An in-source multiple collision-neutral loss filtering based nontargeted metabolomics approach for the comprehensive analysis of malonyl-ginsenosides from Panax ginseng, P. quinquefolius, and P. notoginseng

•An IMC-NLF-based nontargeted metabolomics approach was developed.•It enables the characterization and statistical analysis of target components.•Dual neutral loss filtering facilitates the exact screening of malonyl-ginsenosides.•101 malonyl-ginsenosides were primarily characterized from three Ginseng species.•Ten potential markers were discovered for discriminating three Ginseng species.

The simultaneous identification and quantification of target metabolites from herbal medicines are difficult to implement by the full-scan MS based nontargeted metabolomics approaches. Here an in-source multiple collision-neutral loss filtering (IMC-NLF) based nontargeted metabolomics approach is developed and applied to identify and quantify the variations of malonyl-ginsenosides, a common group of acyl saponins with potential anti-diabetic activity, among Panax ginseng, P. quinquefolius, and P. notoginseng. The key steps of the IMC-NLF strategy are the acquisition of specific high-resolution neutral loss data and the efficient filtering of target precursor ions from the full-scan spectra. Using a hybrid LTQ-Orbitrap mass spectrometer after UHPLC separation, abundant in-source product ions, [M-H-CO2]- (due to the vulnerability of the carboxyl group) and [M-H-Mal.]-, were generated at the energies of 70 V and 90 V, respectively. After spectral deconvolution, the generated peak list was screened by dual NLF using a Neutral Loss MS Finder software (NL of 43.9898 Da for CO2 and 86.0004 Da for the malonyl substituent). By combining the precursor ions list-triggered HCD-MS/MS and basic hydrolysis, a total of 101 malonyl-ginsenosides (including 69 from P. ginseng, 52 from P. quinquefolius, and 44 from P. notoginseng) were identified or tentatively characterized. The variations of 81 characterized malonyl-ginsenosides among 45 batches of Ginseng samples were statistically analyzed disclosing ten potential markers. It is the first systematic analysis of malonyl-ginsenosides. The IMC-NLF approach by a single analytical platform is promising in targeted analyses of modification-specific metabolites in metabolomics and drug metabolism.

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