Research Article | Volume: 5, Issue: 7, July, 2015

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

Sundeep Bath Wael Ebied Hytham Ahmed Fawzy Elbarbry   

Open Access   

Published:  Jul 27, 2015

DOI: 10.7324/JAPS.2015.50720
Abstract

Objective: 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 (R2 >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 7-hydroxycoumarin, respectively. This method was also successfully applied for studying the effect of three phytochemicals on hepatic CYP2A6 activity in rats.


Keyword:     HPLC CYP2A6 Coumarin 7-hydroxy coumarin Resveratrol Sulforaphane Thymoquinone metabolism rats.


Citation:

Sundeep Bath, Wael Ebied, Hytham Ahmed, Fawzy Elbarbry. 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. J App Pharm Sci, 2015; 5 (07): 130-135.

Copyright:The Author(s). This is an open access article distributed under the Creative Commons Attribution Non-Commercial License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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