Drug metabolism in rat liver microsomes

Sprague-Dawley rat liver microsomes containing 20 mg/ml of total protein were obtained from the Rild Research Institute (Shanghai, China). The incubation mixture contained 200 μl of phencynonate racemate at various concentrations, 500 μl of rat liver microsomes at a protein concentration of 2 mg/ml, 200 μl of 5 mM NADPH and 100 μl of a 2 mM solution of MgCl₂. The incubations were carried out with stirring at 37 °C. The final concentrations of phencynonate racemate were 100, 200 and 400 μM.

From 100 μM of phencynonate racemate mixture, 100 μl of the incubation sample was removed after 10, 30, 60, 90 and 120 min. The sampling times were 10, 30, 60, 90, 120, 150 and 180 min in the other two groups. A total of 200 μl of iced methanol was added to stop the incubations immediately upon removing the sample. For the HPLC analysis, the removed sample was vortexed for 1 min, and then centrifuged for 10 min at 10,000 × g. The supernatant was transferred to the HPLC autosampler vials, and directly injected into the HPLC system and analyzed.