title: Isolation, enrichment, and analysis of erythropoietins in anti-doping analysis by receptor-coated magnetic beads and liquid chromatography-mass spectrometry.
abstract: Human erythropoietin (hEPO) is an erythropoiesis stimulating hormone frequently employed in antianemia therapy. Its capability to increase the amount of red blood cells however makes hEPO and its derivatives also attractive to dishonest athletes aiming at an artificial and illicit enhancement of their endurance performance. A major objective of the international antidoping fight is the elimination of drug misuse and prevention of severe adverse effects caused by nontherapeutic administrations of highly potent drugs. The emergence of novel and innovative erythropoietin-mimetic agents (EMAs) has been continuously growing in the last years, and the option of using dedicated monoclonal antibodies (mAb) for analytical and sample preparation approaches is gradually reaching limits. In the present study the common ability and property of all EMAs, to bind on the human erythropoietin receptor (hEPOR), is therefore exploited. An alternative methodology to isolate and analyze EMAs, in particular endogenous EPO and the recombinant forms EPOzeta, darbepoetin alfa, and C.E.R.A., from human urine is described, employing conventional ultrafiltration for preconcentration of the target analytes followed by EMA-specific isolation via hEPOR-bound magnetic beads. Analytical data were generated by means of gel-based electrophoretic analysis and nanoliquid chromatography/high resolution/high accuracy tandem mass spectrometry. Limits of detection enabled by the established sample preparation protocols were approximately 20 pg/mL for EPOzeta, 30 pg/mL for darbepoetin alfa, and 80 pg/mL for C.E.R.A.
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.forename: Mike
authors.initials: M
authors.lastname: Blobel
authors.forename: Andreas
authors.initials: A
authors.lastname: Thomas
authors.forename: Katja
authors.initials: K
authors.lastname: Walpurgis
authors.forename: Wilhelm
authors.initials: W
authors.lastname: Schänzer
authors.forename: Christian
authors.initials: C
authors.lastname: Reichel
authors.forename: Mario
authors.initials: M
authors.lastname: Thevis
mesh_terms: D002853:Chromatography, Liquid; D004300:Doping in Sports; D004921:Erythropoietin / Q000737:chemistry / Q000652:urine*; D006801:Humans; D060328:Magnetic Phenomena; D017467:Receptors, Erythropoietin / Q000737:chemistry*; D053719:Tandem Mass Spectrometry; D016482:Urinalysis / Q000379:methods*
chemical_list: D017467:Receptors, Erythropoietin; D004921:Erythropoietin