Quantitative evaluation of cardiac glycosides and their seasonal variation analysis in Nerium oleander using UHPLC-ESI-MS/MS.

INTRODUCTION: Nerium oleander is an eminent source of structurally diverse cardiac glycosides (CGs), plays a prominent role in the treatment of heart failure, and inhibits the proliferation of cancer cell lines. CGs exert their cardiotonic action by binding to the extracellularly exposed recognition sites on Na+ /K+ -ATPase, an integral membrane protein that establishes the electrochemical gradient of Na+ and K+ ions across the plasma membrane. OBJECTIVE: We aimed to quantitatively determine CGs and their seasonal variation in leaf and stem samples of N. oleander utilizing UHPLC-ESI-MS/MS techniques. METHODS: The UHPLC-ESI-MS/MS analytical method was developed utilizing multiple reaction monitoring (MRM) mode. The Waters BEH C18 (150 mm × 2.1 mm, 1.7 µm) column was used with a 22-min linear gradient consisting of acetonitrile and 5 mM ammonium acetate buffer. RESULTS: In total 21 CGs were quantitatively determined in the seasonal leaf and stem samples of N. oleander along with the absolute quantitation of the three chemical markers odoroside H (244.8 µg/g), odoroside A (231.4 µg/g), and oleandrin (703.9 µg/g). The season-specific accumulation of chemical markers was observed in the order of predominance odoroside A (summer season, stem), odoroside H (winter season, stem), and oleandrin (rainy season, leaf). Besides this, the remaining 18 CGs were relatively quantified in the same samples. CONCLUSION: The developed method is simple and reliable and can be used for the identification and quantification of multiple CGs in N. oleander.

Structural Analysis of Diastereomeric Cardiac Glycosides and Their Genins Using Ultraperformance Liquid Chromatography-Tandem Mass Spectrometry.

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, ß-d-diginose, and ß-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, ß-d-diginose, and ß-d-sarmentose sugars. At the same time, m/z 161, 143, 129, and 87 product ions confirmed the presence of a ß-d-digitalose unit. As a result, the UPLC-ESI/TQD system was successfully utilized for the structure characterization of CGs in Nerium oleander tissues.