In vitro metabolic stability in hepatic microsomes

Metabolic stability was evaluated with male CD-1 mouse and mixed-gender human liver microsomes (XenoTech, Lenexa, KS). Test articles (1 μM) were incubated with pooled liver microsomes (0.5 mg protein/mL) for 0, 5, 10, 20, and 30 min at 37 °C in the presence of NADPH. Aliquots (50 μL) were taken at each sampling time point and extracted with 150 μL ice-cold acetonitrile containing the internal standard (labetalol). Following centrifugation, supernatants were diluted five-fold into 35/65 A/B Mobile Phase and analyzed for the parent compound by LC/MS/MS as described in Supplemental Methods. The metabolic competency of microsomal preparations was established using the control compounds: 7-ethoxycoumarin, propranolol, and verapamil. Values for half-life (t_1/2) were determined with microsomes from each species by plotting the ln((compound peak area)/(internal standard peak area)) vs. time.