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 mass spectrometry (DI/GCE--MS--MS) method has been validated for high-throughput evaluation of cytochrome P450 (CYP) 1A2 inhibition potential using human hepatic microsomes and 96-well microtiter plates. Microsomal incubations were terminated with formic acid, centrifuged, and the resulting supernatants were injected for DI/GCE--MS--MS analysis. Due to the use of an extremely short C(18) guard cartridge, this method offers several advantages such as no sample preparation, excellent on-line extraction, short run time and minimal source contamination and performance deterioration. The DI/GCE--MS--MS method demonstrates acceptable accuracy and precision for the quantification of resorufin, a marker metabolite of ethoxyresorufin mediated by CYP1A2, in microsomal incubations. The inhibition potential of CYP1A2 has been evaluated using its selective inhibitors, alpha-naphthoflavone and furafylline. The IC(50) values (120 nM for alpha-naphthoflavone and 5.1 microM for furafylline) measured by the new method are in agreement with the literature values.
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Department of Metabolic Chemistry, Covance Laboratories, Inc., 3301 Kinsman Boulevard, Madison, WI 53704, USA. haizhi.bu@covance.com
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Magis
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Teitelbaum
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D001571:Benzoflavones / Q000494:pharmacology; D001681:Biological Assay / Q000379:methods; D019388:Cytochrome P-450 CYP1A2 / Q000378:metabolism; D065609:Cytochrome P-450 CYP1A2 Inhibitors; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008862:Microsomes, Liver / Q000187:drug effects* / Q000502:physiology; D010078:Oxazines / Q000378:metabolism*; D013806:Theophylline / Q000031:analogs & derivatives / Q000494:pharmacology
D001571:Benzoflavones; D065609:Cytochrome P-450 CYP1A2 Inhibitors; D004791:Enzyme Inhibitors; D010078:Oxazines; C007649:ethoxyresorufin; C011512:alpha-naphthoflavone; C014180:resorufin; D013806:Theophylline; C050131:furafylline; D019388:Cytochrome P-450 CYP1A2