Characterization of ginsenoside structural isomers from mixtures using in situ methylation with direct analysis in real-time ionization tandem mass spectrometry

Characterization of structural isomers of bioactive molecules is important for recognizing their functions, but it has been challenging due to their highly similar structures. As the main bioactive constituents of Panax ginseng, ginsenosides have different structural isomers attributed to the aglycone structure and glycosylation sites as well as stereochemistry of sugar groups attached. This work demonstrated a simple and robust in situ methylation reaction with tetramethylammonium hydroxide (TMAH) using ambient ionization source of direct analysis in real time (DART) to characterize saponin structural isomers. The DART ion source provides favorable conditions to methylate hydroxyl groups of ginsenoside instantaneously with TMAH, and it can ionize the methylated products at the same time. Methylated ginsenoside stereoisomers even with subtle structure differences generated very different mass signals from full-scan MS and tandem MS. High-resolution mass spectrometry aided the assignment of molecular structures of the various precursor and fragment ions from different ginsenosides, which provided structural information for both the aglycone skeleton and the sugar moieties in ginsenosides. The presented method was successfully used for the identification of ginsenosides in Panax ginseng, and saponin isomers were characterized without the need for chromatographic separation and/or tedious offline sample pretreatment.