Chlorzoxazone Metabolism in Liver Microsomes

Each incubation mixture (200 μL) included liver microsomal protein (0.75 mg. mL^-1), MgCl₂ (10 mM) in 100 mM phosphate buffer (pH7.4) and 25 μM chlorzoxazone. With 5 min pre-incubation, all reactions were initiated by addition of NADPH (1 mM) and were carried out in 37°C water bath for 30 min, and then were stopped by addition of 150 μL ice-cold acetonitrile and internal standard (80 μM phenacetin). After centrifugation at 12000 r · min^-1 for 30 min, 20 μL of the supernatant was injected into the HPLC system, and eluted with methanol–water (47:53) at a flow rate of 1.0 mL · min^-1, UV absorbance was monitored at 287 nm.