High-throughput cytochrome P450 (CYP) inhibition screening via cassette probe-dosing strategy. I. Development of direct injection/on-line guard cartridge extraction/tandem mass spectrometry for the simultaneous detection of CYP probe substrates and their metabolites

A highly efficient direct injection/on-line guard cartridge extraction/tand em mass spectrometry (DI-GCE/ MS/MS) method utilizing electrospray polarity switching was developed for the simultaneous detection of probe substrates and marker metabolites of seven human hepatic cytochrome P450 (CYP) isozym es: CYP1A2, 2A6, 3A4, 2C9, 2C19, 2D6 and 2E1. Microsomal incubations were t erminated with formic acid, centrifuged, and the resulting supernatants wer e injected for analysis by DI-GCE/MS/MS, This method employed an extremely short Cls cartridge (4 mm in length) which allowed rapid cleanup of sample matrices while retaining the analytes an appropriate time (2.0-2.2 min). Fr om 1.5 to 2.7 min the effluent was directed to the mass spectrometer for de tection otherwise diverted to waste. As a result of the efficient online ex traction, matrix (e.g., salts and proteins) suppression was minimized. In a ddition, no visible source contamination was observed and system performanc e (chromatographic and mass spectrometric) did not significantly deteriorat e after 500 consecutive injections. Electrospray polarity switching was str ategically executed on a Micromass Quattro II mass spectrometer by establis hing dummy ion transitions to protect the analytes from the interference of the overwhelming noise which was unavoidable for the first transition scan ned following each polarity switch. This unique strategy led to the simulta neous detection of seven CYP probe substrates and seven corresponding marke r metabolites (12 by positive mode and 2 by negative mode). Copyright (C) 2 000 John Wiley & Sons, Ltd.