Separation, identification, and quantification of selected withanolides in plant extracts of Withania somnifera by HPLC‐UV(DAD) – Positive ion electrospray ionisation–mass spectrometry

This paper describes a method for separation, identification, and quantification of selected withanolides in Withania somnifera plant extracts by HPLC‐UV(DAD)–Mass Spectrometry (HPLC‐MS). Withaferin‐A (WS‐3), 12‐deoxywithastramonolide (WS‐12DS), Withanolide A (WS‐1), and Withanone (WS‐2) were used as external standards. The compounds were isolated from Withania somnifera by repeated column chromatography of the root extract and their identity was established by 1 H‐ and 13 C‐NMR and mass spectral data. The compounds were chromatographed on a Merck (250×4.6 mm ID, 5 μm) column and analyzed by Electrospray Ionization on a mass spectrometer in Selected Ion Mode (SIM). For quantification, [M + Na] + ions were monitored. Linear calibration curves were obtained in the concentration range of 1.50 μg/mL to 6.5 μg/mL. The method was applied successfully to the detection and quantification of the said withanolides in a number of samples.