High-throughput cytochrome P450 inhibition screening via cassette probe-dosing strategy. IV. Validation of a direct injection on-line guard cartridgeextraction/tandem mass spectrometry method for simultaneous CYP3A4, 2D6 and 2E1 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) 3A4, 2D6 and 2E1 inhibition pote ntial via cassette dosing of midazolam, dextromethorphan and chlorzoxazone using human hepatic microsomes and 96-well microtiter plates, Microsomal in cubations were terminated with formic acid, centrifuged, and the resulting supernatants were injected for analysis by DI-GCE/MS/MS. Due to the novel u se of an extremely short C-18 guard cartridge (4 mm in length), this method exhibits several advantages such as no sample preparation, excellent on-li ne extraction, short run time (2.5 min), and minimized source contamination and performance deterioration. The DI-GCE/MS/MS method demonstrates accept able accuracy and precision for the simultaneous quantification of 1'-hydro xymidazolam, dextrorphan and 6-hydroxychlorzoxazone in microsomal incubatio ns. The inhibition potential of CYP3A4, 2D6 and 2E1 has been evaluated usin g their known selective inhibitors. The IC50 values measured by the cassett e dosing approach (high-throughput) are consistent with those observed by a n individual dosing regimen (conventional) and are all in good agreement wi th the literature values, The results suggest that the cassette probe-dosin g strategy may provide an in vitro approach to minimize cost while maximizi ng throughput of CYP inhibition evaluation of new chemical entities in supp ort of drug discovery and development, Copyright (C) 2000 John Wiley & Sons , Ltd.