Human liver microsomes (pooled from 50 mixed sex donors) were purchased from Sekisui
XenoTech, LLC, (Kansas City, KS) whereas human kidney microsomes (pooled from 5 mixed sex
donors) were obtained from BIOIVT (Baltimore, MD). Microsomes (0.5 mg/mL) were
preincubated with CAB (10 μM) at 37 °C in a water bath for 5 min in 100 mM
potassium phosphate buffer, pH 7.4, prior to the addition of a UGT reaction mixture
(Corning Gentest, Discovery Labware, Woburn, MA) containing 25 mM uridine
5′-diphosphoglucuronic acid (UDPGA) and alamethicin. The reaction volume was 250
μL, and reactions were allowed to proceed for 60 min at 37 °C. At the end of
the incubations, the reactions were quenched with 250 μL of acetonitrile followed by
centrifugation at 10 000g at 4 °C for 10 min. Next, the
supernatants were dried under vacuum and subsequently reconstituted in 50 μL of
methanol. Samples were analyzed using LC–MS/MS as described above.
XenoTech, LLC, (Kansas City, KS) whereas human kidney microsomes (pooled from 5 mixed sex
donors) were obtained from BIOIVT (Baltimore, MD). Microsomes (0.5 mg/mL) were
preincubated with CAB (10 μM) at 37 °C in a water bath for 5 min in 100 mM
potassium phosphate buffer, pH 7.4, prior to the addition of a UGT reaction mixture
(Corning Gentest, Discovery Labware, Woburn, MA) containing 25 mM uridine
5′-diphosphoglucuronic acid (UDPGA) and alamethicin. The reaction volume was 250
μL, and reactions were allowed to proceed for 60 min at 37 °C. At the end of
the incubations, the reactions were quenched with 250 μL of acetonitrile followed by
centrifugation at 10 000g at 4 °C for 10 min. Next, the
supernatants were dried under vacuum and subsequently reconstituted in 50 μL of
methanol. Samples were analyzed using LC–MS/MS as described above.