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Simultaneous Detection of CYP3A4, CYP2D6 and CYP2C9 Metabolites with a Single, Sensitive, LC/MS/MS Method

Jason S. Wood, Varian, Inc.

Introduction

Cytochrome P450s (CYPs) are heme-containing enzymes responsible for initial metabolism of xenobiotics within the human body1. CYPs are part of the Phase I (oxidative) metabolism within the body, which introduces a hydrophilic site on the metabolized drug enabling either easy elimination of the drug or further conjugation by Phase II enzymes, such as UDPGTs, sulfotransferases or amino acid-conjugation (among others)2. Recently there has been increased interest in understanding CYPs to discover the underlying mechanisms of drug-drug interactions3, drug side effect(s)4 and toxicity (including hepatotoxicity). Also relevant to CYP testing is the discovery that polymorphisms in the genetic sequences expressing these enzymes leads to populations of people whose ability to metabolize certain classes of drugs may be attenuated in comparison to other races/populations5.

In this application note LC-ESI-MS/MS is used to simultaneously characterize three drug metabolites for the detection of three different cytochrome P450s enzymatic activities; the hydroxylation of midazolam (CYP3A4), hydroxylation of tolbutamide (CYP2C9) and Odemethylation of dextromethorphan (CYP2D6), see Figure 1. This assay could be used to analyze CYP3A4, CYP2D6 and CYP2C9 activity in vitro for kinetic studies (e.g., liver microsome inhibition studies) or possibly for detection of these metabolites in more complex matrices (plasma or urine) after SPE.

Instrumentation

Varian ProStar 210 Solvent Delivery Modules (2)

Varian 1200L LC/MS equipped with an ESI source

Varian ProStar 430 AutoSampler

Materials and Reagents

All chemicals were reagent or HPLC grade from Sigma- Aldrich Corporation (St. Lo
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