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3DJH RI
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)