SPDS SET 2: ASH-01

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:

Withanoside IV

Withanoside V

12-Deoxy withastramonolide

Withanolide A

Withanolide B

Batch No.

Withaferin A

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

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