title: 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.
abstract: 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.
authors.affiliation: Department of Pharmaceutical Sciences, University of Pittsburgh School of Pharmacy, Pittsburgh, PA, USA.
authors.forename: Venkateswaran C
authors.initials: VC
authors.lastname: Pillai
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Strom
authors.forename: Steve N
authors.initials: SN
authors.lastname: Caritis
authors.forename: Raman
authors.initials: R
authors.lastname: Venkataramanan
mesh_terms: D002478:Cells, Cultured; D002853:Chromatography, Liquid / Q000379:methods; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis* / Q000737:chemistry; D004347:Drug Interactions / Q000502:physiology; D004789:Enzyme Activation / Q000502:physiology; D022781:Hepatocytes / Q000201:enzymology*; D006801:Humans; D004364:Pharmaceutical Preparations / Q000032:analysis / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods* / Q000592:standards*
chemical_list: D004364:Pharmaceutical Preparations; D003577:Cytochrome P-450 Enzyme System