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-tand em mass spectrometry (DI/GCE-MS-MS) method has been validated for high-thro ughput evaluation of cytochrome P450 (CYP) 2D6 inhibition potential using h uman hepatic microsomes and 96-well microtiter plates. Microsomal incubatio ns were terminated with formic acid, centrifuged, and the resulting superna tants were injected for DI/GCE-MS-MS analysis. Due to the novel use of an e xtremely short C-18 guard cartridge, this method exhibits several advantage s, such as no sample preparation, excellent on-line extraction, short run t ime (2.5 min), and minimized source contamination and performance deteriora tion. The DI/GCE-MS-MS method demonstrates acceptable accuracy and precisio n for the quantification of dextrorphan, a marker metabolite of dextrometho rphan 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 muM) measured by the new method is in goo d agreement with the literature value (0.22 muM). (C) 2001 Elsevier Science B.V. All rights reserved.