SPDS SET 2: ASH-01

c) Procedure: 20 µ l of each of the standard solutions were injected two times and 20 µ l of

each of the sample solutions were injected two times and Chromatograms were recorded. There are various late eluting compounds and hence run time of the sample

chromatograms were at least for 60 minutes. From the peak area Withanoside IV,

Withanoside V, Withaferin A, 12-Deoxy withastramonolide, Withanolide A, and Withanolide B were calculated. RSD for the peak area, retention time and content of all

the withanolides were calculated.

4.3.7) Calculation:

Calculation for individual withanolides

Areas of Withanoside IV in the sample x Weight of Withanoside IV RS in mg x Sample dilution x Purity of standard x 100 Area of the Withanoside IV RS Standard dilution Sample weight in mg 100

Areas of Withanoside V in the sample x Weight of Withanoside V RS in mg x Sample dilution x Purity of standard x 100 Area of the Withanoside V RS Standard dilution Sample weight in mg 100

Areas of Withaferin A in the sample x Weight of Withaferin-A RS in mg x Sample dilution x Purity of standard x 100 Area of the Withaferin A RS Standard dilution Sample weight in mg 100

Areas of 12-deoxywithastramonolide x Weight of 12-deoxywithastramonolide RS in mg x Sample dilution x Purity of standard x 100 in the sample_______________________________________________________________________________________ Area of the 12-Deoxystramonolide RS Standard dilution Sample weight in mg 100

Areas of Withanolide A in the sample x Weight of Withanolide A RS in mg x Sample dilution x Purity of standard x 100 Area of the Withanolide A RS Standard dilution Sample weight in mg 100

Areas of Withanolide B in the sample x Weight of Withanolide B RS in mg x Sample dilution x Purity of standard x 100 Area of the Withanolide B RS Standard dilution Sample weight in mg 100

4.3.8) Observation:

Table-18: Peak area of Withanoside IV

Withanoside IV (mcg/ml)

Different Concentrations

Dilution -6 (20.64)

Dilution -5 (41.29)

Dilution -4 (82.58)

Dilution -3 (165.15)

Dilution -2 (330.30)

Dilution -1 (660.60)

S. solution (1321.20)

166047

329878

651382

1275513

2493967

4815460

9597190

Replicate – 1

166038

330019

651009

1278440

2493747

4811411

9587035

Replicate – 2

166015

329943

651630

1269142

2491031

4819826

9584608

Replicate – 3

166025

329827

649769

1278566

2492809

4815725

9588582

Replicate – 4

165993

329140

651869

1279224

2497552

4818317

9579369

Replicate – 5

166024

329761

651132

1276177

2493821

4816148

9587357

Mean

0.01

0.11

0.13

0.33

0.10

0.07

0.07

RSD

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