LC-MS/MS-based quantification of clinically relevant intestinal uptake and efflux transporter proteins

Multidrug transporter proteins are crucial determinants in the pharmacokinetics of many drugs. To evaluate their impact on intestinal drug absorption, we developed and validated quantification methods for 10 uptake transporters (OATP1A2, OATP2B1, PEPT1, ASBT, OCT1, OCT3) and efflux transporters (ABCB1, ABCC2, ABCC3, ABCG2) that have been reported to be expressed and to be of clinical relevance in the human intestine. Quantification was performed by targeted liquid chromatography with tandem mass spectrometry (LC-MS/MS)-based quantification of proteospecific peptides after tryptic digestion using stable isotope labeled internal standard peptides. The chromatography of the respective peptides was performed by gradient elution using a reversed phase (C18) column (Kinetex(®), 100 × 3.0 mm, 2.6 μm) and 0.1% formic acid (FA) and acetonitrile with 0.1% FA as mobile phases at a flow rate of 0.5 ml/min. The MS/MS detection was done in the positive multiple reaction monitoring (MRM) mode by monitoring in each case three mass transitions for the transporter-derived peptides and the internal standard peptides. The assays were validated with respect to specificity, linearity (0.1-25 nM), within-day and between-day accuracy and precision as well as stability according to current bioanalytical guidelines. Finally, the developed methods were used to determine the transporter protein content in human intestinal tissue (jejunum and ileum). The methods were shown to possess sufficient specificity, sensitivity, accuracy, precision and stability to measure transporter proteins in the human intestine.