Homolog-focused profiling of ginsenosides based on the integration of step-wise formate anion-to-deprotonated ion transition screening and scheduled multiple reaction monitoring

• Formate anion > deprotonated ion pairing is specific and sensitive for ginsenoside.
• Step-wise MRM was used to detect ginsenosides in ginseng and American ginseng.
• Scheduled MRM was utilized to quantitatively analyze all 221 detected ginsenosides.
• Comparative metabolomics was performed for ginseng and American ginseng.

Homolog-focused profiling is a favored option to bridge targeted metabolomics toward non-targeted metabolomics. In current study, an attempt was made for the large-scale ginsenoside-specific analysis in ginseng (G) and American ginseng (AG). When formic acid (0.1%, v/v) was introduced as the mobile phase additive, formate anion-to-deprotonated ion transitions ([M+HCOO]− > [M−H]−) with an optimal collision energy (−32 eV) could result in satisfactory responses for ginsenosides. Therefore, a step-wise multiple reaction monitoring (MRM)-based method employing [M+HCOO]− > [M−H]− ion pairs was constructed to screen ginsenosides among 501–1250 u (for Q1) with a step-size of 2 u, and MRM also served as a survey experiment to trigger enhanced product ion scans for MS2 spectrum acquisition on a hybrid triple quadrupole-linear ion trap mass spectrometer; then, the identification of those observed ginsenosides was achieved on the basis of the well-defined mass cracking patterns for ginsenosides; afterwards, scheduled MRM was introduced for large-scale relatively quantitative analysis of all detected ginsenosides. Finally, comparative metabolomics were performed to differentiate G, AG, and their processed products. Method validation was carried out using thirteen authentic compounds. A total of 221 ginsenosides, among which 185 ones were annotated, were observed and relatively quantitated. All crude materials were obviously classified into groups I–III. Above all, the MRM-based homolog-focused profiling of ginsenosides could be used as a reliable tool to gain an in-depth view for ginsenoside-enriched herbal products.