High-throughput cytochrome P450 (CYP) inhibition screening via cassette probe-dosing strategy. II. Validation of a direct injection/on-line guard cartridge extraction-tandem mass spectrometry method for CYP2D6 inhibition assessment.
A highly efficient direct injection/on-line guard cartridge extraction-tandem mass spectrometry (DI/GCE-MS-MS) method has been validated for high-throughput evaluation of cytochrome P450 (CYP) 2D6 inhibition potential using human hepatic microsomes and 96-well microtiter plates. Microsomal incubations were terminated with formic acid, centrifuged, and the resulting supernatants were injected for DI/GCE-MS-MS analysis. Due to the novel use of an extremely short C18 guard cartridge, this method exhibits several advantages, such as no sample preparation, excellent on-line extraction, short run time (2.5 min), and minimized source contamination and performance deterioration. The DI/GCE-MS-MS method demonstrates acceptable accuracy and precision for the quantification of dextrorphan, a marker metabolite of dextromethorphan mediated by CYP2D6, in microsomal incubations. The CYP2D6 inhibition assay has been validated using quinidine as a known selective inhibitor of the isoform. The IC50 value (0.20 microM) measured by the new method is in good agreement with the literature value (0.22 microM).
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Department of Metabolic Chemistry, Covance Laboratories, Inc., Madison, WI 53704, USA. haizhi.bu@covance.com
Magis
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Knuth
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Teitelbaum
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D002138:Calibration; D065690:Cytochrome P-450 CYP2D6 Inhibitors; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D008862:Microsomes, Liver / Q000201:enzymology; D015335:Molecular Probes; D015203:Reproducibility of Results
D065690:Cytochrome P-450 CYP2D6 Inhibitors; D004791:Enzyme Inhibitors; D015335:Molecular Probes