Validation of a HPLC method for the determination of p-nitrophenol hydroxylase activity in rat hepatic microsomes

We report a HPLC-UV method for determination of p-nitrophenol (PNP) hydroxylation to 4-nitrocatechol (4NC) as a marker for CYP2E1 activity in rat hepatic microsomes. Proteins were precipitated by addition of 50 μL phosphoric acid (50%, v/v in water) to 500 μL microsomal suspensions. Following vortex mixing and centrifugation the supernatant (20 μL) was injected onto a Supelcosil® C18 column (150 mm × 4.6 mm, 5 μm), and mobile phase (22% acetonitrile, 0.1% trifluoroacetic acetic acid, 0.5% triethylamine) delivered at 1.0 mL/min produced resolved peaks for internal standard, 4NC, and PNP in <11 min. Calibration curves were linear (r2 = 0.999) from 0.1 to 40 μM with intra- and inter-day precision <12% and accuracy >90%. The method's improved sensitivity (LOQ = 0.1 μM) and minimal sample processing allowed rapid monitoring of PNP hydroxylase activity in fetal, neonatal, juvenile, and adult rat livers.