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.
Department of Metabolic Chemistry, Covance Laboratories, Inc., 3301 Kinsman Boulevard, Madison, WI 53704, USA. haizhi.bu@covance.com
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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