Role of high-performance thin-layer chromatography method in separation and analysis of withanosides-withanolides with flavonoid glycoside in Withania somnifera

Withania somnifera (L.) Dunal (WS), known as Ashwagandha, is an Indian herb classified as Rasayana in Ayurveda for rejuvenating and health-promoting activities. A sensitive and robust high–performance thin–layer chromatography (HPTLC) method for the estimation of withaferin A (WFA), withanoside IV (WSIV), withanoside V (WSV), and kaempferol based glucoside (KRG) was developed and validated in the roots and aerial parts of WS. This method reports the separation and simultaneous quantification of three diverse classes of WS with an analytical marker as KRG for effective detection and quantification of the aerial parts separating them from roots. The densitometric analysis was performed for KRG (at 254 nm), WFA, WSIV, and WSV (at 540 nm after derivatization) with characterization by HPTLC-MS/MS. The method was found linear (R² > 0.99), robust with excellent recoveries (80 – 100%). Furthermore, the class of pigment-based compounds in aerial parts at 366 nm in chemical fingerprinting in WS samples was separated with this method.

In this HPTLC-based assessment, WFA, WSIV, WSV, along with 12-deoxywithastramonolide, withanolide A, withanone, and withanolide B could be detected for quality control distinguishing them from other Withania species and other plant species reported earlier as adulterants. At the same time, the presence of flavonoid glycoside as an analytical marker with pigment zones can identify aerial parts, leaves, stems, and fruit with calyx and without calyx in the fingerprint analysis, separating them from root samples for authenticity. Thus, this HPLTC-based DS and MS/MS method were found rapid and economical for the estimation and surety of quality of WS samples.

Publication History

Article published online:
12 December 2022