High-throughput cytochrome P450 (CYP) inhibition screening via cassette probe-dosing strategy: III. Validation of a direct injection/on-line guard cartridge extraction - tandem mass spectrometry method for CYP2C19 inhibitionevaluation

A new direct injection/on-line guard cartridge extraction-tandem mass spect rometry (DI/GCE-MS-MS) method has been validated for high-throughput evalua tion of cytochrome P450 (CYP) 2C19 inhibition potential using human hepatic microsomes and 96-well microtiter plates. Microsomal incubations were quen ched with formic acid, centrifuged, and the resulting supernatants were inj ected for DI/GCE - MS - IVIS analysis. Due to the use of a C-18 guard cartr idge (4 x 2.0 mm I.D.), this method exhibits several advantages such as lit tle sample preparation, rapid on-line extraction, short analysis time (2.5 min), and minimal source contamination and performance deterioration. The D I/GCE -MS - MS method demonstrates an inter-day accuracy ranged from 0.3 to 2.4% with precision ranging from 2.0 to 3.0% for the quantification of 4-h ydroxymephenytoin, a marker metabolite of S-mephenytoin mediated by CYP2C19 , in microsomal incubations. The CYP2C19 inhibition assay has been validate d using tranylcypromine as a known inhibitor of the isoform. The IC50 value (43.5 muM) measured by the new method is in agreement with a reported lite rature value (similar to 30 muM). (C) 2001 Elsevier Science B.V. All rights reserved.