Detection of ESAs in equine urine and blood by SAR‐PAGE

Erythropoiesis‐stimulating agents (ESAs) have been used in horses for doping purposes to increase the performance of these animals in endurance sports. Currently, enzyme‐linked immunosorbent assay (ELISA) and mass spectrometry methods are used to detect ESA abuse in equines. However, the sarcosyl polyacrylamide gel‐electrophoresis (SAR‐PAGE) technique could also be used, since its application in human doping control is well established and has proven to be more sensitive. In this work, the SAR‐PAGE method was used to detect recombinant human erythropoietin (rHuEPO), novel erythropoiesis stimulating protein (NESP), continuous erythropoietin receptor activator (CERA), and fusion protein of erythropoietin with human immunoglobulin heavy chain Fc region (EPO‐Fc) in horse blood and urine. The purification technique for human blood using MAIIA kits worked well for horse samples. The major challenge was horse urine immunopurification, which proved difficult due to filter clogging, but heating and cooling of the horse urine followed by filtration in 30‐kDa molecular weight cut‐off filters solved this problem. The limits of detection (LODs) of 1.3, 1.6, 6.6, and 13.3 pg/mL for rHuEPO, NESP, CERA, and EPO‐Fc, respectively, obtained in spiked urine and 40, 100, 80, and 400 pg/mL for rHuEPO, NESP, CERA, and EPO‐Fc, respectively, acquired in spiked blood are lower than the LODs reported in the literature using liquid chromatography–mass spectrometry (LC–MS) methods. In addition, the presence of ESAs was detected up to 9 days after the administration of microdoses of Hemax (rHuEPO), NESP, and CERA in horse blood and urine. SAR‐PAGE may be implemented in the routine analysis of horse doping control laboratories for screening and confirmation of ESA abuse, mainly due to its high sensitivity for both matrices compared to published mass spectrometric methods. This study used the SAR‐PAGE method to detect rHuEPO, NESP, CERA, and EPO‐Fc in horse samples for doping control purposes. Here the blood immunopurification technique performed for human samples was validated and the urine immunopurification shortcomings were overcome so that both can be applied in equine matrices for detection of ESAs by SAR‐PAGE. An excretion study was conducted to detect ESAs in horses after single microdose administration to evaluate the sensitivity of the method in real samples.