Possible Indirect Detection of rHuEPO Administration in Human Urine by High-Performance Liquid Chromatography Tandem Mass Spectrometry

The present study is based on the assumption that changes in an ADMA–DDAH–NOS (ADMA—asymmetrical dimethylarginine; DDAH—dimethyl-arginine dimethylaminohydrolase; NOS—nitric oxide synthase) system could be employed as indirect markers for recombinant human erythropoietin (rHuEPO) administration in doping control. We assessed a predictive value of four proposed new markers for rHuEPO abuse. Preliminary data showed that concentrations of ADMA, symmetrical dimethylarginine (SDMA), citrulline and arginine in human urine were increased after administration of a single intravenous erythropoietin injection (2000 U day⁻¹, Epocrine, St-Petersburg, Russia). The study of variations of ADMA, SDMA, arginine and citrulline levels before and after rHuEPO administration was performed with two healthy male volunteers. Urine samples were collected before rHuEPO administration and urinary concentrations of ADMA and SDMA were determined at 10.0–40 μg mL⁻¹ and of arginine and citrulline at 0.5–10 μg mL⁻¹. A single dose injection of rHuEPO caused an increase in ADMA, SDMA, arginine and citrulline concentrations up to 40–270 μg mL⁻¹, 40–240 μg mL⁻¹, 10–60 μg mL⁻¹ and 12–140 μg mL⁻¹, respectively. These preliminary results indicated that an indirect approach could be used as a pre-screening of urine samples in order to decrease the number of samples with a low probability of rHuEPO abuse and, thus, save costs and human workload.