Reverter-Branchat Gemma, Ventura Rosa, Ezzel Din Mohammed, Mateus Julián, Pedro Carme, Segura Jordi
Integrative Pharmacology and Systems Neuroscience Research Group, Neurosciences Research Program, IMIM - Hospital del Mar Medical Research Institute, Barcelona, Spain.
Catalonian Antidoping Laboratory, Doping Control Research Group, Neurosciences Research Programme, IMIM - Hospital del Mar Medical Research Institute, Barcelona, Spain.
Drug Test Anal. 2018 Oct;10(10):1496-1507. doi: 10.1002/dta.2418. Epub 2018 Jul 5.
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 48 hours and 17 days 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 16 days 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.
使用干血斑（DBS）进行反兴奋剂检测将有助于增加血样数量，因为它无需专业人员，侵入性极小，成本降低，稳定性好，易于运输和储存。在此，世界反兴奋剂机构（WADA）建立的用于检测促红细胞生成素（ESA）的电泳方法已被调整，以评估其在DBS中的适用性。优化并验证了一种在单个DBS中检测重组促红细胞生成素（rEPO）、新型促红细胞生成蛋白（NESP）和持续促红细胞生成素受体激活剂（CERA）的定性方法。对于rEPO和NESP，分别在一项初步研究和一项给药患者研究的手指刺血DBS中进行了确证。对于CERA，在含有已知浓度该蛋白的模拟血制备的DBS中评估了检测能力。关于DBS采样的主要验证参数，如稳定性、血细胞比容影响和血型（毛细血管血与静脉血），显示出微小差异。运输前现场干燥似乎并非必要。日内和日间变异范围为2.9% - 23.5%。线性得以维持（r≥0.9），ESA回收率高（CV≤20.2%）。经过验证的方法分别在给予rEPO和NESP后48小时和17天检测到了受试对象。CERA药代动力学的重现性表明该方法具有良好的可能性，检测窗口在实际给药后可达16天。因此，此处提供的结果强化了DBS血样采集在体育药物检测中分析ESA滥用情况的适用性。
