In vitro metabolite identification and characterization

Metabolites generated in the pooled HLM assay were tentatively identified and characterized by LC–QToF-MS in manual data processing with following criteria: MS peak area > 1 × 10⁵ cps, mass error of the precursor ion < 5 ppm, signal-to-noise ratio > 3:1, and mass tolerance for fragment ions ± 10 ppm. First of all, a list of hypothetical metabolites was generated on the basis of previous metabolism studies of structurally related SCs [11, 20, 21]. To avoid missing the main metabolites, precursor ions were searched via typical fragment ions in the bbCID data. Based on the LC–HRMS data, an LC–MS/MS MRM-method was developed, comprising the two most abundant ion transitions of each metabolite applying the optimized MS parameters of the parent compound (see Supplementary Material Table S2). The most abundant fragment ions were identified by recording EPI spectra of the metabolites. For EPI scans, EP was set to 10 V and a CE of 35 V with a spread of ± 15 V was applied. The two most abundant in vitro metabolites (M10 and M12) were integrated into an existing LC–MS/MS routine screening method to screen for positive urine samples.