Systematic screening and characterization of novel bufadienolides from toad skin using ultra-performance liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry.

During the discovery process of novel compounds, it is of significant importance to differentiate novel from known compounds in crude extracts before starting the time-consuming process of purification. Bufadienolides are the main active components of the skin of the toad Bufo bufo gargarizans Cantor (toad skin), an important traditional Chinese medicine. The fragmentation behavior and mass spectra profiles of bufadienolides standards were investigated using ultra-performance liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC/ESI-Q-TOFMS). Several fragmentation rules were summarized and applied to characterize novel and known bufadienolides in toad skin. Characteristic substituent groups could be identified by both diagnostic ions and their relative abundance. Bufadienolide stereoisomers could be differentiated from positional isomers by comparing fragment abundance profiles. This was used to characterize new stereoisomers for known bufadienolides. A total of 39 bufadienolides were screened out using a systematic method developed in our laboratory. In addition to 19 known bufadienolides, 20 putative novel compounds, including 8 stereoisomers, were characterized. UPLC/Q-TOFMS was demonstrated to be a powerful tool for the characterization of low-abundance bufadienolides in complex samples. This study provides guidelines for the targeted isolation of novel bufadienolides from natural products.