Metabolite interpretation
Potential metabolites (M1 to M13) identified from the MetabolitePilotTM software (Sciex) are reported inTable 1 . The molecular ions that were detected in negative controls (including 0-minute incubations, negative co-factor controls, and negative ivermectin controls) were excluded from the list. Percentage scores were based on three parameters including mass accuracy, mass defect (Sleno, 2012; Zhang et al., 2009), and MS/MS spectrum (for details see supplementary material). Chemical structures and biotransformation sites of the metabolites (M1 to M13) were interpreted based on two fragment ions for IVM-B1a, m/z 307.2 and 551.3. IVM-B1a metabolites without ion m/z 307.2 indicated that biotransformation occurred in the spiroketal. The presence of ion m/z 307.2 without ion m/z 551.3 indicated that biotransformation occurred in the cyclohexene cyclic ether, and metabolites with changed molecular ions with presence of both ions m/z 307.2 and 551.3 indicated that biotransformation occurred in the disaccharide moiety. Identical results were observed for IVM-B1b metabolites, but with ions m/z 293.2 and 537.3. Interpretations of M1 to M13 are detailed in Table 2. The 60-min microsome reactions for pure IVM-B1a and IVM-B1b substrates confirmed an identical transformation pattern of both compounds (Tables S1 ).