Validation of higher-throughput high-performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry assays to conduct cytochrome P450s CYP2D6 and CYP3A4 enzyme inhibition studies in human liver microsomes

Rapid Commun Mass Spectrom. 2000;14(4):207-14. doi: 10.1002/(SICI)1097-0231(20000229)14:4<207::AID-RCM863>3.0.CO;2-#.

Abstract

In the early stage of drug discovery, thousands of new chemical entities (NCEs) may be screened before a single drug candidate can be identified for development. In order to accelerate the drug discovery process, we have developed higher-throughput enzyme assays to evaluate the inhibition of cytochrome P450 isoforms 2D6 (CYP2D6) and 3A4 (CYP3A4) in human liver microsomes. The assays are based on high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) techniques. The analysis time for each sample was reduced from approximately 20 minutes for the conventional HPLC assay to 30 seconds for the LC/MS/MS assay. For both LC/MS/MS assays, the linearity (r(2) > 0.99), precision (%CV < 15%) and accuracy (% bias <15%) for both inter- and intraday validations were satisfactory. Since the implementation of the LC/MS/MS assays, our sample throughput has increased by over 40-fold.

MeSH terms

  • Atmospheric Pressure
  • Chromatography, High Pressure Liquid / methods*
  • Cytochrome P-450 CYP2D6 / analysis*
  • Cytochrome P-450 CYP2D6 Inhibitors*
  • Cytochrome P-450 CYP3A
  • Cytochrome P-450 Enzyme Inhibitors*
  • Cytochrome P-450 Enzyme System / analysis*
  • Dextrorphan / chemistry
  • Dextrorphan / pharmacology
  • Drug Design
  • Drug Evaluation, Preclinical
  • Enzyme Inhibitors / chemistry
  • Enzyme Inhibitors / pharmacology
  • Evaluation Studies as Topic
  • Humans
  • Hydroxytestosterones / chemistry
  • Hydroxytestosterones / pharmacology
  • In Vitro Techniques
  • Mass Spectrometry / methods*
  • Microsomes, Liver / enzymology*
  • Mixed Function Oxygenases / analysis*
  • Mixed Function Oxygenases / antagonists & inhibitors*
  • Reproducibility of Results
  • Substrate Specificity

Substances

  • Cytochrome P-450 CYP2D6 Inhibitors
  • Cytochrome P-450 Enzyme Inhibitors
  • Enzyme Inhibitors
  • Hydroxytestosterones
  • Dextrorphan
  • 6 beta-hydroxytestosterone
  • Cytochrome P-450 Enzyme System
  • Mixed Function Oxygenases
  • CYP3A protein, human
  • Cytochrome P-450 CYP2D6
  • Cytochrome P-450 CYP3A
  • CYP3A4 protein, human