Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T., Hong Kong, China.
Anal Bioanal Chem. 2010 Apr;396(7):2513-21. doi: 10.1007/s00216-010-3455-8. Epub 2010 Feb 11.
Recombinant human erythropoietin (rhEPO), darbepoetin alfa (DPO) and methoxy polyethylene glycol-epoetin beta (PEG-EPO) are synthetic analogues of the endogenous hormone erythropoietin (EPO). These erythropoiesis-stimulating agents have the ability to stimulate the production of red blood cells and are commercially available for the treatment of anaemia in humans. These drugs are understood to have performance-enhancing effects on human athletes due to their stimulation of red blood cell production, thereby improving delivery of oxygen to the muscle tissues. Although their effect on horses has not been proven, these substances were thought to be similarly performance enhancing and have indeed been applied covertly to horses. As such, these protein-based drugs are prohibited by authorities in both human and equine sports. The method officially adopted by the International Olympic Committee (IOC) and World Anti Doping Agency (WADA) for the confirmation of rhEPO and/or DPO (rhEPO/DPO) in human urine is based on electrophoresis in combination with Western blotting. A shortcoming of the WADA method is the lack of definitive mass spectral data for the confirmation of a positive finding. Recently, a liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for the detection and confirmation of rhEPO/DPO in equine plasma was reported. However, we have not been successful in achieving the reported sensitivity. This paper presents a method for the detection and confirmation of rhEPO/DPO, as well as the newly released PEG-EPO, in equine plasma. The procedures involve immunoaffinity extraction using anti-rhEPO antibody-coated Dynabeads followed by trypsin digestion. The injected extract was further purified and concentrated using an on-line trap column in the nano-LC system. Detection and confirmation were achieved by monitoring a unique peptide segment of rhEPO/DPO/PEG-EPO using nano-liquid chromatography-tandem mass spectrometry equipped with a nanospray ionisation source operated in the selected reaction monitoring mode. rhEPO, DPO and PEG-EPO can be confirmed at 0.1, 0.2 and 1.0 ng/mL, respectively, in equine plasma.
重组人促红细胞生成素（rhEPO）、达贝泊汀α（DPO）和甲氧基聚乙二醇-促红细胞生成素β（PEG-EPO）是内源性激素促红细胞生成素（EPO）的合成类似物。这些红细胞生成刺激剂能够刺激红细胞的生成，并且可用于治疗人类贫血。由于它们刺激红细胞的生成，这些药物被认为对人类运动员具有增强表现的作用，从而改善肌肉组织的氧气输送。尽管它们对马的影响尚未得到证实，但这些物质被认为具有类似的增强表现作用，并且确实被秘密地应用于马匹。因此，这些基于蛋白质的药物在人类和马类运动中都被当局禁止。国际奥委会（IOC）和世界反兴奋剂机构（WADA）正式采用的用于确认人类尿液中的 rhEPO 和/或 DPO（rhEPO/DPO）的方法是基于电泳结合 Western 印迹。WADA 方法的一个缺点是缺乏用于确认阳性结果的明确质谱数据。最近，报道了一种用于检测和确认马类血浆中 rhEPO/DPO 的液相色谱-串联质谱（LC/MS/MS）方法。然而，我们未能达到报道的灵敏度。本文介绍了一种用于检测和确认马类血浆中的 rhEPO/DPO 以及新发布的 PEG-EPO 的方法。该方法涉及使用抗 rhEPO 抗体包被的 Dynabeads 进行免疫亲和提取，然后进行胰蛋白酶消化。通过使用在线阱柱在纳升 LC 系统中进一步对注入的提取物进行纯化和浓缩。通过使用配备纳升喷雾离子源的纳升液相色谱-串联质谱在选择反应监测模式下监测 rhEPO/DPO/PEG-EPO 的独特肽段片段来实现检测和确认。rhEPO、DPO 和 PEG-EPO 可分别在马类血浆中以 0.1、0.2 和 1.0 ng/mL 的浓度确认。
