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Recombinant human erythropoietin (rHuEPO) is used as doping a substance. Anti‐doping efforts include urine and blood testing and monitoring the athlete biological passport (ABP). As data on the performance of these methods are incomplete, this study aimed to evaluate the performance of two common urine assays and the ABP. In a randomized, double‐blinded, placebo‐controlled trial, 48 trained cyclists received a mean dose of 6000 IU rHuEPO (epoetin β) or placebo by weekly injection for eight weeks. Seven timed urine and blood samples were collected per subject. Urine samples were analyzed by sarcosyl‐PAGE and isoelectric focusing methods in the accredited DoCoLab in Ghent. A selection of samples, including any with false presumptive findings, underwent a second sarcosyl‐PAGE confirmation analysis. Hematological parameters were used to construct a module similar to the ABP and analyzed by two evaluators from an Athlete Passport Management Unit. Sensitivity of the sarcosyl‐PAGE and isoelectric focusing assays for the detection of erythropoietin abuse were 63.8% and 58.6%, respectively, with a false presumptive finding rate of 4.3% and 6%. None of the false presumptive findings tested positive in the confirmation analysis. Sensitivity was highest between 2 and 6 days after dosing, and dropped rapidly outside this window. Sensitivity of the ABP was 91.3%. Specificity of the urine assays was high; however, the detection window of rHuEPO was narrow, leading to questionable sensitivity. The ABP, integrating longitudinal data, is more sensitive, but there are still subjects that evade detection. Combining these methods might improve performance, but will not resolve all observed shortcomings., World Anti‐Doping Agency methods for detection of recombinant human erythropoietin were evaluated in cyclists. Detection in urine by sarcosyl‐PAGE had a sensitivity of 63.8% and isoelectric focusing of 58.6%, sensitivity peaked between 2 and 6 days after dosing, rapidly dropping outside this window. False presumptive finding rates were 4.3% and 6%, respectively, but none of the false presumptive findings tested positive in the confirmation analysis. Sensitivity of the athlete biological passport that integrates longitudinal hematological data was 91.3%.

Background: The intake of erythropoietins as performance-enhancing drugs is banned in sport. The current method for their detection is based on advanced electrophoretic techniques (IEF-PAGE and SDS-PAGE/sarcosyl-PAGE) with double-blotting and chemiluminescence detection, requiring at least 2.5 days to be completed. Methodology & Results: The proposed procedure, based on vacuum-driven blotting technology, drastically reduces the time necessary to complete the analysis, while still fulfilling the criteria of the World Anti-Doping Agency. Validation was carried out on urine samples spiked with different recombinant erythropoietins, as well as on urine samples obtained following controlled excretion studies and on anonymized urine samples from antidoping tests. Conclusion: The proposed approach, allowing a faster turnaround time, could be very advantageous on the occasion of major sport international events (i.e., Olympic Games).