*2.7. LC–MS*/*MS Analysis*

All metabolites and the IS were separated on a Kinetex XB-C18 column (100 × 2.10 mm, 2.6 μm, 100 Å; Phenomenex, Torrance, CA, USA) and analyzed using a Shimadzu LCMS 8060 triple-quadrupole mass spectrometer coupled with a Nexera X2 ultra high-performance liquid chromatography system (Shimadzu, Kyoto, Japan) equipped with an electrospray ionization interface. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The elution condition was set as 8% B (0–0.5 min), 8% →60% B (0.5–5 min), 60% B (5–6 min), 60% →8% B (6–6.1 min) and 8% B (6.1–9 min) for the analysis of metabolites of P450 probe substrates and set as 0% →40% B (0–1 min), 40% →50% B (1–5 min), 50% →0% B (5–5.1 min), and 0% B (5.1–8 min) for the analysis of metabolites of UGT probe substrates. The flow rate was 0.2 mL/min. Electrospray ionization was performed in positive-ion mode at 4000 V or in negative-ion mode at −3500 V. The optimum operating conditions were determined as follows: vaporizer temperature, 300 ◦C; capillary temperature, 350 ◦C; collision gas (argon) pressure, 1.5 mTorr. Quantitation was conducted in selected reaction monitoring (SRM) modes with the precursor-to-product ion transition for each metabolite (Tables 1 and 2).
