Development and Validation of a New Simple Liquid Chromatographic Method for Determination of Coumarin and its 7-Hydroxy Metabolite as a Marker of Cytochrome P450 2A6 in Rats

Abstract

In this study, a simple and reliable high performance liquid chromatographic method with UV detection was developed and validated for rapid determination of coumarin hydroxylase activity in rat hepatic microsomes. Materials and Methods: The chromatographic separation was achieved using Zorbax Eclipse XDB C18 column (150×4.6 mm, 5 μm), which was kept at 40°C. The isocratic mobile phase consisted of methanol and 1% glacial acetic acid mixture (35:65, v/v) with a flow rate of 0.6 ml/min. The effluent was monitored at 320 nm using photodiode array detector (PDA). 6-hydroxychlorzoxazone (6-OH CZX) was used as internal standard. Results and Conclusion: The method exhibited good linearity (R 2 >0.999) for both coumarin (COUM) and its 7-hydroxy metabolite (7-OH COUM) over the assayed concentration range (0.025-5.0 μM) and demonstrated good intra-day and inter-day precision and accuracy (relative standard deviations and the deviation from predicted values were less than 15%). The detection limits were 0.001 and 0.005 μM for coumarin and 7hydroxycoumarin, respectively. This method was also successfully applied for studying the effect of three phytochemicals on hepatic CYP2A6 activity in rats.