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 wer...

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Bibliographic Details
Published in:European journal of pharmaceutical sciences 2001-02, Vol.12 (4), p.447-452
Main Authors: Bu, Hai-Zhi, Knuth, Kim, Magis, Lisa, Teitelbaum, Philip
Format: Article
Language:English
Subjects:
Analysis
Benzoflavones - pharmacology
Biological and medical sciences
Biological Assay - methods
CYP1A2
Cytochrome P-450 CYP1A2 - metabolism
Cytochrome P-450 CYP1A2 Inhibitors
Direct injection
Enzyme Inhibitors - pharmacology
General pharmacology
High-throughput
Humans
Inhibition
Mass Spectrometry - methods
Medical sciences
Microsomes, Liver - drug effects
Microsomes, Liver - physiology
On-line extraction
Oxazines - metabolism
Pharmacology. Drug treatments
Theophylline - analogs & derivatives
Theophylline - pharmacology
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Summary: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, α-naphthoflavone and furafylline. The IC 50 values (120 nM for α-naphthoflavone and 5.1 μM for furafylline) measured by the new method are in agreement with the literature values.
ISSN:0928-0987
1879-0720
DOI:10.1016/S0928-0987(00)00190-1