Estimation of microsomal CYP1A2 activity by high performance liquid chromatography

Abstract

Phenacetin has been reported to be a reliable probe for the estimation of microsomal CYP1A2 activity. Phenacetin is converted to paracetamol by CYP1A2. A high pressure liquid chromatographic assay for the estimation of CYP1A2 activity in microsomes, was evaluated with caffeine as the internal standard. An RP C-18 supelcosil (150 × 4.6 mm, 5 µm) column was used for the assay. The mobile phase composition was acetonitrile:water (15:85), with a flow rate of 0.7 ml/min, injection volume of 100 µl, and detection at 240 nm. The retention times for paracetamol, caffeine and phenacetin were found to be 4.9, 8.6 and 15.5 min, respectively. The limit of detection was 0.1 µM for phenacetin, and the limit of quantitation was 0.2 µM for paracetamol. The percent coefficient of variation associated with paracetamol determination after duplicate estimation, was found to be in the range 0.02 to 7.7% for buffer matrix, and 0.8 to 9.7% for microsomal matrix. The mean rate of paracetamol formation in rat liver and guinea pig liver microsomes was 0.027 and 0.161 nmoles/min/nmole of P450, respectively. No interference was observed from incubation mixture components.