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High‐throughput cytochrome P450 inhibition screening via cassette probe‐dosing strategy. IV. Validation of a direct injection on‐line guard cartridge extraction/tandem mass spectrometry method for simultaneous CYP3A4, 2D6 and 2E1 inhibition assessment
Author(s) -
Bu HaiZhi,
Knuth Kim,
Magis Lisa,
Teitelbaum Philip
Publication year - 2000
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/1097-0231(20001030)14:20<1943::aid-rcm116>3.0.co;2-f
Subject(s) - chemistry , chromatography , chlorzoxazone , tandem mass spectrometry , cyp3a4 , cartridge , mass spectrometry , microsome , cyp2e1 , cytochrome p450 , biochemistry , enzyme , mechanical engineering , engineering
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) 3A4, 2D6 and 2E1 inhibition potential via cassette dosing of midazolam, dextromethorphan and chlorzoxazone using human hepatic microsomes and 96‐well microtiter plates. Microsomal incubations were terminated with formic acid, centrifuged, and the resulting supernatants were injected for analysis by DI‐GCE/MS/MS. Due to the novel use of an extremely short C 18 guard cartridge (4 mm in length), 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 simultaneous quantification of 1′‐hydroxymidazolam, dextrorphan and 6‐hydroxychlorzoxazone in microsomal incubations. The inhibition potential of CYP3A4, 2D6 and 2E1 has been evaluated using their known selective inhibitors. The IC 50 values measured by the cassette dosing approach (high‐throughput) are consistent with those observed by an individual dosing regimen (conventional) and are all in good agreement with the literature values. The results suggest that the cassette probe‐dosing strategy may provide an in vitro approach to minimize cost while maximizing throughput of CYP inhibition evaluation of new chemical entities in support of drug discovery and development. Copyright © 2000 John Wiley & Sons, Ltd.