Synthesis, characterisation, and mass spectrometric detection of a pegylated EPO-mimetic peptide for sports drug testing purposes

Erythropoietin (EPO) and other erythropoiesis-stimulating agents possess a high misuse potential in elite sport due to their ability to increase the oxygen transport capacity, which plays a vital role in enhancing endurance performance. Currently, a new generation of EPO-mimetic peptides is under development from which peginesatide (also referred to as Hematide™), a pegylated homodimeric peptide of approximately 45 kDa with no structural relationship to erythropoietin, is the most advanced drug candidate undergoing phase-III clinical trials. Since preventive doping research aims at the development of detection methods before a drug receives clinical approval, a selective and sensitive assay has to be established knowing that conventional doping control assays for EPO will not succeed in detecting peginesatide. Thus, a pegylated EPO-mimetic peptide simulating the structure and properties of the lead drug candidate peginesatide was synthesised as a model compound for this new class of emerging drugs and characterised by means of gel electrophoresis, matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry, as well as liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS) after proteolytic digestion. Based on these results, a mass spectrometric detection method of the product in plasma was developed targeting a pentapeptide fragment after protein precipitation and subtilisin digestion. Its fitness for purpose was evaluated by the determination of selected method characteristics focusing particularly on specificity, recovery (ca. 60%), and limit of detection (1 ng/mL).