Development and validation of an UPLC-MS/MS assay for quantitative analysis of the ghrelin receptor inverse agonist PF-5190457 in human or rat plasma and rat brain

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PF-5190457 is a ghrelin receptor inverse agonist that is currently undergoing clinical development for the treatment of alcoholism. Our aim was to develop and validate a simple and sensitive assay for quantitative analysis of PF- 5190457 in human or rat plasma and rat brain using liquid chromatography-tandem mass spectrometry. The analyte and stable isotope internal standard were extracted from 50 µL plasma or rat brain homogenate by protein precipitation using 0.1%formic acid in acetonitrile. Chromatography was carried out on an Acquity UPLC BEH C18 (2.1mm×50mm) column with 1.7 µm particle size and 130 Å pore size. The flow rate was 0.5 mL/min and total chromatographic run time was 2.2 min. The mobile phase consisted of a gradient mixture of water: acetonitrile 95:5 % (v/v) containing 0.1 % formic acid (solvent A) and 100 % acetonitrile containing 0.1 % formic acid (solvent B). Multiple reaction monitoring was carried out in positive electro-spray ionization mode using m/z 513.35?209.30 for PF-5190457 and m/z 518.47? 214.43 for the internal standard. The recovery ranged from 102 to 118 % with coefficient of variation (CV) less than 6 % for all matrices. The calibration curves for all matrices were linear over the studied concentration range (R2=0.998, n=3). The lower limit of quantification was 1 ng/mL in rat or human plasma and 0.75 ng/g in rat brain. Intra- and inter-run mean percent accuracies were between 85 and 115 % and percent imprecision was =15 %. The assays were successfully utilized to measure the concentration of PF- 5190457 in pre-clinical and clinical pharmacology studies of the compound.

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Analytical and Bioanalytical Chemistry