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., Delta(16)anhydrogitoxigenin, Delta(16)adynerigenin, 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, beta-D-diginose, and beta-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, beta-D-diginose, and beta-D-sarmentose sugars. At the same time, m/z 161, 143, 129, and 87 product ions confirmed the presence of a beta-D-digitalose unit. As a result, the UPLC-ESI/TQD system was successfully utilized for the structure characterization of CGs in Nerium oleander tissues.

Automated summary

This study utilized UPLC-ESI-MS/MS for the structural analysis of 38 naturally occurring cardiac glycosides in Nerium oleander, revealing the chemical diversity of CGs and differentiating diastereomers based on RRt and reference standards. The in-source fragmentation of CGs and MS/MS of disaccharide daughter ions provided intrinsic structural information on the sugar units, confirming the successful application of UPLC-ESI/TQD system for CG structure characterization in Nerium oleander tissues.