ASH-001_AOAC_Withania_Submission

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c) Procedure: 20µl of each of the standard solutions were injected two times and 20µl 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
Different Concentrations Withanoside IV
(mcg/ml)
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)
Replicate – 1 166047 329878 651382 1275513 2493967 4815460 9597190
Replicate – 2 166038 330019 651009 1278440 2493747 4811411 9587035
Replicate – 3 166015 329943 651630 1269142 2491031 4819826 9584608
Replicate – 4 166025 329827 649769 1278566 2492809 4815725 9588582
Replicate – 5 165993 329140 651869 1279224 2497552 4818317 9579369
Mean 166024 329761 651132 1276177 2493821 4816148 9587357
RSD 0.01 0.11 0.13 0.33 0.10 0.07 0.07