Identification of erythropoietin mimetic peptide 1 linear form in a sealed vial and its administration study in horses for doping control purpose

The erythropoietin mimetic peptide 1 linear form (EMP1‐linear), GGTYSCHFGPLTWVCKPQGG‐NH2, was identified in an unknown preparation consisting of white crystalline powder contained in sealed glass vials using ultrahigh performance liquid chromatography‐high‐resolution mass spectrometry (UPLC‐HRMS). The white crystalline powder, allegedly used for doping racehorses, was found to contain around 2% (w/w) of EMP1‐linear. EMP1‐linear can be cyclised in equine plasma at physiological temperature of 37°C by forming an intramolecular disulfide bond to give EMP1, which is a well‐known erythropoiesis stimulating agent that can bind to and activate the receptor for cytokine erythropoietin (EPO). Thus, EMP1‐linear is a prodrug of EMP1, which is a performance‐enhancing doping agent that can be misused in equine sports. In order to identify potential target(s) for detecting the misuse of EMP1‐linear in horses, an in vitro metabolic study using horse liver S9 fraction was performed. After incubation, EMP1‐linear mainly existed in its cyclic form as EMP1, and four N‐terminus truncated in vitro metabolites TYSCHFGPLTWVCKPQGG‐NH2 (M1), SCHFGPLTWVCKPQGG‐NH2 (M2), WVCKPQGG‐NH2 (M3) and VCKPQGG‐NH2 (M4) were identified. An intravenous administration study with the preparation of white crystalline powder containing EMP1‐linear was also conducted using three retired thoroughbred geldings. EMP1 was detectable only in the postadministration plasma samples, whereas the four identified in vitro metabolites were detected in both postadministration plasma and urine samples. For controlling the misuse of EMP1‐linear in horse, its metabolite M3 gave the longest detection time in both plasma and urine and could be detected for up to 4 and 27 h postadministration, respectively. The erythropoietin mimetic peptide 1 linear form (EMP1‐linear) was identified in an unknown preparation consisting of white crystalline powder contained in sealed glass vials, which allegedly had been used for doping racehorses in an overseas country. To better control the misuse of EMP1‐linear, in vitro and in vivo metabolism studies of the preparation of white crystalline powder containing EMP1‐linear were conducted to investigate its metabolism in horses and to identify target analytes with the longest detection time for doping control.