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.