Brazilian Doping Control Laboratory 'LBCD-LADETEC', Av. Horácio Macedo 1281 Cidade Universitária, 21941-598, Rio de Janeiro, Brazil.
Brazilian Doping Control Laboratory 'LBCD-LADETEC', Av. Horácio Macedo 1281 Cidade Universitária, 21941-598, Rio de Janeiro, Brazil.
J Pharm Biomed Anal. 2021 Feb 20;195:113870. doi: 10.1016/j.jpba.2020.113870. Epub 2020 Dec 30.
Considering the huge amount of substances associated with athletic performance improvement, current doping control analysis requires a comprehensive screening method, which leads to the detection of prohibited substances of different physico-chemical properties. This comprehensiveness associated with instrumental approaches based on high resolution mass spectrometry has allowed the development of extremely sensitive and selective detection methods. Furthermore, it is desirable the method to be simple, fast and straightforward. Mildronate is a highly polar quaternary amine, classified as metabolic modulator by the World Anti-Doping Agency (WADA). The inclusion of mildronate in the screening strategy is a challenge considering its singular physicochemical properties, compared to numerous doping agents of low and medium polarity. For this purpose, a method combining solid-phase extraction (SPE) and dilute-and-shoot approach has been developed and validated, allowing the detection of mildronate and other 332 prohibited substances. In the sample preparation protocol, the enzymatic deconjugation step and SPE conditions were stressed to enable the recovery of mildronate without jeopardizing the detection of other doping agents. The C/18% SPE cartridge without any type of ionic interaction, associated with the dilute-and-shoot approach proved to be effective for all monitored substances. The instrumental method employed was based on liquid chromatography using a reversed-phase column in a 12-minute gradient coupled to a high-resolution mass spectrometry in full scan with positive and negative switching and fragmentation in the positive mode, for the most critical detection compounds. The performance of the method was evaluated regarding selectivity, precision, recovery, carry-over, limit of detection and stability, following the recommendations of WADA.
考虑到与运动表现提升相关的物质数量庞大，当前的兴奋剂控制分析需要一种全面的筛选方法，这导致了对不同物理化学性质的禁用物质的检测。这种全面性与基于高分辨率质谱的仪器方法相结合，使得开发出了极其灵敏和选择性的检测方法成为可能。此外，该方法还应简单、快速且直接。米屈肼是一种高度极性的季铵盐，被世界反兴奋剂机构（WADA）归类为代谢调节剂。考虑到其独特的物理化学性质，与低极性和中极性的众多兴奋剂相比，将米屈肼纳入筛选策略是一项挑战。为此，开发并验证了一种结合固相萃取（SPE）和稀释-进样方法，该方法允许检测米屈肼和其他 332 种禁用物质。在样品制备方案中，强调了酶去结合步骤和 SPE 条件，以在不影响其他兴奋剂检测的情况下实现米屈肼的回收。未进行任何类型离子相互作用的 C/18% SPE 柱与稀释-进样方法相结合，被证明对所有监测物质都有效。所采用的仪器方法基于使用反相柱在 12 分钟梯度中的液相色谱法，并结合具有正、负切换和在正模式下碎裂的全扫描高分辨率质谱法，用于最关键的检测化合物。该方法的性能是根据 WADA 的建议，通过选择性、精密度、回收率、携带污染、检测限和稳定性进行评估的。
