Enhancing ginsenoside isomers annotation by integrated analysis of electron-activated dissociation and collision-induced dissociation tandem mass spectrometry.

Natural products, with their diverse structures and biological activities, are a critical source for new drug development. Advances in mass spectrometry have significantly enhanced the accurate and rapid identification of natural products. However, differentiating isomers remains a challenge. This study establishes a complementary approach combining collision-induced dissociation (CID) and electron-activated dissociation (EAD) to effectively distinguish isomers, exemplified by ginsenoside isomers. The CID mode clearly distinguishes the structural types of ginsenosides, while EAD mode identifies the substitution sites (e.g., C-3, C-6, C-20) of the glycosyl groups. Mass spectrometry molecular network (MN) analysis showed that, compared to CID-MN, the EAD-MN demonstrated more robust clustering of ginsenosides and more accurately reflected structural similarities of ginsenosides. The developed method enables precise characterization of unknown ginsenoside isomers in Panax species including Panax ginseng, Panax notoginseng, and Panax quinquefolius, and reveals species-specific features, thereby facilitating improved quality control. The proposed strategy effectively addresses the challenge of distinguishing ginsenoside isomers by integrated analysis of CID and EAD tandem mass spectrometry to identify diagnostic ions specific to each isomer. This approach enhances the accuracy of natural product annotation.