Detection of pegylated epoetin β in horse plasma after intravenous administration

Abstract

Methoxy polyethylene glycol–epoetin β (pegylated epoetin β, peg-epoetin β, Mircera^®) is an erythropoiesis-stimulating agent that is synthesized by pegylating epoetin β. It is prohibited in racehorses and humans because it enhances their performance. It is detectable in human urine and plasma using the isoelectric focusing–double-blotting (IEF–DB) method that has been widely applied in human doping control laboratories to detect doping with recombinant erythropoietins (EPOs). Fifty micrograms of peg-epoetin β were administered intravenously to a 450 kg female Thoroughbred, and blood was collected for 48 h. The plasma was processed by immunopurification followed by IEF–DB. The analytical procedures required about 2 days. No peg-epoetin β was detected in the baseline plasma collected prior to drug administration. It was readily detected in samples collected at 2, 4, 6, 8, 24 and 48 h. The isoforms showed a characteristic pattern that differed from epoetin β and was unlike any other known erythrocyte-stimulating agent or recombinant EPO. No peg-epoetin β was detected in plasma from five control horses. The plasma samples were also tested by a rapid, automated and chemiluminescent immunometric assay for EPO (Immulite^®). The samples collected from the treated horse at 2–48 h contained the equivalent of approximately 200–800 pg ml^− 1 of peg-epoetin β. The samples from the ten control horses were negative. These data show that a small dose of peg-epoetin β is detectable for 48 h by two different methods that are based on two different principles (chemiluminescence immunoassay and IEF–DB). The Immulite^® assay is suitable as a screen and the IEF–DB assay as a confirmation method. These studies need to be expanded to other horses. The screen and confirmation tests, if applied, will probably eliminate the use of peg-epoetin β as a horse doping agent.