Various Multicharged Anions of Ginsenosides in Negative Electrospray Ionization with QTOF High-Resolution Mass Spectrometry.

When characterizing components from ginseng, we found a vast number of multicharged anions presented in the liquid chromatography-mass spectrometry (LC-MS) chromatograms. The source of these anions is unclear yet, while ginsenosides, the major components of ginseng, are the main suspected type of molecules because of their sugar moiety. Our investigation using 14 pure ginsenosides affirmed that the multicharged anions were formed by ginsenosides rather than other types of ingredients in ginseng. Various anions could be observed for each ginsenoside. These anions contain ions ([M-2H]2-, [M+Adduct]2-), as well as those formed by polymerization of at least two ginsenosides, such as [nM-2H]2-, [nM-H+Adduct]2-, and [nM-3H]3-. The presence of so different types of ions from a ginsenoside explains the reason for the large number of anions in the LC-MS analysis of ginseng. We further found that formation of [nM-2H]2- ions was influenced by the number of sugar chains: ginsenosides containing two sugar chains produced all [nM-2H]2- ion types, whereas ginsenosides containing one sugar chain did not produce [2M-2H]2-. Thus, [2M-2H]2- and [3M-2H]2- can be utilized to rapidly identify monodesmosidic and/or bidesmosidic ginsenosides as joint diagnostic anions. The position of the glycosyl radical might be the key factor affecting the formation of multicharged multimer ions from monodesmosidic ginsenosides. Consequently, three groups of ginsenoside isomers were differentiated by characteristic [nM-2H]2- anions. Using concentration-dependent characteristics and collision-induced dissociation (CID), we confirmed that [nM-2H]2- ions are non-covalently bound multimers whose aggregation has marked distinction between monodesmosidic and bidesmosidic ginsenosides, accounting for the differentiated formation of [nM-2H]2- between them. Graphical Abstract.