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SOFOSBUVIR

SPECIFICATION SAMPLING AND HANDLING


STANDARD PACK#
1 to 50KG packed in double bag, and hermetically sealed, clear PE bags enclosed in a
triple laminated kept in a fiber drum.
STORAGE REQUIREMENTS
Store in well-closed, at room temperature.

SAMPLING
Sample equal quantity from each container. Sample a minimum of 5 g from each of
the containers into individual, non-toxic, self-sealing, transparent polythene bag
bearing Sample for analysis label kept in another black self-sealing polythene bag
bearing Sample for analysis label.
QUANTITY OF COMPOSITE SAMPLE FOR ANALYSIS
10g
REANALYSIS PERIOD ##
One year
CATEGORY
Antiviral
HAZARDS AND PRECAUTIONS, IF ANY
Wear nose mask and hand gloves while sampling. Avoid inhalation or exposure to
skin and reseal the containers immediately after sampling.

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Xiamen Halosyntech Co., Ltd.

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SOFOSBUVIR SPECIFICATION STANDARDS

Molecular formula: C22H29FN3O9P


Molecular weight: 529.45
Chemical name:

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(S)-isopropyl-2-((S)-(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hy
droxy-4-methyltetrahydrofuran-2-yl)methoxy)-(phenoxy)phosphorylamino)propanoate

DESCRIPTION:
A white to off white powder.

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SOLUBILITY:
Slightly soluble in water (pH 1.2-7.7), freely soluble in ethanol and acetone, soluble in
2-propanol, and insoluble in heptane.

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IDENTIFICATION:
A) <By Infrared absorption spectrophotometry>
The Infrared absorption spectrum is concordant with the spectrum obtained from
the similar preparation of Sofosbuvir Test Stands /Working Standard.
B) <By High performance liquid chromatography>
The principal peak in the chromatogram obtained with the test solution is similar in
retention time and size to the principal peak in the chromatogram obtained with
reference solution.

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WATER:
Not more than 0.50% w/w.

CLARITY AND COLOUR OF SOLUTION


1% sample solution in Ethanol should not more intense than reference solution B9
and reference suspension II.
HEAVY METALS:
Not more than 0.002%
MELTING POINT
120~128.
RESIDUAL SOLVENTS: <By Gas chromatography>
Dichloromethane: Not more than 0.06%
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Tetrahydrofuran: Not more than 0.072%


Methanol: Not more than 0.1%
Acetic acid: Not more than 0.3%
Ethyl acetate: Not more than 0.1%
CHROMATOGRAPHIC PURITY: <By High performance liquid chromatography>
Rp-isomer: Not more than 0.2%
Impurity ANot more than 0.3%
Impurity BNot more than 0.15%
Impurity CNot more than 0.2%
Impurity DNot more than 0.15%
Impurity ENot more than 0.2%
Any other impurity: Not more than 0.1%
Total impurities: Not more than 1.0%

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ASSAY: <By High performance liquid chromatography>


Sofosbuvir contains not less than 98.0% w/w and not more than 102.0%w/w of
C22H29FN3O9P calculated on anhydrous sample.








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SOFOSBUVIR SPECIFICATION TESTS AND METHODS


1. DESCRIPTION
Examine the individual sample taken.
#Carry out Visual inspection on the individual sample.
## Examine the individual samples. Carry out identification on all individual sampled
containers.
Reporting: Report as Complies/Does not Comply.

2. SOLUBILITY
Dissolve 1g of accurately weighed sample to 10ml of ethanol and acetone,
respectively, check the solubility; Dissolve 1g of accurately weighed sample to 30ml
of 2-propanol, check the solubility; Dissolve 0.1g of accurately weighed SOF to 100ml
of water, check the solubility; Dissolve 0.1g of accurately weighed SOF to 1000ml of
heptane, check the solubility.
Reporting: Report as Complies/Does not Comply.

3. IDENTIFICATION

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A)<By Infrared absorption spectrophotometry>


Examine the Sample in potassium bromide dispersion and compare with the
spectrum obtained from the similar preparation of Sofosbuvir Test Standard /
Working Standard
ReportingReport as Complies/Does not Comply.
B) <By High performance liquid chromatography>:
Compare the retention time of the principal peak obtained in the chromatogram of
the Test solution with that of the Standard solution as obtained in the test for Assay.
ReportingReport as Complies/Does not Comply.

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4.

WATER

Determine on 0.2 g of the sample, accurately weighed, using methanol as solvent.


Reporting: Report as a value in% w/w.

5.

CLARITY AND COLOUR OF SOLUTION

a) Compare the colour of 1% of sample solution in Ethanol with the reference


solution B9.
b) Compare the clarity of 1% of sample solution in Ethanol with the reference
suspension II. Compare the solution visually in 50ml nessler's cylinders.
ReportingReport as Complies/Does not Comply.

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6.

HEAVYMETALS

Determine on 1 g of the sample. Prepare the reference solution using 2 mL of lead


standard solution.
ReportingReport as Complies/Does not Comply.

7.

MELTING POINT

Reduce the substance to a very fine powder Introduce into a capillaryglass tube, a
sufficient quantity of the dry powder to form acompact column 4 to 6 mm high. Heat
the bath until the temperature is about 10 below the expected melting temperature.
the temperature to rise at a rate of about 10 per minute.
Reporting: Report as a value in .

8. RESIDUAL SDLVENTS:<By Gas chromatography>


Solvents in determination
Name of solvent
Dichloromethane

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Methanol
Tetrahydrofuran

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Ethyl acetate
Acetic acid

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Class of solvent
Class 2
Class 2
Class 2
Class 3
Class 3

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Testing of other Class 12 and 3 residual solvents and other organic solvents is not
required since these solvents are not used during manufacturing process.
Report the same as `Complies` with remark as `Testing of other Class 12 and 3
residual solvent and other organic solvents is not required as these solvents are not
used during manufacturing process;
Instrument: A Gas chromatograph capable of temperature programming, equipped
with a capillary column, split/splitless injector, a flame ionization detector.
Chromatoaraphic conditions:
Chromatographic Conditions:
Chromatographic Column: 30m 0.53mm 3m, DB-624
Column temperature: Hold at 40for 8min, then increase at a rate of 15 per min
to 150, Further increase at a rate of 30/min to 210 and hold for 5min.
Flow rate: 5.0ml/min
Split ratio: 1:10
Carrier gas: Nitrogen
Purge flow: 3.0ml/min
Injection port temperature: 200C
Detector temperature: 250C
Injection volume: 1.0l
Reference Stock Solution I Transfer about 0.20ml of 2-pentanone, accurately
measured, to a 1000ml volumetric flask as internal standard. Dilute with a mixture

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of dimethyl sulfoxide to volume, and mix.


Reference Stock Solution II Weigh accurately about 36 mg of Tetrahydrofuran, and
30 mg of Dichloromethane, 50 mg of Methanol, 150 mg of Ethyl acetate, 50 mg of
acetic acid into a 100 ml volumetric flask containing about 50 ml of reference stock
solution I. Dilute to volume with diluent and shake vigorously.
Reference Standard Solution Transfer 10ml of reference stock solution II, accurately
measured, to a 100ml volumetric flask, dilute with reference stock solution I to
volume, and mix to prepare reference standard solution.
Test solution Transfer about 0.25g of SOF, accurately weighed, to a 5 ml volumetric
flask, then dilute with reference stock solution I to volume, mix.
Procedure Inject 1.0l of the reference standard solution into the chromatograph,
record the chromatograms and measure the peak areas. System suitability is
checked by that the resolution, R, between each of two adjacent peaks from
concerned solvents and internal standard (2-pentanone), is NLT 1.5. Then inject 1.0ul
of the test solution, and measure the peak areas. Calculate the percentage of each
residual solvent in the portion of SOF taken by the formula:

( AT / ATI ) C S
100%
( AS / ASI ) CT

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where,
CS = the concentration of the respective analyte in the Standard solution, in mg / ml;
CT =the concentration of test solution, in mg /ml;
AT =the peak area of respective analyte in the test solution;
ATI = the peak area obtained from the internal standard in the test solution;
AS = the peak area of respective analyte in the standard solution;
ASI =the peak area obtained from the internal standard solution in the standard
solution.
Reporting: Report as a value in ppm
If no peak is observed at the expected retention time of specified solvent, then
report as "N.D".

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9. CHROMATOGRAPHIC PURITY: <By High performance liquid


chromatography>

Chromatographic conditions:
Standard column type
C18,4.6*150mm,5 um
Detection
spectrophotometer at 260 nm.
Flow rate
1.2 ml/min
Column oven temperature
35
Injection volume
20 ul
Diluent
DMSO
Mobile phase A
0.05%TFA IN(5%MEOH)
Mobile phase B
0.05%TFA IN(95%MEOH)
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The Gradient Program is as follows:

0
7
15
30
35
50
55
60
65

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98
51
51
30
30
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0
98

2
2
49
49
70
70
100
100
2

Preparation of sample solution: Weigh accurately about 50 mg of the sample and


transfer into a 25 ml volumetric flask. Dissolve in 15 ml of diluent and dilute to
volume with diluent and mix.
Preparation of Reference solution:
Dilute 1.0 ml of sample solution to 100.0 ml with the diluent and mix. Further, dilute
1.0 ml of this solution to 10.0 ml with the diluent and mix.
System suitability solution:
Solution (A): Weigh accurately about 10.0mg of Rp-isomer sample and transfer into a
100 ml volumetric flask.
Solution( B): Weigh accurately about 50.0mg of sofosbuvir sample and transfer into a
5 ml volumetric flask .
Mix 2.0 mL of solution A and 1.0 mL of solution (B) and dilute to 10.0 ml with
diluent.
Note: Prepare reference solution and sample solution in duplicate. Inject freshly
prepared solution.
Procedure: Separately inject equal volumes of solutions as per Sequence of injection
into the chromatograph and record the peak area responses for the major peaks and
check for the System suitability requirements.
Sequence of injection:
1) Blank
2) System suitability A1,.,6
3) Blank
4) Reference solution A
5) Reference solution B
6) Blank
7) Sample solution A
8) Sample solution B
System suitability requirements:
The test is not valid unless
1) The Relative Standard Deviation for the peak area response and retention time for
the peak of Sofosbuvir for replicate injections of Standard solution A is not more than
5.00% and 1.00% respectively.
2) The Tailing factor for the peak of Sofosbuvir obtained in the chromatogram of

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individual Standard solution A injected in replicates and Standard solution B is not


more than 2.0 (Report the value obtained from the last injection of replicate of
Standard solution A).
3) The Column efficiency for the peak of Sofosbuvir obtained in the chromatogram of
individual Standard solution A injected in replicates and Standard solution B is not
less than 2000 theoretical plates.
4) The resolution: minimum 1.5 between the peaks due to the Rp-isomer and
Sofosbuvir.
Calculations:
%specified impurity
=

A CS
100%
B CT

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%Total impurities=%Sum of all specified impurities and unspecified impurities.


Where,
A=Peak area response of Impurity obtained in the chromatogram of sample
solution.
B=Average peak area response of Sofosbuvir obtained in the chromatogram of
Reference solution.
CS= the concentration of Standard solution, in mg/ml;
CT = the concentration of Test solution, in mg/ml; Reporting:
a) If the value of the impurity is detected, then report in %.
b) If no peak is observed at the expected retention time of specified impurity, then
report as "N.D".
c) While calculation the values of total impurity, report total impurity in %. If no
impurity, are observed, report as "N.D".

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10. ASSAY: <By High performance liquid chromatography>

10.1 Chromatographic conditions


Standard column type
Detection
Flow rate
Column oven temperature
Injection volume
Mobile phase A
Mobile phase B
Mode
Run time
Diluent
Preparation of standard
solution

C18,4.6*150mm,5um
spectrophotometer at 260 nm.
1.2 ml/min
35
10 ul
0.05%TFA IN(5%MEOH)
0.05%TFA IN(95%MEOH)
Isocatic ( Mobile phase A: Mobile phase B=52:48)
About 20 min
DMSO
Weigh accurately about 25 mg of Sofosbuvir Test
Standard / Working Standard and transfer into a 25 ml
volumetric flask. Dissolve and dilute to volume with

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diluent and mix.


Preparation of sample solution: Weigh accurately about 25mg of the Sample and
transfer into a 25 ml volumetric flask. Dissolve and dilute to volume with diluent and
mix.
Note: Prepare standard solution and sample solution in duplicate
Procedure: Separately inject equal volumes of solutions as per Sequence of injection
into the chromatograph and record the peak area responses for the major peaks and
check for the System suitability requirements.
Sequence of injection:
1)Blank
2) Standard solution A1.....5
3) Standard solution B
4) Blank
5) Sample solution A
6)sample solution B
System suitability requirements:
The test is not valid unless,
1)The Relative Standard deviation for the peak area response and retention time for
the peak of Sofosbuvir for replicate injections of Standard solution A is not more than
2.00% and 1.00% respectively.
2) The Tailing factor for the peak of Sofosbuvir obtained in the chromatogram of
individual Standard solution A injected in replicates and Standard solution B is not
more than 2.0 (Report the value obtained from the last injection of replicate of
Standard solution A).
3) The Column efficiency for the peak of Sofosbuvir obtained in the chromatogram of
individual Standard solution A injected in replicates and Standard solution B is not
less than 2000 theoretical plates.
(Report the value obtained from the last injection of replicate of Standard solution A)
Calculations:

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AW1 P

BW2

Where:
A=Peak area response of T Sofosbuvir obtained in the chromatogram of sample
solution.
B= Average peak area response of Sofosbuvir obtained in the chromatogram of
Standard solution A.
W1 = Weight of Sofosbuvir Test /Working Standard in Standard solution A in mg.
W2=Weight of Sample in mg.
P=%w/w Assay of Sofosbuvir Test Standard/Working Standard on as such basis.
If the assay values obtained are within the limit, calculate the %variation between
both the assay values using the formula:
=2X

(*+,,-.-/0- 1-23--/ 234 56657 859:-6)


(<+=>-. 56657 859:-?943-. 56657 859:-)

x 100

The assay results are acceptable only if the variation is less than
Xiamen Halosyntech Co., Ltd. Page 9 of 10

2.0%.If the acceptable criteria for %variation is met, report the mean assay value
using the formula:
=

@6657 859:- (@)?@6657 859:- (A)


B

Reporting: report as a value in %w/w.


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