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Synthesis, characterisation, and mass spectrometric detection of a pegylated EPO‐mimetic peptide for sports drug testing purposes
Author(s) -
Möller Ines,
Thomas Andreas,
Geyer Hans,
Schänzer Wilhelm,
Thevis Mario
Publication year - 2011
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.5109
Subject(s) - chemistry , chromatography , mass spectrometry , peptide , erythropoietin , electrospray ionization , protein precipitation , pentapeptide repeat , tandem mass spectrometry , liquid chromatography–mass spectrometry , biochemistry , medicine
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). Copyright © 2011 John Wiley & Sons, Ltd.