Profiling and structural analysis of cardenolides in two species of Digitalis using liquid chromatography coupled with high-resolution mass spectrometry

Plants of the Digitalis genus contain a cocktail of cardenolides commonly prescribed to treat heart failure. Cardenolides in Digitalis extracts have been conventionally quantified by high-performance liquid chromatography yet the lack of structural information compounded with possible co-eluents renders this method insufficient for analyzing cardenolides in plants. The goal of this work is to structurally characterize cardiac glycosides in fresh-leaf extracts using liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) that provides exact masses. Fragmentation of cardenolides is featured by sequential loss of sugar units while the steroid aglycon moieties undergo stepwise elimination of hydroxyl groups, which distinguishes different aglycones. The sequence of elution follows diginatigenin[->]digoxigenin[->]gitoxigenin[->]gitaloxigenin[->]digitoxigenin for cardenolides with the same sugar units but different aglycones using a reverse-phase column. A linear range of 0.8-500 ng g-1 has been achieved for digoxigenin, {beta}-acetyldigoxin, and digitoxigenin with limits of detection ranging from 0.09 to 0.45 ng g-1. A total of 17 cardenolides have been detected with lanatoside A, C, and E as major cardenolides in Digitalis lanata while 7 have been found in Digitalis purpurea including purpurea glycoside A, B, and E. Surprisingly, glucodigifucoside in D. lanata and verodoxin and digitoxigenin fucoside in D. purpurea have also been found as major cardenolides. As the first MS/MS-based method developed for analyzing cardenolides in plant extracts, this method serves as a foundation for complete identification and accurate quantification of cardiac glycosides, a necessary step towards understanding the biosynthesis of cardenolide in plants.