Optimizing detection of erythropoietin receptor agonists from dried blood spots for anti-doping application

The World Anti-Doping Agency (WADA) has recently implemented dried blood spots (DBSs) as a matrix for doping control. However, specifications regarding the analysis of the class of prohibited substances called erythropoietin (EPO) receptor agonists (ERAs) from DBSs are not yet described. The aim of this study was to find optimal conditions (sample volume and storage) to sensitively detect endogenous erythropoietin (hEPO) and prohibited ERAs from DBSs and compare detection limits to WADA-stipulated minimum required performance levels (MRPLs) for ERAs in serum/plasma samples. Venous whole blood was spotted onto Whatman 903 DBS cards with primarily 60 mu l of blood, but various volumes from 20 to75 mu l were tested. All samples were immunopurified with MAIIA EPO Purification Gel kit (EPGK) and analysed with sodium N-lauroylsarcosinate polyacrylamide gel electrophoresis (SAR-PAGE) and Western blot. Sixty-microliter DBSs allowed the detection of the four main ERAs (BRP, NESP, CERA and EPO-Fc) at concentrations close to WADA's MRPLs described for 500 mu l of serum/plasma. Different storage temperatures, from -20 degrees C to 37 degrees C, were evaluated and did not affect ERA detection. A comparison of the detection of endogenous EPO from the different anti-doping matrices (urine, serum and DBSs produced from upper arm capillary blood) from five participants for 6 weeks was performed. Endogenous EPO extracted from DBSs showed intra-individual variations in male and female subjects, but less than in urine. Doping controls would benefit from the stability of ERAs on DBSs: It can be a complementary matrix for ERA analysis, particularly in the absence of EPO signals in urine.

Automated summary

This study investigated the use of dried blood spots (DBS) as a matrix for doping control and found optimal conditions for detecting prohibited erythropoietin receptor agonists (ERAs) and endogenous erythropoietin (EPO) from DBS. The results showed that 60 μl DBS could detect the main ERAs at concentrations close to the minimum required performance levels (MRPLs) set by WADA for serum/plasma samples. Storage temperature did not affect ERA detection. Compared with urine, DBS showed less variation in the detection of endogenous EPO.