In vitro and in vivo metabolite identification by UHPLC-QTOF MS
Thirteen IVM metabolites (M1-M13) were identified from 60 minute -incubated microsomes as shown in the metabolite chromatogram inFigure 2A. All metabolites were more polar than the IVM parent compound. M1, M3, M5, and M6 were found in the medium of 24hour-exposed primary human hepatocytes (Figure 2B) . M1, M3, and M6 were also found in 24hour-post-treatment volunteer blood (Figure 2C).Reference MS/MS spectra of IVM-B1a and IVM-B1b used in the library search were generated from analysis of 100 ng/mL of IVM standard solution. IVM- B1b (m/z 878.5) eluted 0.9 min earlier than IVM-B1a (m/z 892.5) as shown in the extract ion chromatogram (Figure 3A). Major product ions of IVM-B1b were m/z 293.2, 537.3, and 555.3 (Figure 3B) and those of IVM-B1a were m/z 307.2, 551.3, and 569.3 (Figure 3C) . The fragment pattern of the ammonium adduct ion of IVM-B1a and IVM-B1b is presented in Figure 3D. MS/MS spectra of M1 to M13 (Figure 4) were used to define the molecular structure of the metabolites.