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.