Reliable high-throughput method for inhibition assay of 8 cytochrome P450 isoforms using cocktail of probe substrates and stable isotope-labeled internal standards

Drug Metab Pharmacokinet. 2012;27(5):520-9. doi: 10.2133/dmpk.dmpk-12-rg-014. Epub 2012 Apr 10.


A significant number of new chemical entities (NCEs) disappear due to cytochrome P450 (CYP)-mediated clinical drug-drug interactions in drug discovery. Therefore, a high throughput assay of CYP activities is necessary in order to evaluate the inhibitory or inducible potencies of CYP isoforms with NCEs in early drug discovery. Here, we developed and validated a high-throughput assay to simultaneously monitor the in vitro activities of 8 CYP isoforms. A cocktail of 9 probe substrates for the 8 major CYPs (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4/5) was incubated with human liver microsomes. Each substrate-derived metabolite was simultaneously analyzed by multiple reactions monitoring with a single ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) run using stable isotope-labeled internal standards. The ultra-fast UPLC gradient allowed each metabolite to be separated within 1 min, providing quantitative linearity of over 2 orders of magnitude. CYP inhibition by 8 well-known inhibitors was confirmed by comparing single substrates with the substrate cocktail. The inhibition curve profiles and IC₅₀ values for all CYPs in the cocktail substrate were similar to those of single substrates. UPLC-MS/MS using a CYP substrate cocktail is a reliable and robust high-throughput method to accurately assess CYP inhibition potencies of newly developed drugs.

MeSH terms

  • Chromatography, High Pressure Liquid / methods
  • Cytochrome P-450 Enzyme Inhibitors*
  • Cytochrome P-450 Enzyme System / metabolism*
  • Drug Discovery / methods
  • Drug Evaluation, Preclinical / methods*
  • Enzyme Inhibitors / pharmacology*
  • High-Throughput Screening Assays / methods*
  • Humans
  • Isoenzymes
  • Isotope Labeling / methods
  • Microsomes, Liver / drug effects
  • Microsomes, Liver / metabolism
  • Reference Standards
  • Reproducibility of Results
  • Substrate Specificity
  • Tandem Mass Spectrometry / methods


  • Cytochrome P-450 Enzyme Inhibitors
  • Enzyme Inhibitors
  • Isoenzymes
  • Cytochrome P-450 Enzyme System