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3DJH RI
d) Procedure:
20
µ
l of each of the standard solution, 20
µ
l of each concentration of the
sample solutions were injected and Chromatograms were recorded. From the peak
area individual withanolides content were calculated. The results are given below: -
Table-54:
Batch No.
Withanoside
IV
Withanoside
V
Withaferin A
12-Deoxy
withastramonolide
Withanolide
A
Withanolide
B
Sum
WS/06Lot08
0.798
0.700
0.644
0.278
0.226
0.067
2.715
WS/06Lot10
0.804
0.684
0.606
0.266
0.208
0.064
2.631
WS/05Lot20
0.053
0.044
0.069
0.019
0.020
0.005
0.211
WS/05Lot21
0.399
0.319
0.413
0.123
0.107
0.030
1.390
RD/1170
0.746
0.644
0.690
0.335
0.239
0.079
2.733
RD/1045
0.552
0.416
0.408
0.253
0.137
0.045
1.812
RD/1162 RM
0.131
0.095
0.089
0.019
0.023
0.004
0.360
ERH-46 RM
0.155
0.151
0.130
0.037
0.039
0.010
0.523
For standard chromatograms Enclosure – 5
Enclosure-10:
Initial analysis sample chromatograms
(8 Pages).
Spike recovery studies:
a) Sample Preparation for Spike recovery:
1) Samples of WS/05Lot21 (453.70 mg) was weighed in to 250 ml beaker and 0.91mg of
Withanoside IV, 2.53mg of Withanoside V, 1.03mg of Withaferin A, 1.09mg of 12-Doxy
withastramonolide, 1.03mg of Withanolide A and 0.90mg of Withanolide B were added to the
same beaker. The mixture was
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.
20
µ
l of standard solution dilution-3 (Refer 4.2.4) and sample solutions were
injected and chromatograms were recorded. From the obtained chromatograms
individual
withanolides content
were calculated.
2) Samples of WS/06Lot10 (
328.90
mg) was weighed in to 250 ml beaker and 1.08mg of
Withanoside IV, 2.38mg of Withanoside V, 1.00mg of Withaferin A, 1.00mg of 12-Doxy
withastramonolide, 0.91mg of Withanolide A and 1.15mg of Withanolide B were added to the
same beaker. The mixture was
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