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Keywords:

  • multiplexed MRM quantification of proteins;
  • transporters;
  • membrane transporter;
  • cytochrome P450;
  • UDP-glucuronosyltransferases;
  • nano-LC;
  • LC–MS/MS;
  • mass spectrometry;
  • proteomics

Abstract

Mass spectrometry (MS)-based multiplexed multiple reaction monitoring quantification of proteins has recently evolved as a versatile tool for accurate, absolute quantification of proteins. The purpose of this study was to examine the validity of the present method with regard to standard bioanalytical criteria for drug transporters, cytochrome P450 (CYP) enzymes and uridine 5′-diphospho-glucuronosyltransferases (UGTs). Membrane preparations from human liver tissue were used for target protein quantification. As a result, the determination coefficients (r2) of all targets were greater than 0.986. In the absence of matrix, inaccuracy values (expressed as % deviation) were −8.1% to 20.3%, whereas imprecision values (expressed as % coefficient of variation) were within 15.9%. In the presence of matrix, which consisted of digested plasma membrane fraction for transporters and digested microsomal membrane fraction for CYP enzymes and UGTs, respectively, the inaccuracy was −15.3%–8.1%, and the imprecision were within 18.9%. Sufficient sample stability of membrane fraction was shown for three freeze–thaw cycles, 32 days at −20°C, and in processed samples for 7 days at 10°C. In conclusion, this study demonstrated, for the first time, that the MS-based assay with nano-liquid chromatography provides adequate reliability and robustness for the quantification of selected drug transporters, P450 enzymes and UGTs. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:4037–4043, 2011