Development and full validation of six inhibition assays for five major cytochrome P450 enzymes in human liver microsomes using an automated 96-well microplate incubation format and LC-MS/MS analysis

J Pharm Biomed Anal. 2007 May 9;44(1):211-23. doi: 10.1016/j.jpba.2007.02.034. Epub 2007 Mar 3.


Substrate inhibition assays for five of the major CYP enzymes (phenacetin for CYP1A2, diclofenac for CYP2C9, (S)-mephenytoin for CYP2C19, dextromethorphan for CYP2D6 and midazolam and testosterone for CYP3A4) in human liver microsomes were developed. Fully automated incubations were conducted in a 96-well format under optimized enzyme kinetic conditions. Metabolites of probe substrates were analyzed with rapid LC-MS/MS methods. The assays were fully validated following the procedure for validating bioanalytical methods recommended by regulatory agencies. Quality control samples and a positive control CYP inhibitor were included in each assay. The IC(50) values determined for typical CYP inhibitors were reproducible and consistent with those reported in the literature. The high quality and throughput of these assays make them ideally suited for providing information for decision making in late drug discovery and early development and for providing labeling input for new drug registrations.

MeSH terms

  • Aryl Hydrocarbon Hydroxylases / antagonists & inhibitors
  • Aryl Hydrocarbon Hydroxylases / chemistry
  • Autoanalysis
  • Biological Assay
  • Chromatography, Liquid / methods*
  • Cytochrome P-450 CYP1A2 / chemistry
  • Cytochrome P-450 CYP1A2 Inhibitors
  • Cytochrome P-450 CYP2C19
  • Cytochrome P-450 CYP2C9
  • Cytochrome P-450 CYP2D6 / chemistry
  • Cytochrome P-450 CYP2D6 Inhibitors
  • Cytochrome P-450 CYP3A
  • Cytochrome P-450 Enzyme Inhibitors*
  • Cytochrome P-450 Enzyme System / chemistry
  • Enzyme Inhibitors / pharmacology*
  • Humans
  • Inhibitory Concentration 50
  • Isoenzymes / antagonists & inhibitors
  • Kinetics
  • Microsomes, Liver / enzymology*
  • Mixed Function Oxygenases / antagonists & inhibitors
  • Mixed Function Oxygenases / chemistry
  • Molecular Structure
  • Reproducibility of Results
  • Substrate Specificity
  • Tandem Mass Spectrometry / methods*


  • Cytochrome P-450 CYP1A2 Inhibitors
  • Cytochrome P-450 CYP2D6 Inhibitors
  • Cytochrome P-450 Enzyme Inhibitors
  • Enzyme Inhibitors
  • Isoenzymes
  • Cytochrome P-450 Enzyme System
  • Mixed Function Oxygenases
  • CYP2C9 protein, human
  • Cytochrome P-450 CYP2C9
  • Aryl Hydrocarbon Hydroxylases
  • CYP2C19 protein, human
  • Cytochrome P-450 CYP1A2
  • Cytochrome P-450 CYP2C19
  • Cytochrome P-450 CYP2D6
  • Cytochrome P-450 CYP3A
  • CYP3A4 protein, human