Detection of erythropoiesis-stimulating agents in a single dried blood spot.

Abstract

The use of dried blood spots (DBS) for anti-doping purposes would facilitate an increase in the number of blood samples because it eliminates the need for specialized personnel and involves minimal invasiveness, reduced costs, stability, and easy transportation and storage. Here, the electrophoretic methodology established by the World Anti-Doping Agency (WADA) to detect erythropoiesis-stimulating agents (ESAs) has been adapted to evaluate their applicability to DBS. A qualitative procedure to detect recombinant erythropoietin (rEPO), novel erythropoiesis-stimulating protein (NESP), and continuous erythropoietin receptor activator (CERA) in a single DBS was optimized and validated. For rEPO and NESP, confirmation was performed in finger-prick DBS from a pilot study and an administration patients study, respectively. For CERA, detection capabilities were evaluated in DBS prepared with modeled-blood spiked with known concentrations of the protein. Main validation parameters concerning DBS sampling such as stability, hematocrit influence, and blood type (capillary vs. venous) described minor variations. Onsite drying appeared not to be essential before transport. Intra- and inter-day variation range was 2.9%-23.5%. Linearity was maintained (r≥0.9) and ESAs were robustly recovered (CV≤20.2%). The validated method permitted the detection of treated subjects after 48hours and 17days of rEPO and NESP administration, respectively. The reproduction of a CERA pharmacokinetics showed good possibilities for the method with a detection window that could reach 16days after its actual administration. Thus, results provided here reinforce the suitability of DBS blood sampling for the analysis of ESA misuse in sports drug testing.