Incubation conditions
The final incubation conditions with HLM and recombinant CYPs were optimized, which were within the linear range for the concentrations of major metabolite that formed. The HLM incubation system, with a total volume of 200 μL, contained potassium phosphate buffer (10 mM, pH 7.4), NADPH (1 mM), and liver microsomes (0.3 mg protein·mL−1). The total volume of the incubation system with recombinant CYPs was set at 100 μL. All rivaroxaban samples in this study were obtained by a series of dilutions from a stock solution (100 mM in DMSO). The final organic solvent concentration was no more than 0.5% (v/v). After 5 minutes of incubation at 37 °C, the reaction was initiated by the addition of NADPH and the resulting mixture incubated at 37 °C on a vibrating mixer for 60 minutes. The reaction was terminated by the addition of cold methanol in a volume equivalent to that of the reaction system. The reaction mixture was then centrifuged at 2000 x g for 15 minutes. Aliquots of the supernatants were stored at –20 °C until analysis by HPLC. The reactions that were incubated without NADPH, substrate or enzymes were designed to confirm the formation of the metabolite was dependent on enzymes and NADPH.