NOTE: This is a summary of the foundational USP analytical method for analysis of withanolides from ashwagandha root or root powder by High Performance Liquid Chromatography (HPLC). It is provided for background and informational purposes only. Modifications related to plant part and root extract were provided in the 2019 monograph. Details on the method in entirety is only available from www.usp.org.
Composition
Content of Withanolides:
Follow the complete method instructions for preparation of standard solutions with USP Withanolide A RS and USP Withanoside IV RS dissolved in methanol.
Mobile Phase: Follow the gradient table provided in the method.
Chromatographic System:
Mode: LC
Detector: UV 227 nm
Column: 4.6-mm x 25-cm, end-capped, packing L1
Temperature: 27 +/- 1 degrees C
Flow rate: 1.5 mL/min
Injection size: 20 microliters
System suitability:
- Following the method, identify the relative retention times of the withanolide aglycones and glycosides provided in the table: Withanoside IV, Physagulin D, 27-hydroxywithanone, Withanoside V, Withanoside VI, Withaferin A, Withastramonolide, Withanolide A, Withanone, Withanolide B.
- The chromatogram for Standard solution C is similar to the reference chromatogram provided with the lot of USP Powdered Ashwagandha Root Extract RS being used.
- The resolution and tailing factors must meet those specified in the method.
- The relative standard deviation (RSD) shall be NMT 2.0% for withanolide A peak, based on replicate injections of Standard solution A.
Analysis:
Samples: Standard solution A, Standard solution B, Standard solution C, and Sample solution.
Calculate the percentage of withanolide aglycones in the portion of Ashwagandha Root taken based on the calculation.
Result = 5(rT/rS)(CS/W), where rT= sum of the peak responses for withaferin A, withastramonolide, withanolide A, withanone, and withanolide B from the Sample solution, rS = peak response of withanolide A from Standard solution A, CS = concentration of USP Withanolide A RS in Standard solution A (mg/mL), and w = weight of Ashwagandha Root taken to prepare the Sample solution (g).
Calculate the percentage of withanolide glycosides in the portion of Ashwagandha Root taken based on the calculation.
Result = 5(rT/rS)(CS/W), where rT = sum of the peak responses for withanoside IV, withanoside V, and withanoside VI from the Sample solution
rS = peak response of USP Withanoside IV from
Standard solution B, CS = concentration of USP Withanoside IV RS in Standard solution B (mg/mL), and W = weight of Ashwagandha Root taken to prepare the Sample solution (g).
Acceptance criteria: The sum of the percentages of withanolide aglycones and withanolide glycosides is NLT 0.3%, calculated on the dried basis. [NOTE—Because of inherent variations, some of the withanolides mentioned in this test may be present in minor quantities or may be totally absent. The sample will be deemed compliant if the sum of the total withanolides is NLT 0.3%.]
LABELING: The label states the Latin binomial and, following the official name, the part of the plant contained in the article.