Characterization of in vitro generated metabolites of selected peptides <2 kDa prohibited in sports

With an increasing number of prohibited substances in doping controls, knowledge about their metabolism is crucial for efficient analysis. While for low molecular mass molecules, standard protocols for in vitro metabolism experiments are well established, the situation with peptidic drugs has been shown to be substantially more heterogeneous and complex. Two principle strategies aiming at simulating the metabolism of lower molecular mass peptides in vitro are presented within this study. The prohibited peptides ARA-290, GHRP-3, and Peforelin, with a to-date unknown metabolism, were chosen as model compounds for these experiments and metabolism after incubation with different blood specimens (EDTA-, heparin-, citrate-plasma, and serum) and exposure to recombinant amidase were investigated. The characterization of in vitro generated drug-derived peptidic analytes was accomplished by means of liquid chromatography coupled to high resolution mass spectrometry. Identification of the generated metabolites was ensured by dedicated high resolution product ion experiments after liquid chromatographic separation. While extensive exopeptidase-driven metabolism was observed for ARA-290 (with one main metabolite PyrEQLERALN), GHRP-3 and Peforelin were found to exhibit a considerable metabolic stability with a low tendency for deamidation only. Copyright © 2017 John Wiley & Sons, Ltd.