Separation and identification of the major metabolite M1
The HPLC conditions to simultaneously detect and separate the major metabolite M1 were methanol:pure water:formic acid = 60:40:0.2 (v/v/v), flow rate 0.5 mL·min−1 and column temperature 30 °C. As shown in Figure 2A, when rivaroxaban was incubated with HLM without NADPH, the HPLC peak appeared at 12.839 min. When rivaroxaban was incubated with HLM and NADPH, a new single peak (M1) appeared with a retention time of 10.831 min. The M1 peak was absolutely separate from rivaroxaban and did not interfere with the quantitative analysis, which indicated that the method could be used to simultaneously detect and conduct quantitative analysis of M1 and rivaroxaban.
LC-MS analysis of the major metabolite that was separated and collected from the HPLC was conducted in positive ion mode. Results showed the major metabolite with m/z 452.9 was the most abundant component with an intensity of > 7 × 107 cps (Figure 2B). This molecular weight was consistent with results reported in the prior literature; thus, we can confirm that the major metabolite of rivaroxaban was morpholinone 2- hydroxylation (M1) (Figure 1) [8].