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