title: Analytics of nonpeptidic erythropoietin mimetic agents in sports drug testing employing high-resolution/high-accuracy liquid chromatography-mass spectrometry.
abstract: Since its release as anti-anemic drug, recombinant erythropoietin (rEPO) gradually entered the illicit way to sports competitions as endurance-enhancing drug. Novel modifications biopharmaceutically introduced into the rEPO molecule in the form of carbohydrate or polyethylene glycol moieties made robust and sensitive test methods vital to doping controls in order to provide the necessary tools enabling the conviction of dishonest athletes. Modern protein analysis by means of gel electrophoretic separation and western blotting represents the status quo in rEPO anti-doping analysis. However, new therapeutically promising erythropoietin receptor activating compounds have been developed that exhibit cytokine hormone-mimicking properties but lack any protein structure. Progression to evade parenteral application and substitute for rEPO by low molecular mass and orally available compounds is still one of the major objectives in pharmaceutical research. In this approach, four promising in-house synthesized nonpeptidic erythropoietin mimetic agents, namely compound 129, compound 163, A1B10C1, and A5B10C4 were thoroughly evaluated by employing high-resolution/high-accuracy liquid chromatography tandem mass spectrometry experiments. Characteristic product ions were determined supporting the identification of these drugs and putative metabolites as well as related compounds in future doping controls. Test methods employing direct urine injection and receptor affinity purification strategies were assessed, which demonstrated that EPO receptor purification is of limited utility for nonpeptidic EPOR agonists while direct urine injection allowed for comprehensive method characterization. Thereby, achieved limits of detection were 1 ng/mL for compounds 129/163 and 5 ng/mL for A1B10C1/A5B10C4.
authors.affiliation: Center for Preventive Doping Research/Institute of Biochemistry, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Vogel
authors.affiliation: Center for Preventive Doping Research/Institute of Biochemistry, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany.
authors.forename: Josef
authors.initials: J
authors.lastname: Dib
authors.affiliation: Center for Preventive Doping Research/Institute of Biochemistry, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany.
authors.forename: Laura
authors.initials: L
authors.lastname: Tretzel
authors.affiliation: Center for Preventive Doping Research/Institute of Biochemistry, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Piper
authors.affiliation: Center for Preventive Doping Research/Institute of Biochemistry, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Thomas
authors.affiliation: Center for Preventive Doping Research/Institute of Biochemistry, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany.
authors.forename: Wilhelm
authors.initials: W
authors.lastname: Schänzer
authors.affiliation: Center for Preventive Doping Research/Institute of Biochemistry, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany. thevis@dshs-koeln.de.|European Monitoring Center for Emerging Doping Agents (EuMoCEDA), Cologne/Bonn, Germany. thevis@dshs-koeln.de.
authors.forename: Mario
authors.initials: M
authors.lastname: Thevis
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004300:Doping in Sports; D004921:Erythropoietin / Q000031:analogs & derivatives* / Q000378:metabolism / Q000652:urine*; D005260:Female; D006801:Humans; D057230:Limit of Detection; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism; D017467:Receptors, Erythropoietin / Q000378:metabolism; D015813:Substance Abuse Detection / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D017467:Receptors, Erythropoietin; D004921:Erythropoietin
keywords: Cytokine receptor agonists; EPO; Liquid chromatography tandem mass spectrometry; Nonpeptidic erythropoietin mimetic agents; Sports drug testing