Chemicals and instruments
Rivaroxaban (> 98%) was obtained from Shanghai Yuanye Bio-Technology Co., Ltd. Danazol was purchased from Med Chem Express (New Jersey, USA). All other CYPs inhibitors were purchased from Sigma-Aldrich (Missouri, USA). PBS was prepared from dipotassium bisulfate and potassium dihydrogen sulfate in the appropriate proportions and stored at 4 °C until use. Analytical reagent grade and HPLC grade solvents were from Tedia, Inc. (Ohio, USA). Pooled human liver microsomes were purchased from BioreclamationIVT (Maryland, USA). cDNA-expressed recombinant human CYP3A4 and CYP3A5 were obtained from Cypex Ltd (Dundee, UK). cDNA-expressed CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP2J2, CYP3A7, CYP4F2, CYP4F3 and CYP46A1 derived from baculovirus-infected insect cells were obtained from BD Gentest Corp. (Massachusetts, USA). cDNA-expressed CYP2C19 fromEscherichia coli was purchased from New England Biolabs Ltd. (Beijing, China). All microsomal samples and recombinant human P450 isoforms were stored at –80 °C until use.
High-performance liquid chromatography (HPLC) was performed using an Agilent MSD/MS system controller, two 1260 series pumps, a 1200 series autosampler and a 1200 series variable wavelength detector. Other instrumentation included a constant temperature vibrating mixer (Hangzhou ALLSHENG Instrument Co. Ltd, China), scroll machine, centrifuge and elite ODS-BP analysis column (4.6 × 200 mm, 5 μm). An API 3200 triple-quadrupole mass spectrometer (Applied Biosystems, Ontario, Canada) was used for LC-MS analysis. Ionization was conducted using an electron spray interface in the positive ion mode for detecting metabolites.