A sensitive and specific CYP cocktail assay for the simultaneous assessment of human cytochrome P450 activities in primary cultures of human hepatocytes using LC-MS/MS

J Pharm Biomed Anal. 2013 Feb 23:74:126-32. doi: 10.1016/j.jpba.2012.10.016. Epub 2012 Oct 22.


A sensitive and specific CYP cocktail assay for simultaneous measurement of the activities of major human cytochrome P450 enzymes (CYP1A2 (phenacetin), CYP3A4/5 (midazolam), CYP2C9 (diclofenac), CYP2C19 (S-mephenytoin) and CYP2D6 (dextromethorphan)) in primary cultures of human hepatocytes, was developed and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Hepatocyte incubation medium was processed by a solid phase extraction (SPE) using Oasis SPE extraction cartridges prior to chromatography. The metabolites derived from each of the substrates were simultaneously quantitated using the corresponding stable isotope-labeled internal standards by a positive electrospray ionization mode using multiple reactions monitoring with a single eight minute run. The mean accuracy was in the range of 98-114%. The interday and intraday precision over the concentration ranges evaluated for all the analytes were lower than 15%, and 14%, respectively. All the generated metabolites were stable under the conditions used for sample analysis. Additionally, the interaction of a cocktail substrate on other CYP substrates was also analyzed. Due to substantial inter-substrate interaction, chlorzoxazone (CYP2E1) and bupropion (CYP2B6) were removed from the initial seven probes CYP cocktail assay. Therefore, the final CYP cocktail assay consisting of five probes provides a robust method to simultaneously measure activities of CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4/5 in primary cultures of human hepatocytes.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Cells, Cultured
  • Chromatography, Liquid / methods
  • Cytochrome P-450 Enzyme System / analysis*
  • Cytochrome P-450 Enzyme System / chemistry
  • Drug Interactions / physiology
  • Enzyme Activation / physiology
  • Hepatocytes / enzymology*
  • Humans
  • Pharmaceutical Preparations / analysis
  • Pharmaceutical Preparations / chemistry
  • Tandem Mass Spectrometry / methods*
  • Tandem Mass Spectrometry / standards*


  • Pharmaceutical Preparations
  • Cytochrome P-450 Enzyme System