Detection of recombinant human EPO and darbepoetin administrated to horses using EPO WGA MAIIA and LC-MS / MS

Doping of horses with recombinant human erythropoietin to enhance their endurance capacity is sometimes occurring in horse-races and has been used and reported on various occasion in Europe, USA, Australia and New Zeeland. Besides unhealthy increased blood viscosity, the horse might start to produce antibodies against the injected human EPO, which can crossreact with the endogenous EPO and lead to severe anaemia and even death. Two recently developed methods have been compared for their capability to detect administration with epoetin alpha (Eprex) or darbepoetin alpha (Aranesp) in horses in both plasma and urine samples. The easy-to-use and rapid EPO WGA MAIIA test distinguishes epoetin and darbepoetin from endogenous equine EPO by their different carbohydrate motifs on the protein. LC-MS/MS differentiates human and equine EPO due to the size of some selected peptide structures obtained after trypsin digestion. Seven horses were s.c. administrated with 40 IU Eprex/kg during six days and one horse obtained a single dose of 0.39 μg Aranesp/kg. It was found that EPO WGA MAIIA could detect (CL 99%) the presence of Eprex four days after last injection for four of the seven tested horses, while LC-MS/MS identified Eprex up to one day after last injection. EPO WGA MAIIA detected (CL 99.9%) the presence of Aranesp up to eight days, while LC-MS/MS could identify Aranesp up to five days after last injection. EPO WGA MAIIA seems to be much more sensitive than LC-MS/MS, while the latter has the mass spectral data required for equine forensic drug testing. Before the advent of more sensitive MS analytical techniques the use of EPO WGA MAIIA might be a valuable means for supressing EPO doping of horses. Abbreviations used: ESA, erythropoiesis stimulating agents; IEF, isoelectric focusing; NGlcNAc, N-Acetylglucosamine; PMI, percentage of migrated isoforms; rhEPO, recombinant human EPO; WGA, wheat germ agglutinin