SPDS SET 2: ASH-01

5.0] STEP-2: STABILITY OF SAMPLE SOLUTIONS

5.1) Assessment: Stability of in solution was determined by checking the assay of a sample using freshly prepared solution of that sample and comparing it with the assay after 24

hours of preparing the solution.

5.2) Acceptance Criteria: The longest duration at which the assay shows variation ± 4.0%.

5.3) Experiment: Batch No. WS/05Lot21 prepared and injected to the HPLC immediately after

the preparation. The sample solution was left in the auto injector vial and again injected exactly after 24 hours. The assays were calculated in 0, 6, and 24 hours.

a) Standard solution preparation: Refer 4.2.4 – Dilution-6.

b) Sample preparation: Sample of WS/05Lot021 (2612.50mg) was weighed in 250 ml beaker, extracted with 100ml of methanol, boiling on water bath for 10-15 minutes and repeated the procedure 3-4 times till the material is completely extracted or till the extracts turn colourless. Combined all the fractions, concentrated and made up the volume to 50ml with methanol. Filtered through 0.45 microns membrane filter paper.

5.4) Procedure: 20 µ l of each standard solution was injected and chromatogram was recorded.

20 µ l of sample solution at 0 and 24 hours were injected and chromatograms were

recorded. Withanolides content were calculated using the peak area, sample and

standard.

Table-76: Withanolides content at various intervals Duration Withanoside IV (%w/w) Withanoside V (%w/w) Withaferin A

12-Deoxy withastramonolide

Withanolide A

Withanolide B

Sum

Initial result

0.399

0.319

0.413

0.123

0.107

0.030

1.390

After 24 hours

0.409

0.350

0.415

0.122

0.110

0.030

1.435

The percentage variation is calculated using the following formula: -

The difference between two results x 100 = % The initial result

1.435– 1.390 x 100 =

3.237%

1.390

Conclusion: The solution is stable for 24 hours.

Enclosure-13: Standard and sample chromatograms (4 Pages)

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