Pillai VC, Strom SC, Caritis SN, Venkataramanan RA 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 74: 126-132

Department of Pharmaceutical Sciences, University of Pittsburgh School of Pharmacy, Pittsburgh, PA, USA.
Journal of pharmaceutical and biomedical analysis (Impact Factor: 2.98). 02/2013; 74:126-32. DOI: 10.1016/j.jpba.2012.10.016
Source: PubMed


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.

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Available from: Stephen Strom, Aug 12, 2014
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    • "So-called 'cocktail' assays are particularly useful (Feidt et al., 2010) in rapidly assessing possible DDIs. Although a number of such assays have been published in recent years (Pillai et al., 2013; De Bock et al., 2012; Otten et al., 2011; Feidt et al., 2010), none have been reported that involve simultaneous determination of the seven metabolites acetaminophen, hydroxy-bupropion, n-desethylamodiaquine , 4′-hydroxy-diclofenac, 4′-hydroxy-mephenytoin, dextrorphan and 1′-hydroxy-midazolam in a single-run process. This study presents an efficient cocktail approach that uses low protein concentration and minimizes compound-to-compound interactions among substrates as well. "
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