Separation and identification of dammarane-type ginsenoside isomers from Panax ginseng using cyclic ion mobility and tandem mass spectrometry.

BACKGROUND: Dammarane-type ginsenosides (DTGs) exhibit extensive isomerism including aglycone isomers, glycosylation site isomers, double-bond positional isomers, and R/S configurational isomers. Many of these isomers co-elute in liquid chromatography and are difficult to distinguish by MS/MS alone, compromising the accuracy of identification in Panax ginseng extract. Consequently, a more systematic approach is needed to separate and identify these isomers.

RESULTS: Multi-pass cyclic ion mobility (cIMS) was validated on both reference standards and Panax ginseng extracts for its ability to resolve challenging DTG isomers. Following optimization of the trap collision energy, a systematic putative identification strategy was established by integrating MS/MS data, CCS values of intact DTGs, and CCS values of their characteristic aglycone fragments. This approach enables the differentiation of aglycone isomers, sugar type isomers, glycosidic linkage isomers, glycosylation site isomers, R/S configurational isomers and double-bond positional isomers, markedly improving the reliability of identification.

SIGNIFICANCE: The use of characteristic CCS values of homologous aglycones offers a complementary approach for inferring C-20 R/S configurations, which may help to address a key limitation of conventional MS in certain cases. While the current study focuses on ginsenosides, this workflow-particularly the integration of MS/MS with aglycone CCS data-may serve as a useful reference for isomer identification in other natural products with similar isomeric complexity.