High-throughput cytochrome P450 (CYP) inhibition screening via a cassette probe-dosing strategy - V. Validation of a direct injection/on-line guard cartridge extraction-tandem mass spectrometry method for CYP1A2 inhibition assessment

An efficient direct injection/on-line guard cartridge extraction-tandem mas s spectrometry (DI/GCE-MS-MS) method has been validated far high-throughput evaluation of cytochrome P450 (CYP) 1A2 inhibition potential using human h epatic microsomes and 96-well microtiter plates. Microsomal incubations wer e terminated with formic acid, centrifuged, and the resulting supernatants were injected for DI/GCE-MS-MS analysis. Due to the use of an extremely sho rt C-18 guard cartridge, this method offers several advantages such as no s ample preparation, excellent on-line extraction, short run time and minimal source contamination and performance deterioration. The DI/GCE-MS-MS metho d demonstrates acceptable accuracy and precision for the quantification of resorufin, a marker metabolite of ethoxyresorufin mediated by CYP1A2, in mi crosomal incubations. The inhibition potential of CYP1A2 has been evaluated using its selective inhibitors, alpha -naphthoflavone and furafylline. The IC50 values (120 nM for alpha -naphthoflavone and 5.1 muM for furafylline) measured by the new method are in agreement with the literature values. (C ) 2001 Elsevier Science B.V. All rights reserved.