Erythropoiesis-stimulating agents (ESAs) have frequently been confessed to be 
illicitly used in elite sports due to their endurance enhancing effects. 
Recently, peginesatide, the first representative of a new generation of ESAs, 
referred to as Erythropoietin (EPO)-mimetic peptides, obtained approval in the 
USA under the trade name Omontys(®) for the treatment of anaemic patients. 
Lacking sequence homology with EPO, it consists of a pegylated homodimeric 
peptide of approximately 45 kDa, and thus, specific approaches for the 
determination of peginesatide in blood were developed as conventional detection 
assays for EPO do not allow for the analysis of the EPO-mimetic peptides. 
However, as urine specimens are the most frequently provided doping control 
samples and pharmacokinetic studies conducted in rats and monkeys revealed the 
excretion of the pegylated peptide into urine, a detection method for 
peginesatide in urine would be desirable. A mass spectrometric assay in human 
urine was developed consisting of protein precipitation with acetonitrile 
followed by proteolytic digestion after the removal of the acetonitrile fraction 
under reduced pressure. Purification and concentration of the resulting 
proteotypic target peptide was accomplished by means of solid-phase extraction 
on strong cation-exchange resin prior to liquid chromatographic-tandem mass 
spectrometric analysis. Method validation was performed for qualitative purposes 
and demonstrated specificity, precision, linearity as well as sufficient 
sensitivity (limit of detection: 0.5 ng/ml) while proof-of-concept for the 
applicability of the assay for the determination of peginesatide in authentic 
urine samples was obtained by analyzing animal in vivo specimens collected after 
a single i.v. administration of peginesatide over a period of 4 days.