Current Pharmaceutical Analysis

Anastasios Economou
Department of Chemistry
Laboratory of Analytical Chemistry
University of Athens
Athens
Greece
Email: cpa@benthamscience.org

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Development and Validation of HPLC Methods for the Determination of CYP2D6 and CYP3A4 Activities

Author(s): Yan Pan, Joon Wah Mak and Chin Eng Ong

Affiliation: Jeffrey Cheah School of Medicine and Health Sciences, Monash University Sunway Campus, Jalan Lagoon Selatan, 46150 Bandar Sunway, Selangor Darul Ehsan, Malaysia.

Keywords: Cytochrome P450, CYP2D6, CYP3A4, HPLC, Validation, drug metabolism, pharmacokinetics, Dextromethorphan, drug-drug interactions, corticosterone

Abstract:

Employment of in vitro experimentation to measure the effect of new chemical entities on human cytochrome P450 (CYP) marker activities represents a convenient approach in studying drug metabolism and pharmacokinetics. In this study, simple and accurate high performance liquid chromatograhic (HPLC) methods were developed and validated for quantitative analysis of CYP2D6-mediated dextromethorphan O-demethylation and CYP3A4-mediated testosterone 6β-hydroxylation. Both the assays showed a good linearity in the substrate concentration range of 0.05 – 20.0 μM and 0.01 – 100.0 μM with limit of detection (LOD) of 0.01 μM and 0.001 μM for CYP2D6 and CYP3A4, respectively. The intra- and inter-day precisions were from 7.21% to 12.22% and 3.09% to 14.60% for CYP2D6; and from 4.77% to 9.19% and 3.65% to 11.84% for CYP3A4. Assay accuracy for CYP2D6 ranged from 85.3% to 104.9% over dextrorphan concentrations of 0.05-5.0 μM; and that of CYP3A4 was 105.1% to 109.6% at hydroxytestosterone concentrations of 0.01-50 μM. Enzyme kinetic parameters obtained (Km and Vmax) using the two assays were within reported ranges. Thus, the assays were able to serve as activity markers in the assessment of pharmacokinetic drug interaction and metabolism mediated by CYP2D6 and CYP3A4.

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Article Details

VOLUME: 8
ISSUE: 3
Page: [219 - 224]
Pages: 6
DOI: 10.2174/157341212801619342