Erythropoietin (EPO) and its recombinant analogues are suspected to be illicitly 
administered to horses for performance enhancing purposes and, consequently, 
prohibited in equine sports. Recently, a new erythropoiesis-stimulating agent, 
peginesatide (Omontys, formerly referred to as Hematide), belonging to the 
upcoming class of EPO-mimetic peptides, received approval for the treatment of 
anaemia in humans with chronic kidney disease on dialysis. As the pegylated 
dimeric peptide of approximately 45 kDa without sequence homology to EPO is not 
detectable by conventional EPO detection assays, specific methods are bound to 
be established for horse sports drug testing. Thus, by fortifying equine serum 
with peginesatide, an approach consisting of a proteolytic digestion with 
subtilisin after protein precipitation was developed, eventually targeting a 
proteotypic and xenobiotic pentapeptide which is easily accessible to liquid 
chromatography- tandem mass spectrometry analysis. The method was validated for 
qualitative purposes and demonstrated to be specific, precise (relative standard 
deviations below 14%), sensitive (limit of detection 10 ng mL(-1)) and linear. 
Being simple, cost-effective and readily transferable to other doping control 
laboratories, a mass spectrometric assay for the detection of therapeutic 
concentrations of peginesatide in equine serum is, in terms of preventive doping 
research, applicable to routine analysis shortly after approval of the drug.