Detection of Darbepoetin Alfa Misuse in Urine and Blood

Abstract

Darbepoetin alfa is a modified erythropoietin (EPO) molecule with a longer serum half-life than recombinant human erythropoietin (rhEPO). Because the detection period of rhEPO in urine is only 2-3 d after the last injection, blood algorithms have been developed in order to expand the detection window of rhEPO misuse. The main objectives were to establish the period of detection of darbepoetin alfa by isoelectric focusing (IEF) and examine the applicability of blood algorithms and individual variations in blood variables in an antidoping context. Six recreationally active males and six recreationally active females had 0.78 microg.kg(-1).wk(-1) of darbepoetin alfa administered for 3 wk. Blood and urine samples were collected continuously during and after administration. Urine samples were analyzed by IEF and immunoblotting for darbepoetin alfa, and blood samples were analyzed for erythropoietic sensitive blood variables on a hematological analyzer. Darbepoetin alfa was detected in 8 of 12 samples at 10 d after the last injection. Ten subjects showed variations in hemoglobin concentration [Hb] > 10%, whereas only three males and one female exceeded suggested upper [Hb] limits of 17.0 and 16.0 g.dL(-1), respectively. Four subjects exceeded the 1:1000 ON- as well as the OFF-model cutoff limit. The large number of samples containing detectable amounts of darbepoetin alfa at 10 d into the washout period stipulate the possibility of a 7-d window of detection after administration, wherein a sample would be regarded as an adverse analytical finding. The marked variations in all examined blood parameters could be used for the targeting of urine samples. These preliminary findings open up for larger scale studies with more frequent urine sampling in the washout period on elite athletes.