Görgens Christian, Guddat Sven, Dib Josef, Geyer Hans, Schänzer Wilhelm, Thevis Mario
Institute of Biochemistry - Center for Preventive Doping Research, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany.
European Monitoring Center for Emerging Doping Agents (EuMoCEDA), Cologne/Bonn, Germany.
Drug Test Anal. 2015 Nov-Dec;7(11-12):973-9. doi: 10.1002/dta.1788. Epub 2015 Apr 5.
To date, substances such as Mildronate (Meldonium) are not on the radar of anti-doping laboratories as the compound is not explicitly classified as prohibited. However, the anti-ischemic drug Mildronate demonstrates an increase in endurance performance of athletes, improved rehabilitation after exercise, protection against stress, and enhanced activations of central nervous system (CNS) functions. In the present study, the existing evidence of Mildronate's usage in sport, which is arguably not (exclusively) based on medicinal reasons, is corroborated by unequivocal analytical data allowing the estimation of the prevalence and extent of misuse in professional sports. Such data are vital to support decision-making processes, particularly regarding the ban on drugs in sport. Due to the growing body of evidence (black market products and athlete statements) concerning its misuse in sport, adequate test methods for the reliable identification of Mildronate are required, especially since the substance has been added to the 2015 World Anti-Doping Agency (WADA) monitoring program. In the present study, two approaches were established using an in-house synthesized labelled internal standard (Mildronate-D3 ). One aimed at the implementation of the analyte into routine doping control screening methods to enable its monitoring at the lowest possible additional workload for the laboratory, and another that is appropriate for the peculiar specifics of the analyte, allowing the unequivocal confirmation of findings using hydrophilic interaction liquid chromatography-high resolution/high accuracy mass spectrometry (HILIC-HRMS). Here, according to applicable regulations in sports drug testing, a full qualitative validation was conducted. The assay demonstrated good specificity, robustness (rRT=0.3%), precision (intra-day: 7.0-8.4%; inter-day: 9.9-12.9%), excellent linearity (R>0.99) and an adequate lower limit of detection (<10 ng/mL).
迄今为止，诸如米屈肼（Meldonium）之类的物质尚未被反兴奋剂实验室列入监测范围，因为该化合物未被明确列为违禁物质。然而，抗缺血药物米屈肼显示出可提高运动员的耐力表现、改善运动后的恢复情况、抵御压力以及增强中枢神经系统（CNS）功能的激活。在本研究中，米屈肼在体育领域使用的现有证据（可以说并非完全基于医学原因）得到了明确的分析数据的证实，这些数据有助于估计职业体育中滥用该药物的流行程度和范围。此类数据对于支持决策过程至关重要，尤其是在体育赛事中禁用药物方面。由于越来越多关于其在体育领域被滥用的证据（黑市产品和运动员陈述）出现，因此需要可靠鉴定米屈肼的适当检测方法，特别是因为该物质已被纳入2015年世界反兴奋剂机构（WADA）的监测计划。在本研究中，使用内部合成的标记内标（米屈肼-D3）建立了两种方法。一种旨在将分析物纳入常规兴奋剂检测筛选方法，以便在实验室尽可能低的额外工作量下对其进行监测，另一种方法则适用于该分析物的特殊特性，可使用亲水作用液相色谱-高分辨率/高精度质谱（HILIC-HRMS）明确确认检测结果。在此，根据运动药物检测的适用规定进行了全面的定性验证。该检测方法显示出良好的特异性、稳健性（相对保留时间rRT = 0.3%）、精密度（日内：7.0 - 8.4%；日间：9.9 - 12.9%）、出色的线性（R>0.99）以及足够低的检测限（<10 ng/mL）。
