AOAC SPDS Set 2 ERP

Method Review Form - AOAC INTERNATIONAL Standards Development

Name of Reviewer: Anton Bzhelyansky Title of Method: Withanolide Glycosides and Aglycones of Ashwagandha ( W. somnifera ) AOAC Candidate Method Number: ASH-01

Applicable SMPR : 2015.007

I.

SUMMARY OF METHOD

Withanolides in ashwagandha raw material and various extracts are determined following methanolic extraction and using an acetonitrile gradient against phosphate buffer on a conventional octadecylsilyl column, with UV detection at 227 nm. REVIEW OF THE METHOD ONLY: 1. Does the applicability of the method support the applicability of the SMPR? If no, please explain what is missing. Method does not explicitly state LOQs for individual withabolides. Through the back-of-the- envelope calculations, I arrived at about 5 μg/mL for aglycones, and about 15 μg/mL for glycosides. It may, however, be mentioned that the 10-ppm (0.001%) LOQ may be considered unnecessarily low, considering that ashwagandha root typically contains 0.25 – 0.35% of withanolides. Besides, a cursory look at the very busy chromatographic baseline of the plant at 227 nm would defy determination of withanolides at the concentrations specified in SMPR; a similar exercise with the standards will be just a purely analytical exercise. The degree of granularity requested in the SMPR Table 2, for accuracy, repeatability and reproducibility at four different levels was likewise not addressed in method validation, but is likely not necessary. 2. Does the analytical technique(s) used in the method meet the SMPR? If no, please specify what how it differs from what is stated in the SMPR. HPLC is likely to be a technique of choice for fulfilling the requirements of this SMPR, save for, perhaps, CE. Therefore, the chosen methodology is appropriate.

II.