y and Acyclovir USP RS AC-D.
4. Test data：See table 3.2.S.3-3, Fig. 3.2.S.3-3 and Fig. 3.2.S.3-4.
Table 3.2.S.3-3 IR data of Acyclovir
Absorption peak (cm-1) Strength of
Vibration
absorption Group
AC-Y AC-D type
peak
Alcohol hydroxy（Intramolecular
3521.7 3521.6 M Stretching
hydrogen chain) O-H
3469.4,3441.3 3469.4,3441.5 S Stretching Free amino (bimodal) N-H
3299.9 3305.2 S Stretching Alcohol hydroxyl O-H
3182.4 3183.8 S Stretching Amide N-H
2856.2 2857.9 M Stretching Alkyl (-CH2-) C-H
2709.2 2716.5 M Stretching Alkyl (-CH2-O-CH2-) C-H
1715.1 1714.6 S Stretching Ketone (hexatomic ring) C=O
Double bond conjugation in two
1633.1 1633.2 S Stretching
rings C=C
1577.0 1578.0 M Stretching Nitrogen heterocyclic C=C+ C=N
1541.0 1541.4 M Stretching Purine C=C+ C=N
Deformatio
1484.5 1485.1 M Alkyl -CH2-
n
1388.7 1388.9 M Stretching Amides C-N
1348.9 1346.8 M Primary amine on the ring C-N
1183.5 1183.6 M Stretching Alcohol C-OH
1105.5 1106.1 S Stretching Ether R-O-R
1049.1 1049.2 M Stretching Primary alcohol C-OH
5. Analysis
(1) 3521.7 cm-1、3299.9 cm-1、1183.5 cm-1、1049.1 cm-1 are the vibrational absorption peaks of
hydroxyl, and 3521.7 cm-1 is stretching vibration of hydrogen bonds in hydroxyl, 3299.9 cm-1 is
stretching vibration of O-H in hydroxyl, 1183.5 cm-1 is stretching vibration of C-OH in alcohols,
1049.1 cm-1 is stretching vibration of C-OH in primary alcohol, which shows there is alcohol
10
hydroxyl in structure.
(2) 3469.4 cm-1, 3441.3 cm-1 and 1348.9 cm-1 are the vibrational absorption peaks of amine, 3469.4
cm-1 and 3441.3 cm-1 are stretching vibration of free -NH2, 1348.9 cm-1 is the vibrational absorption
peaks of primary amine, which shows there is -NH2 in structure.
(3) 3182.4 cm-1, 1715.1 cm-1, 1633.1 cm-1, 1577.0 cm-1, 1541.0 cm-1 and 1388.7 cm-1 are the
vibrational absorption peaks of guanine ring, 3182.4 cm-1 is stretching vibration of N-H bond in
ring, 1715.1 cm-1 is stretching vibration of hexacyclic ketone carbonyl (Note: The carbonyl has both
ketone and amide properties), 1633.1 cm-1 is stretching vibration of C=C conjugate double bond
between two rings, 1577.0 cm-1 is stretching vibration of C=C+C=N nitrogen heterocyclic, 1541.0
cm-1is stretching vibration of C=C+ C=N purine or pyrimidine ring, 1388.7 cm -1 is the vibrational
absorption peaks of C-N amide in ring, which shows there is guanine ring in structure (the structure
of the ring is complex, see nuclear magnetic part).
(4) 2856.2 cm-1 and 1484.5 cm-1 are the vibrational absorption peaks of alkyl, 2856.2 cm-1 is
stretching vibration of -CH2-, 1484.5 cm-1 is deformation vibration of -CH2-, 1105.5 cm-1 is
stretching vibration of R-O-R aliphatic ether, which shows there is alkyl in structure.
(5) 2709.2 cm-1 and 1105.5 cm-1 are the vibrational absorption peaks of ether, 2709.2 cm-1 is
stretching vibration of C-H connected to oxygen, 1105.5 cm-1 is stretching vibration of R-O-R
aliphatic ether, which shows there is ether bond in structure.
Seen from the IR spectrums, the spectrum of sample Acyclovir AC-Y is correspond to that of the
Acyclovir RS AC-D, both have the same groups in structure.
C. Ultraviolet absorption spectrum (UV)
1. Test instrument: Shimadzu UV-2550 UV spectrophotometer.
2. Solvent: methanol.
3. Test sample:Acyclovir sample AC-Y and Acyclovir RS AC-D.
4. Test data: See table 3.2.S.3-3, Fig. 3.2.S.3-5 and Fig. 3.2.S.3-6.
Table 3.3-4 UV data and analysis of Acyclovir
Name λmax-1 (Methanol) (nm)
Sample AC-Y 253
RS AC-D 253
5. Analysis
There are two absorption peak in the sample UV spectrum, the biggest absorption wavelength is
about 253nm, the 242nm absorption peak is mainly produced by π-π* transition of a large conjugate
system formed by guanine ring, that is, the K band absorption of the system, which is consistent
with the structural characteristics of the sample.
11
Seen from the UV spectrums, the spectrum of sample Acyclovir AC-Y is correspond to that of the
Acyclovir RS AC-D, and there is conjugate system in structure, conform to the structure of the
sample submitted for inspection.
D. NMR spectroscopy (1H NMR, 1H-1H COSY)
1. Instrument: Varian mercury VX-300.
2. Test conditions: D6-DMSO
3. Test sample: Acyclovir sample AC-Y and Acyclovir RS AC-D.
4. Test result: See table 3.2.S.3-5~ table 3.2.S.3-6, Fig. 3.2.S.3-7 ~ Fig. 3.2.S.3-12.
Table 3.2.S.3-5 1H NMR data of Acyclovir

Chemical shift (ppm) Proton Corresponding
Multiplicity
AC-Y AC-D number proton
3.458 3.426 Multiple peak 2 C-11
3.472 3.472 Multiple peak 2 C-12
4.729 4.723 Single peak 1 OH
5.358 5.355 Single peak 2 C-10
6.569 6.548 Single peak 2 NH2
7.843 7.836 Single peak 1 C-2
10.764 10.720 Single peak 1 NH
1 1
Table 3.2.S.3-6 H- H COSY data of Acyclovir
Chemical shift (ppm) Related proton chemical shift (ppm) Related proton
Proton No.
AC-Y AC-D AC-Y AC-D No.
C-11 3.458 3.426 3.472 3.472 C-12
C-12 3.472 3.472 3.458, 4.729 3.426, 4.723 C-11, OH
OH 4.729 4.723 3.472 3.472 C-12
C-10 5.358 5.355 -------- -------- --------
NH2 6.569 6.548 -------- -------- --------
C-2 7.843 7.836 -------- -------- --------
NH 10.764 10.720 -------- -------- --------
1 1 1
5. Analysis of H NMR and H- H COSY
12
1
H NMR of Acyclovir sample shows that there are 7 proton hydrogen, the number of protons
represented by the seven signal peaks was 11, which was consistent with the number of hydrogen in
acyclovir molecule. The spectra of heavy water exchange show that three groups of four proton
signal peaks decrease or even disappear, indicating that there are four active hydrogen in the
structure which can exchange deuterium in heavy water, all of which are consistent with the
structure of acyclovir. Seen from the 1HNMR, 1H- 1H COSY and HSQC spectrum of sample, the 11
protons’ signals are attributed as follows:
(1) The signal peak with chemical shift 3.472ppm presents the multiple signal peak with 2 protons,
COSY shows that the signal is correlated with 3.458ppm and 4.729ppm proton signal peak, the
signal peak of 4.729ppm disappears when it is exchanged with heavy water and should be hydroxyl
hydrogen alcohol in acyclovir structure, therefore, the protons are marked as the hydrogen signal
peak of C-12 in structure.
(2) The signal peak with chemical shift 3.458ppm presents the multiple signal peak with 2 protons,
COSY shows that the signal is correlated with 3.472ppm proton signal peak, therefore, the protons
are marked as the hydrogen signal peak of C-11 in structure.
(3) The signal peak with chemical shift 3.458ppm presents the broad peak with 1 proton, COSY
shows that the signal is correlated with 3.472ppm proton signal peak and disappears when
exchanged with heavy water, it should be the active hydrogen signal peak. HSQC shows that there
is no correlation with any carbon, therefore, the protons are marked as the alcohol hydroxyl
hydrogen signal peak of C-12 in structure.
(4) The signal peak with chemical shift 5.358ppm presents the single signal peak with 2 protons,
COSY shows that there is no correlation with any other proton signal peaks. HSQC shows that there
is a correlation with a secondary carbon, therefore, the protons are marked as hydrogen signal peak
of C-10 in structure.
(5) The signal peak with chemical shift 6.569ppm presents the broad peak with 2 proton, the signal
peak with chemical shift 10.764ppmppm presents the broad peak with 1 proton, COSY shows that
there is no correlation with any other proton signal peaks and disappears when exchanged with
heavy water, it should be the active hydrogen signal peak. HSQC shows that there is no correlation
with any carbon, therefore, the 2 protons are marked as the hydrogen signal peak of NH 2 and NH in
structure.
(6) The signal peak with chemical shift 5.358ppm presents the single signal peak with 1 protons,
from the chemical shift value, it may be a proton signal peak connected with benzene ring or other
aromatic carbon atoms. COSY shows that there is no correlation with any other proton signal peaks.
This structural feature is consistent with the only proton on the acyclovir imidazole ring, so the
13
signal should be attributed to the proton signal linked to the structural marker C-2.
The hydrogen NMR spectra of the samples basically coincide with the reference substance, which
all conform to the structure of acyclovir.
E. Nuclear magnetic resonance carbon spectrum (13C-NMR, HMQC, HMBC)
1. Instrument: Varian mercury VX-300.
2. Test conditions: D6-DMSO
4. Test result: See table 3.2.S.3-7~ table 3.2.S.3-9, Fig.3.2.S.3-13~ Fig.3.2.S.3-20.
Table 3.2.S.3-7 13C-NMR and DEPT data of Acyclovir

Chemical shift(ppm) DEPT
Carbon attribution
AC-Y AC-D Types of carbon
60.349 60.350 Secondary carbon C-12
116.857 116.879 Quaternary carbon C-4
138.315 138.280 Tertiary carbon C-2
Table 3.2.S.3-8 HSQC data of Acyclovir
Hydrogen chemical shift (ppm) Related carbon chemical shift (ppm）
Carbon No.
AC-Y AC-D AC-Y AC-D
3.458 3.426 60.349 60.350 C-12
3.472 3.472 70.826 70.826 C-11
5.358 5.355 72.498 72.492 C-10
-------- -------- 116.857 116.879 C-4
7.843 7.836 138.315 138.280 C-2
-------- -------- 151.899 151.892 C-9
14
--------- -------- 154.314 154.312 C-5

--------- -------- 157.362 157.324 C-7
Table 3.2.S.3-9 HMBC data of Acyclovir
Carbon chemical shift (ppm) Related hydrogen chemical shift (ppm）
Carbon No.
AC-Y AC-D AC-Y AC-D
116.857 116.879 7.843（strong） 7.836（strong） C-4
151.899 151.8925.358，7.843（strong） 5.355，7.836（strong） C-9
154.314 154.312 -------- -------- C-5
157.362 157.324 7.843（weak） 7.836（weak） C-7
13
5. Comprehensive analysis of C NMR, DEPT, HSQC and HMBC
13
C NMR of sample shows 8 carbon signals, DEPT shows 3 secondary carbon, 1 tertiary carbon and
4 quaternary carbon, the number of carbon signals and the type of carbon are consistent with the
structure of acyclovir. Through analysis of the 13C NMR, DEPT, HSQC and HMBC, the 8 carbon
signal peaks are attributed as follows:
(1) The carbon signal peak with chemical shift 60.349ppm is secondary carbon signal peak, and
HSQC shows it is correlated with 3.458ppm proton signal peak, therefore, this signal peak is
attributed to C-12 carbon signal peak in structure.
(4) The carbon signal peak with chemical shift 138.315ppm is tertiary carbon signal peak, and
(5) The carbon signal peak with chemical shift 151.899ppm is quaternary carbon carbon signal peak,
and HSQC shows it is correlated with 7.843ppm proton signal peak, therefore, this signal peak is
(6) The carbon signal peak with chemical shift 116.857ppm is quaternary carbon signal peak, and
15
13
The C NMR, DEPT, HSQC and HMBC spectra of the samples basically coincide with the
reference substance, which all conform to the structure of acyclovir.
F. High Resolution Mass Spectrometry (Hi-Mass)
1. Instrument:Varian 320-MS mass analyzer
2. Determination condition: EI ion source
Ion source temperature:200 ℃
Electron energy: 70 eV
4. Test result: See details in Table 3.2.S.3-10 and Fig.3.2.S.3-21~Fig.3.2.S.3-24.
Table 3.2.S.3-10 MS data of Acyclovir
AC-Y AC-D
m/z Relative abundance m/z Relative abundance
225.1 18 225.1 32
180.2 9 179.9 14
164.0 26 164.2 27
150.9 100 150.9 100
134.1 4 134.1 7
109.0 2 109.0 5
5. The possible cleavage mechanism of the fragment peak is as follows:
m/z 225
O O
•
N N
HN HN
N OH N OH
H2 N N H2 N N
O O
m/z 225
m/z 180
16
According to the MS spectrum, monitored under negative ion, m/z 225.1 signal peak is shown and
it is quasi-molecular ion peaks of this substances, m/z 180.2、m/z 164.0、m/z 150.9、m/z 134.1and
m/z 109.0 signal peak are the debris peaks in the structure of the substance, as shown above. The
mass spectrogram date conform to the molecular weight of the structure.
G.Thermal analysis
1. Test instrument: PerkinElmer TG/DTA6300
2. Test content: DTA
3. Test sample: Acyclovir AC-Y and Acyclovir RS AC-D.
4. Test results: See details in Table 3.2.S.3-11 and Fig.3.2.S.3-25~Fig.3.2.S.3-26.

Table 3.2.S.3-11 The DTA data of Acyclovir
AC-Y AC-D
Onset(0C) 0
Peak( C) Onset(0C) Peak(0C)
254.4 264.6 254.0 265.2
5. Analysis:
From DTA, the melting point of Acyclovir sample AC-Y is 254.40C, the melting point of Acyclovir
RS AC-D is 254.00C. The result of Acyclovir sample AC-Y corresponds to that of Clindamycin
Acyclovir RS AC-D.
H. Powder X-ray Diffraction Spectrum (XRD)
1. Test instrument: Holland PANalytical X’Pert Pro X - ray diffraction
2. Test condition: Cu target Kα X-ray; Pipe pressure 40KV; flow rate 80mA; slit DS 1°, RS 0.15mm,
SS1.
4. Test result: See Fig.Fig.3.2.S.3-27~Fig.3.2.S.3-28.
5. Analysis:
The result of powder X-ray diffraction of Acyclovir sample AC-Y and Acyclovir RS AC-D
shows that they are crystalline compounds, the powder X -ray diffraction of them are same,
which shows the sample and RS are the same crystalline compounds.
XRD tests were performed on three validated batches of acyclovir, the product crystal pattern was
consistent without any change.
I. Comprehensive Analysis
Through the elemental analysis, UV and IR spectra, MS and NMR, Acyclovir sample AC-Y
and Acyclovir RS AC-D are mostly same. Acyclovir sample AC-Y corresponds to its’ structure.
Based on the above-mentioned analysis and discussion, the final structural formula of the product
should be：
22
O
N
HN
OH
N N
H2N
O
Conclusion: the chemical structure of the test sample is Acyclovir.

3.2.S.3.1.2 Physical and chemical properties
Appearance: White to off-white, crystalline powder.
Solubility: Slightly soluble in water, very slightly soluble in ethanol (96 per cent), practically
insoluble in heptane. It dissolves in dilute solutions of mineral acids and alkali hydroxides.
Acyclovir is soluble when the PH of aqueous solution is 14, sparingly soluble when the PH of
aqueous solution is 1 and 13, slightly soluble when the PH of aqueous solution is between 2and 12.
Melting Point: Melts at temperatures higher than 250℃, with decomposition.
pH: Not applicable
Optical Rotation: No optical rotation.
Crystal Form: Form V (2/3 hydrate)
pKa (25℃): 2.52, 9.35
Particle size distribution: D10≤15μm, D50≤45μm, D90≤90μm
Isomerism:
There is one isomerism likely to be found in Acyclovir, it is the impurity C in EP. The name is
7-isomer acyclovir, chemical name is 2-amino-7-[(2-hydroxyethoxy) methyl]-1,7-dihydro-
6H-purin-6-one, the chemical structure is:
Stoichiometric ration between the API under the form it is presented and its
pharmacodynamically active compound: not applicable
Refractive index: NA
Partition coefficient: 0.33(water: n-butanol=1:1)
UV maxima absorption: A254±2nm=0.3~0.6, 10μg/mL in water.
Hygroscopicity: very slightly hygroscopic.
Solubility in physiological pH (37℃): pH1.2: 13.2mg/mL; pH4.0: 3.0mg/mL; pH6.8: 2.6mg/mL;
water: 2.3mg/mL.
Polymorphism: According to the reference documents, Acyclovir shows polymorphism. It has 6
kinds of crystal forms, which are anhydrous forms (Form I, Form II, Form III and Form IV), Form
V(2/3 hydrate), Form VI(2 hydrate). The crystal form of Acyclovir manufactured in our company is
Form V (2/3 hydrate).
23
3.2.S.3.2 Impurities
3.2.S.3.2.1 List of impurities in acyclovir
Table 3.2.S.3.2-1 The impurities in Acyclovir
Whether in
Name Structure Origin Limit
specification
N-2,9-Diacetyl
guanine Starting material 0.05% No
(Impurity L)
Impurity A Process impurity - No
Impurity B Process impurity & Yes, as specified

0.7%
(Guanine) degradation product impurity
Impurity C Process impurity - No
Impurity F Process impurity - No
Impurity G Intermediate I - No
Impurity I Process impurity - No
Impurity J Process impurity - No
Impurity K Process impurity - No
Impurity M Process impurity - No
Impurity N unknown unknown - No

Impurity O unknown unknown - No
Impurity P Process impurity - No
Impurity Q Process impurity - No
24
Impurity R Process impurity - No
Toluene Residual solvent 0.089% Yes
Methanol CH3OH Residual solvent 0.3% Yes

Ethanol CH3CH2OH Residual solvent 0.5% Yes
Benzene Residual solvent 0.0002% No
OH
Acetic acid Residual solvent 0.5% Yes
O
Glycol Raw material 0.062% No
Heavy metals - Inorganic impurities 10ppm No
O
Methyl S O Genotoxic impurities 0.18ppm No
p-toluenesulfonate
O
O
Ethyl S O Genotoxic impurities 0.18ppm No
p-toluenesulfonate
O
Cd - Elemental impurities 0.2ppm No
Pb - Elemental impurities 0.5ppm No
As - Elemental impurities 1.5ppm No
Hg - Elemental impurities 0.3ppm No
Co - Elemental impurities 0.5ppm No
V - Elemental impurities 1ppm No
Ni - Elemental impurities 2ppm No
Cu - Elemental impurities 30ppm No
25
3.2.S.4 Control of Drug Substance

3.2.S.4.1 Specification
Items Specification
Appearance White or almost white crystalline powder
Corresponds to IR spectra of the reference.
HPLC identification
Identification The retention time of the major peak in the chromatogram of the
sample solution corresponds to that in the chromatogram of the
Standard preparation, as obtained in the Assay.
Ordinary impurities Examine by thin-lager chromatography ≤1%
Limit of Guanine NMT 0.7%.
Water NMT 6.0%
Methanol NMT 0.3%
Residual Ethanol NMT 0.5%
solvent Toluene NMT 0.089%
Acetic acid NMT 0.5%
Acyclovir contains NLT 98.0% and NMT 101.0% of C8H11N5O3,
Assay
calculated on anhydrous basis.
3.2.S.4.2 Analytical Procedures
Appearance: White or almost white crystalline powder.
Identification:
a) IR Corresponds to IR spectra of the reference.
b) HPLC The retention time of the major peak of the samples corresponds to that of the reference
standard, as obtained in Assay.
Ordinary impurities:
Examine by thin layer chromatograph
Thin layer board: Silicon GF254 board (coating thickness is about 0.25mm)
Developing solvent: Chloroform: methanol: ammonia (80:20:2)
Examine wavelength: 254nm
About 100 mg (exact sample) of the substance is placed in a 100 ml volumetric flask, dissolved in
dimethylsulfoxide; the volume of the solution is adjusted to the mark with a dimethylsulfoxide and
mixed.
26
Test solution: Accurately weigh 100mg of the substance and place in 10ml-volumetric flask, then
add DMSO to dissolve and dilute to the mark, the test solution is obtained.
Standard solution A (1%): 1.0 ml of the resulting solution is placed in a 10 ml volumetric flask; the
volume of the solution is adjusted to the mark with a dimethylsulfoxide and mixed (about 100 µg of
acyclovir /ml).
Standard solution B (0.5%):5.0 ml of the resulting solution is placed in a 100 ml volumetric flask;
the volume of the solution is adjusted to the mark with a dimethylsulfoxide and mixed (about 50 µg
of acyclovir /ml)
Application: the test solution , the standard solution A and standard solution B should be separately
applied to the chromatographic plate start line.
The plate with the applied samples should be dried in a warm air stream, cooled, then placed in a
chromatographic chamber, chromatographed using an ascending technique; when the front of the
plate passes about ¾ of the length of the plate, it should be removed, dried until the solvent traces
are removed.
Detection:
Evaluate in UV light with the wave length 254 nm.
Any spot on the chromatogram of the test solution, except the main spot, should not exceed the
main spot in the chromatogram of the standard solution (not more than 1.0%) in terms of the total
amount and intensity of absorption.
Guanine limit and assay:
Prepare the solutions immediately before use.
Chromatographic system
Mobile phase: 0.1% glacial acetic acid aqueous solution
Column: padding L1, 4.6mm×250mm, 10μm
Detector wavelength: 254nm
Flow rate: 3.0mL/min
Injection: 20μl
Solution preparation:
System suitability 1 solution: respectively take 20mg Acyclovir reference substance and guanine to
27
200ml graduated flask, dissolve with appropriate 0.1mol/L sodium hydroxide solution and dilute to
scale with water. Shake well.
System suitability 2 solution: take 8.75mg guanine to 500ml graduated flask, dissolve with 50ml
0.1mol/L sodium hydroxide solution and dilute to scale with water. Shake well. Then accurately
take 2 ml solution to 50ml graduated flask, dilute to scale with water. Shake well.
Guanine reference solution: same as System suitability 2 solution.
Acyclovir reference solution: accurately take 25mg Acyclovir reference substance to 50ml
graduated flask, dissolve with 5ml 0.1mol/L sodium hydroxide solution and dilute to scale with
water. Shake well. Then accurately take 10ml solution to 50ml graduated flask, dilute to scale with
0.1mol/L sodium hydroxide solution. Shake well.(Prepare 2 solutions)
Sample solution: accurately take 100mg sample to 200ml graduated flask, dissolve with 20ml
0.1mol/L sodium hydroxide solution and dilute to scale with water. Shake well. Then accurately
take 10ml solution to 50ml graduated flask, dilute to scale with 0.1mol/L sodium hydroxide
solution. Shake well.(Prepare 2 solutions: a and b)
Guanine limit:
System suitability injection sequence
One blank solution;
Six system suitability 1 solution;
Six system suitability 2 solution;
In the first needle of solution chromatography, the resolution between guanine and acyclovir peak
should not be less than 2.0, the trailing factor should not exceed 2, the RSD of 6 needle acyclovir
peak area should not exceed 2.0%.
The RSD of guanine peak area should not more than 2.0%.
Injection sequence
Six acyclovir reference solution 1;
Two acyclovir reference solution 2 ;
One acyclovir reference solution 1;
One sample solution 1a;
One sample solution 1b;

28
One sample solution 2a;
One sample solution 2b;
……
Starting with injection of sample solution, the acyclovir reference solution 1 is repeated with 1
injection for each interval of not more than 2 hours. At the end of the experiment, acyclovir
reference solution was given 1 injection.
The RSD of 6 needle acyclovir reference solution 1 peak area should NMT 2.0%.
Recovery :
W1  A2
100%
W2  A1
W1——weight of acyclovir reference substance 1, mg；

W2——weight of acyclovir reference substance 2, mg；
A1——Average area of main peak of 6 needle acyclovir reference substance 1, pA；
A2——Average area of main peak of 2 needle acyclovir reference substance 2, pA；
After each injection of acyclovir reference solution 1, the RSD should be calculated together with
the peak area of acyclovir reference solution 1 in front, and the result was not more than 2.0%.
Calculation of RSD:
 (x − x )
n 2
i =1 i
RSD= n −1  100%
x
−
x —The average value determined;
n—Sum of sample or determination times;
xi—The value of the i times determined.
Note: The average area of acyclovir reference solution 1 as the peak area of acyclovir (Two
injection before and after the injection of solution next to the sample) .
Calculation:
Guanine limit formula:
Guanine%=（ru/rs）×(Cs/Cu) ×P×100%
ru——Peak area of guanine in sample solution, mAU;
rs——Peak area of guanine in guanine reference solution, mAU;
29
Cs——Concentration of guanine in guanine reference solution, ug/ml;
Cu——Concentration of acyclovir in sample solution, ug/ml;
P——Guanine reference substance content, %;
Note: Guanine reference peak area is average area of six injection in guanine reference solution,
peak area of sample calculated with sample solution 1a or 1b.
Assay formula:
Assay（%）=（ru/rs）×(Cs/Cu) ×P×100%
ru——Main peak area in sample solution，mAU；
rs——Main peak area in reference solution，mAU；
Cs——Concentration of acyclovir in reference solution，mg/ml；
Cu——Concentration of acyclovir in sample solution，mg/ml；
P——Acyclovir reference substance content，%；
Allowable deviation : The relative average deviation of the results of 2 injection per sample is not
more than 2.0%.
Acceptance criteria: Acyclovir contains NLT 98.0% and NMT 101.0% of C8H11N5O3, calculated
on anhydrous basis.
Water: ≤6.0%, determined on 0.5g of the sample with Karl-Fisher method.
Residual solvent:
a) Determination for Methanol, Ethanol and Toluene
Detector: FID
Column: DB-624, 30m×0.32mm×1.8μm
Temperature of injection port: 220 ℃
Temperature of detector: 300 ℃
Temperature of column: Keep at 60℃for 5min, heat to 200℃ at the rate of 10℃/min，then keep for
10min.
The distribution ratio of flow: 1:30
The flow rate of H2: 40mL/min
The flow rate of air: 400mL/min
The flow rate in column: 1.0mL/min
30
Temperature of equilibrium: 85℃
Equilibrium time: 40 min
Solution preparation
Blank solution: Dimethyl Sulfoxide (DMSO).
Standard solution: separately weigh and transfer about 0.3g of methanol, 0.5g of ethanol and 0.089g
of toluene to a 100mL volumetric flask, and dilute with DMSO to the volume, and then shake well.
Transfer 10mL of this solution to a 100mL volumetric flask, and dilute with DMSO to volume, and
then shake well; and transfer 2mL of this solution to a 20mL headspace bottle and seal with lid.
Sample solution: Weigh and transfer about 1.0g of sample to a 10mL volumetric flask, dissolve and
dilute it with DMSO to volume, shake well, and transfer 2mL of this solution to a 20mL headspace
bottle and seal with lid.
Procedure: Separately inject the blank solution (1 or 2 injection), standard solution (6 injections)
and the sample solution (1 injection), record the chromatogram.
In the chromatogram obtained with 6 injections of standard solution, the RSD of peak area for each
solvent is not more than 10.0%.
In the chromatogram obtained with the first standard solution, the theoretical plate number is not
less than 5000, the tailing factor is not more than 2.0, and the resolution between any residual
solvent peaks is not less than 2.0.
Calculate the residual solvent content by the formula:
AS i  c Ri
Ci (%)= ×100%
ARi  c S
In which: Ci = Assay of residual solvent i in the sample, %.
ASi =peak area of residual solvent i in the sample solution.
ARi =peak area of residual solvent i in the standard solution.
cRi = concentration of residual solvent i in the standard solution, mg/mL.
cS = concentration of the sample solution, mg/mL.
b) Determination for Acetic acid
Detector: FID
31
Column: DB-ALC2, 30m×0.53mm×2μm
Temperature of injection port: 200 ℃
Temperature of detector: 280 ℃
Temperature of column: Keep at 60℃for 2min, heat to 100℃ at the rate of 10℃/min, then keep for
4min, heat to 220℃ at the rate of 30℃/min, then keep for 10 min.
The distribution ratio of flow: 1:10
The flow rate of H2: 40mL/min
The flow rate of air: 350mL/min
The flow rate in column: 5.0mL/min
Injection: 1.0μL
Solution preparation
Reference solution: Weigh and transfer about 0.05g of acetic acid to a 100mL volumetric flask, and
dilute with DMSO to volume, and then shake well.
Sample solution: Weigh and transfer about 1.0g of sample to a 10mL volumetric flask, dissolve it
with DMSO, and dilute with DMSO to volume, and then shake well.
Test methods:
Separately chromatograph blank solution for 1 injection (or 2 injections), reference solution for 6
injections, sample solution for 1injection, and record the chromatogram.
In the chromatogram obtained with 6 injections of reference solution, the RSD of peak area is not
more than 10.0%.
In the chromatogram obtained with the first reference solution, the theoretical plate number is not
less than 5000, the tailing factor is not more than 2.0.
The residual solvent in the sample solution is calculated by the following formula.
(Note: the peak area of acetic acid reference solution needs to be calculated with the average value
of 6 injections of acetic acid)
Calculation:
AS i  c Ri
Ci (%)= ×100%
ARi  c S
32
In which: Ci = Assay of residual solvent acetic acid in the sample, %.
ASi =peak area of residual solvent acetic acid in the sample solution.
ARi =peak area of residual solvent acetic acid in the reference solution.
cRi = concentration of residual solvent acetic acid in the reference solution, mg/mL.
cS = concentration of the sample solution, mg/mL.

3.2.S.4.3 Validation of Analytical Procedures
3.2.S.4.3.1 Validation of acyclovir residual solvents
3.2.S.4.3.1.1 Validation of residual solvents (methanol, ethanol, acetone and toluene)

1. Abstract
1.1 According to the requirements of ICH for the content of residual solvent in medicine, we
validate the analytical method for the methanol, ethanol, acetone and toluene solvents used in the
production process.
1.2 The analytical method belongs to the quantitative analysis of impurities, therefore, the contents
needed to be validated are: system suitability, precision, specificity, linearity, range, Detection limit,
quantitation limit, accuracy, robustness, solution stability, specific parameters and standards are
shown below.
Validation
Acceptance criteria Results
contents
Theoretical plates of every solvent peak are more than
RSD of solvents peak area≤10.0%;
System 5000, resolution between each other is more than 1.5,
theoretical plates (N) ≥5000;tailing
suitability RSD% of peak areas are all less than 10.0%, it met
factor (T) ≤2.0;resolution (R) ≥1.5
requirements of system suitability.
There was no significant There was no interference from blank solution in
interference peak near the peak determination of residual solvents, resolution between
position of the solvent in the blank residual solvents were all more than 1.5; it met the
Specificity
solution; resolution of the solvent requirements of specificity.
peak and the adjacent peak should
be not less than 1.5.
Solvent Range, µg/mL Linear equation r
The linear regression equation of Methanol 10.309~360.2 Y=1975.15X+26 0.9989
the residual solvent concentration 76 23.87
Linearity and
and peak area is listed, and the Ethanol 8.236~600.52 Y=2425.7X+257 0.9985
range
correlation coefficient is not less 7.3
than 0.99. Acetone 2.007~601.70 Y=12591X-2818 0.9987
1
33
Toluene 1.824~108.55 Y=12448X-458.5 0.9982

4
When chromatographic condition have little change,
Change the chromatographic
Robustness resolution between each solvent peaks are all more than
system lightly, resolution (R) ≥1.5
1.5; it meets requirements of robustness.
Solvent LOQ ,g/mL LOD, g/mL
-5
LOD : S/N is within the range 2~4; Methanol 1.031×10 3.093×10-6
LOD and
LOQ :S/N is within the range Ethanol 8.236×10-6 2.471×10-7
LOQ
9~11. Acetone 2.007×10-6 6.021×10-7
Toluene 1.824×10-6 5.472×10-7
Single recovery rate for each
Single recovery rate for each solvent is in the range of
Accuracy solvent should be 80% ~ 120%,
80%~120%, RSD of the recovery rate for each solvent is
(recovery) RSD of the recovery rate for each
less than 10.0%, meet the requirements of accuracy.
solvent should be less than 10.0%.
RSD of determination value for
Precision/Rep RSD of determination value for each residual solvent
each solvent should be less than
eatability, n≥6 <10.0%, meet the requirements of repeatability.
10.0%.
Precision RSD of determination value for RSD of determination value for each solvent tested at
/Intermediate each solvent tested at different date different date is less than 10.0%, meet the requirements of
precision should be less than 10.0%. intermediate precision.
In 12 h, RSD of peak area for each solvent in the standard
solution is NMT 10.0%, the solution was basically stable;
Solution RSD of peak area for each solvent
no solvent was detected in the sample solution within 12
stability is not more than 10%.
hours, no degradation solvent is produced, the solution
was basically stable.
2. Chromatographic system and Chromatographic condition
2.1 Chromatographic system
Item Instruments
GC GC7890B
Workstation and version number Openlab EZchrom
Correction expiry date Expiry date: September,5,2017
2.2 Chromatographic condition

GC parameter
Column temperature Keep 60℃for 5min, heat to 200℃10℃/min, and keep 1min.
Detector temperature 300℃ Temperature of injection port 220℃
Carrier gas N2 Carrier gas flow rate 1.0 ml/min
Hydrogen flow rate 40 ml/min Air flow rate 400 ml/min
34
Split ratio 30:1 Detector type FID

Column size 30m×0.32mm×1.8μm Column mode DB-624
Headspace equilibrium temperature 85℃ Equilibrium time 40 min.
3 Validation test
3.1 System suitability
Objective: Evaluating the complete system combined with analysis equipment, the experiment
operation, and the samples for analysis
Acceptance criteria:
Calculate the testing parameters of target peak in Chromatogram of standard solution:
RSD of target peak area in chromatogram for 6 successive injections of standard solution is not
more than 10%.
Number of theoretical plates (N): ≥5000
Resolution (R): ≥1.5(between residual solvents)
Tailing factor(T): ≤2.0
3.1.1 Solution preparation
Stock solution:
Methanol stock solution: Weighing and transferring 3.0023g of methanol solution to 100ml
volumetric flask, and dilute with DMSO to volume, and then shake well.
Ethanol stock solution: Weighing and transferring 5.0043g of ethanol to 100ml volumetric flask,
and dilute with DMSO to volume, and then shake well.
Acetone stock solution: Weighing and transferring 5.0142g of acetone to 100ml volumetric flask,
and dilute with DMSO to volume, and then shake well.
Toluene stock solution:Weighing and transferring 0.9046g of toluene to 100ml volumetric flask, and
dilute with DMSO to volume, and then shake well.
Standard solution:Transferring 1ml of each stock solution to 100ml Volumetric flask, and dilute
with DMSO to volume, and then shake well; and transfer 2ml of it to headspace bottle and seal with
lid.
Sample solution: Weighing and transferring 1.0g of sample to 10ml volumetric flask, heat and
35

DMF For Acyclovir: Index

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DMF For Acyclovir: Index

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DMF FOR ACYCLOVIR

3.2.S.1 General Information ...................................................................................................................................... 2

3.2.S.1 General Information

Chemical Name: 2-amino-9-[(2-hydroxyethoxy)methyl]-1,9-dihydro-6H-purin-6-one

CAS Registry Number: 59277-89-3

Molecular Formula: C8H11N5O3

Molecular Weight: 225.20

3.2.S.1.3 General Properties

Melting Point:Melts at temperatures higher than 250℃, with decomposition.

pH: Not applicable

Optical Rotation: No optical rotation.

Crystal Form: Form V (2/3 hydrate)

Particle size distribution: D10≤15μm, D50≤45μm, D90≤90μm

7-isomer acyclovir, chemical name is 2-amino-7-[(2-hydroxyethoxy)methyl]-1,7-dihydro-

6H-purin-6-one, the chemical structure is:

pharmacodinamically active compound: not applicable

Partition coefficient: 0.33(water: n-butanol=1:1)

UV maxima absorption: A254±2nm=0.3~0.6, 10μg/ml in water.

Hygroscopicity: very slightly hygroscopicity

Solubility in physiological pH (37℃): pH1.2: 13.2mg/ml; pH4.0: 3.0mg/ml; pH6.8: 2.6mg/ml;

Polymorphism: According to the reference documents, Acyclovir show polymorphism. It has 6

Form V (2/3 hydrate).

Name: Hubei Yitai Pharmaceutical Co., Ltd

and testing of Acyclovir.

Name: Hubei Yitai Pharmaceutical Co., Ltd

and testing of Acyclovir.

3.2.S.2.2 Description of manufacturing process and process control

M.F.:C9 H 9 N 5 O 3 M.W.:235.2 M.F.:C7 H 8 O 5 M.W.:176.2 M.F.:C12H 15N 5O 5 M.W.:309.3

Diacetyl Acyclovir(Intermediate I) Sodium Hydroxide Acyclovir Sodium(Intermediate II)

Acyclovir Sodium(Intermediate II) Acetical acid Acyclovir

Equipment number PFA R02201

The key operation Atmospheric

Stirring for 30~40 mins,

Stirring and static Stirring for 30~40 mins, static

Stirring and static Jog for 3 times, static standing

Unfinished, next page

Remark：PFA is the abbreviation of Paraformaldehyde

Continued from previous page Acetic

Sodium acetate Cooling and

Unfinished, next page

Acyclovir sodium Weight 437~727kg

Unfinished, next page

Neutralization, refining, drying and packaging:

Purified Purified water

Total mixing* 45~60min

D-class Inner packing

reference 40.37 5.271 29.33

Table 3.2.S.3-5 1H NMR data of Acyclovir

Table 3.2.S.3-7 13C-NMR and DEPT data of Acyclovir

--------- -------- 154.314 154.312 C-5

4. Test results: See details in Table 3.2.S.3-11 and Fig.3.2.S.3-25~Fig.3.2.S.3-26.

Conclusion: the chemical structure of the test sample is Acyclovir.

Impurity A Process impurity - No

Impurity B Process impurity & Yes, as specified

Impurity C Process impurity - No

Impurity F Process impurity - No

Impurity I Process impurity - No

Impurity J Process impurity - No

Impurity K Process impurity - No

Impurity M Process impurity - No

Impurity N unknown unknown - No

Impurity P Process impurity - No

Impurity Q Process impurity - No

Impurity R Process impurity - No

Toluene Residual solvent 0.089% Yes

Methanol CH3OH Residual solvent 0.3% Yes

Benzene Residual solvent 0.0002% No

3.2.S.4 Control of Drug Substance

Appearance: White or almost white crystalline powder.

a) IR Corresponds to IR spectra of the reference.

standard, as obtained in Assay.

Examine by thin layer chromatograph

Developing solvent: Chloroform: methanol: ammonia (80:20:2)

Examine wavelength: 254nm

applied to the chromatographic plate start line.

Evaluate in UV light with the wave length 254 nm.