Identification of cardiac glycosides in fractions from Periploca forrestii by high-performance liquid chromatography/diode-array detection/electrospray ionization multi-stage tandem mass spectrometry and liquid chromatography/nuclear magnetic resonance

Cardiac glycosides are a class of naturally occurring compounds that are characterized by some interesting biological activities and are widely distributed in the plant kingdom and can also be found in some animals. There is an interest in the chemical characterization of these molecules due to their toxicity and their use in medicines. In the study reported here, a combination of electrospray ionization tandem mass spectrometry with high-performance liquid chromatography equipped with diode-array detector (HPLC-DAD/ESI-MSⁿ), and hyphenation to both liquid chromatography and nuclear magnetic resonance spectroscopy (HPLC/NMR) were utilized for the on-line analyses of cardiac glycosides from Periploca forrestii. The fragmentation patterns and ¹H NMR spectra of nine isolated cardiac glycosides were investigated; their fragmentation rules and ¹H NMR spectral characteristics were summarized and applied to the structural identification of similar constituents in fractions from P. forrestii. As a result, a total of nine trace cardiac glycosides were tentatively determined by analyses of accurate molecular masses, representative fragment ions and characteristic ¹H NMR signals provided by HPLC/high-resolution mass spectrometry (HRMS), HPLC-DAD/ESI-MSⁿ and HPLC/¹H NMR experiments, respectively. Of these, eight (2–9) are new compounds and one (1) is reported from P. forrestii for the first time. Results of the present study can benefit the rapid identification and targeted isolation of new cardiac glycosides from crude plant extracts.