Ultraperformance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UPLC–ESI-MS/MS) is an economical and indispensable tool in natural product research to investigate novel metabolites, biomarker discovery, chemical diversity exploration, and structure elucidation. In this study, the structural analysis of 38 naturally occurring cardiac glycosides (CGs) in various tissues of Nerium oleander was achieved by the extensive use of mass spectrometry. The chemical diversity of CGs was described on the basis of characteristic MS/MS fragmentation patterns, accurate mass measurement, and published scientific information on CGs from Nerium oleander. It was observed that only six genins, viz., Δ16anhydrogitoxigenin, Δ16adynerigenin, gitoxigenin, oleandrigenin, digitoxigenin, and adynerigenine, produce 38 diverse chemical structures of CGs. Among them, 20 were identified as diastereomers having a difference in a sugar (l-oleandrose, β-d-diginose, and β-d-sarmentose) unit. However, the differentiation of diastereomeric CGs was not possible by only MS/MS fragments. Thus, the diastereomer’s chromatographic elution order was assigned on the basis of the relative retention time (RRt) of two reference standards (odoroside A and oleandrin) among their diastereomers. Besides this, the in-source fragmentation of CGs and the MS/MS of m/z 325 and 323 disaccharide daughter ions also exposed the intrinsic structure information on the sugar units. The daughter ions m/z 162, 145, 113, 95, and 85 in MS/MS spectra indicated the abundance of l-oleandrose, β-d-diginose, and β-d-sarmentose sugars. At the same time, m/z 161, 143, 129, and 87 product ions confirmed the presence of a β-d-digitalose unit. As a result, the UPLC–ESI/TQD system was successfully utilized for the structure characterization of CGs in Nerium oleander tissues.

Rationale Cajanus scarabaeoides, belonging to the Fabaceae family, is an underutilized herb and traditionally used to treat several ailments. However, it is not well explored phytochemically. Therefore, mass spectrometry (MS)–based phytochemical analysis was carried out to investigate the bioactive ingredients of the herb. Methods A ultra‐performance liquid chromatography (UPLC) coupled to photodiode array detection (PDA) and electrospray ionization (ESI) tandem mass spectrometry (UPLC‐PDA‐ESI–MS/MS) system was used for the qualitative and quantitative analysis of phytochemicals. The chromatographic separation was achieved on the Acquity BEH C18 column (150 × 2.1 mm, 1.7 μm) using a gradient system consisting of three solvents, acetonitrile, methanol, and 0.1% formic acid, used at a flow rate of 0.300 ml/min. Results Sixteen bioactive ingredients (gallic acid, gallocatechin, epigallocatechin, catechin, procyanidin dimer, epicatechin, procyanidin trimer, isoorientin, orientin, vitexin, isovitexin, quercetin‐mono‐O‐glycoside, isoquercitrin, luteolin‐7‐O‐glucoside, quercetin, and luteolin) were identified and structurally characterized. Consequently, 12 compounds were reported for the first time from C. scarabaeoides, and 13 were quantitatively determined in different seasons. Isoorientin (10.2–7.1% w/w) and orientin (5.78–5.17% w/w) were the most abundant constituents in the dry weight of plant material, followed by vitexin and isovitexin in the rainy season. Conclusions The phytochemical investigation has revealed that C. scarabaeoides could be a potential alternate source of bioactive ingredients, namely, isoorientin, orientin, vitexin, and isovitexin, contributing to further exploration of its biological activity. In addition, analytical methods can be used for the rapid identification and quantification of bioactive ingredients in C. scarabaeoides.

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