Comparative Profiling of Withanolides in Tissues of Withania somnifera and Datura metel Using UHPLC–MS/MS

Withania somnifera and Datura metel, two medicinally significant members of the Solanaceae family, are known for their content of pharmacologically active withanolides. This study investigates the structural diversity and tissue-specific distribution of withanolides in these species using UHPLC-tandem mass spectrometry (MS/MS). Identified 36 distinct withanolides based on characteristic MS/MS fragmentation patterns and accurate mass measurements. Glycosidic linkages in withanosides were recognized through neutral losses of 162, 324, and 486 Da, indicating the presence of mono-, di-, and tri-glycone moieties, whereas two to three water losses were commonly observed from the genin units. A total of 21 isomeric compounds were differentiated by their chromatographic elution profiles and MS/MS fragmentation. However, Withanoside IV, Withaferin A, Withanoside V, Withanone, and Withanolide B were confirmed with the reference standards. Notably, leaves exhibited the highest structural diversity among the analyzed tissues (leaves, stems, fruits, and roots). Given the commercial importance yet non-renewable parts, Withania roots, and the traditional use of Datura leaves in ethnomedicine, this study highlights the leaf as a rich and sustainable source of structurally diverse withanolides for potential therapeutic applications and exploration.