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/tandem 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) isozymes: CYP1A2, 2A6, 3A4, 2C9, 2C19, 2D6 and 2E1. Microsomal incubations were terminated with formic acid, centrifuged, and the resulting supernatants were injected for analysis by DI-GCE/MS/MS. This method employed an extremely short C(18) cartridge (4 mm in length) which allowed rapid cleanup of sample matrices while retaining the analytes an appropriate time (2. 0-2.2 min). From 1.5 to 2.7 min the effluent was directed to the mass spectrometer for detection otherwise diverted to waste. As a result of the efficient on-line extraction, matrix (e.g., salts and proteins) suppression was minimized. In addition, no visible source contamination was observed and system performance (chromatographic and mass spectrometric) did not significantly deteriorate after 500 consecutive injections. Electrospray polarity switching was strategically executed on a Micromass Quattro II mass spectrometer by establishing dummy ion transitions to protect the analytes from the interference of the overwhelming noise which was unavoidable for the first transition scanned following each polarity switch. This unique strategy led to the simultaneous detection of seven CYP probe substrates and seven corresponding marker metabolites (12 by positive mode and 2 by negative mode).
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Department of Metabolic Chemistry, Covance Laboratories, Inc., 3301 Kinsman Boulevard, Madison, WI 53704, USA.
Magis
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Knuth
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Teitelbaum
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D002851:Chromatography, High Pressure Liquid; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005561:Formates / Q000737:chemistry; D006801:Humans; D007202:Indicators and Reagents; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D013058:Mass Spectrometry; D008862:Microsomes, Liver / Q000737:chemistry / Q000201:enzymology; D009862:Online Systems; D004364:Pharmaceutical Preparations / Q000032:analysis
D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D005561:Formates; D007202:Indicators and Reagents; D007527:Isoenzymes; D004364:Pharmaceutical Preparations; D003577:Cytochrome P-450 Enzyme System