The development of a new class of erythropoietin mimetic agents (EMA) for 
treating anemic conditions has been initiated with the discovery of 
oligopeptides capable of dimerizing the erythropoietin (EPO) receptor and thus 
stimulating erythropoiesis. The most promising amino acid sequences have been 
mounted on various different polymeric structures or carrier molecules to obtain 
highly active EPO-like drugs exhibiting beneficial and desirable pharmacokinetic 
profiles. Concomitant with creating new therapeutic options, erythropoietin 
mimetic peptide (EMP)-based drug candidates represent means to artificially 
enhance endurance performance and necessitate coverage by sports drug testing 
methods. Therefore, the aim of the present study was to develop a strategy for 
the comprehensive detection of EMPs in doping controls, which can be used 
complementary to existing protocols. Three model EMPs were used to provide 
proof-of-concept data. Following EPO receptor-facilitated purification of target 
analytes from human urine, the common presence of the cysteine-flanked core 
structure of EMPs was exploited to generate diagnostic peptides with the aid of 
a nonenzymatic cleavage procedure. Sensitive detection was accomplished by 
targeted-SIM/data-dependent MS(2) analysis. Method characterization was 
conducted for the EMP-based drug peginesatide concerning specificity, linearity, 
precision, recovery, stability, ion suppression/enhancement, and limit of 
detection (LOD, 0.25 ng/mL). Additionally, first data for the identification of 
the erythropoietin mimetic peptides EMP1 and BB68 were generated, demonstrating 
the multi-analyte testing capability of the presented approach.