Mass spectrometric detection of peginesatide in human urine in doping control analysis

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.