Center for Preventive Doping Research - Institute of Biochemistry, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany.
Drug Test Anal. 2014 Nov-Dec;6(11-12):1197-205. doi: 10.1002/dta.1680. Epub 2014 Jun 9.
Since January 2014, the anti-anginal drug trimetazidine [1-(2,3,4-trimethoxybenzyl)-piperazine] has been classified as prohibited substance by the World Anti-Doping Agency (WADA), necessitating specific and robust detection methods in sports drug testing laboratories. In the present study, the implementation of the intact therapeutic agent into two different initial testing procedures based on gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) is reported, along with the characterization of urinary metabolites by electrospray ionization-high resolution/high accuracy (tandem) mass spectrometry. For GC-MS analyses, urine samples were subjected to liquid-liquid extraction sample preparation, while LC-MS/MS analyses were conducted by established 'dilute-and-inject' approaches. Both screening methods were validated for trimetazidine concerning specificity, limits of detection (0.5-50 ng/mL), intra-day and inter-day imprecision (<20%), and recovery (41%) in case of the GC-MS-based method. In addition, major metabolites such as the desmethylated trimetazidine and the corresponding sulfoconjugate, oxo-trimetazidine, and trimetazidine-N-oxide as identified in doping control samples were used to complement the LC-MS/MS-based assay, although intact trimetazidine was found at highest abundance of the relevant trimetazidine-related analytes in all tested sports drug testing samples. Retrospective data mining regarding doping control analyses conducted between 1999 and 2013 at the Cologne Doping Control Laboratory concerning trimetazidine revealed a considerable prevalence of the drug particularly in endurance and strength sports accounting for up to 39 findings per year.
自 2014 年 1 月以来，抗心绞痛药物曲美他嗪[1-(2,3,4-三甲氧基苄基)-哌嗪]已被世界反兴奋剂机构（WADA）列为禁用物质，因此需要在运动药物检测实验室中使用特定且强大的检测方法。在本研究中，报告了将完整的治疗剂纳入两种不同初始测试程序的实施情况，这两种程序基于气相色谱-质谱（GC-MS）和液相色谱-串联质谱（LC-MS/MS），并通过电喷雾电离-高分辨率/高精度（串联）质谱对尿液代谢物进行了表征。对于 GC-MS 分析，尿液样品经过液液萃取样品制备，而 LC-MS/MS 分析则通过已建立的“稀释和注入”方法进行。两种筛选方法均针对曲美他嗪进行了特异性、检测限（0.5-50ng/mL）、日内和日间精密度（<20%）以及 GC-MS 方法的回收率（41%）验证。此外，在兴奋剂控制样品中鉴定出的主要代谢物，如去甲基曲美他嗪和相应的磺酸盐、氧代曲美他嗪和曲美他嗪-N-氧化物，用于补充基于 LC-MS/MS 的测定方法，尽管在所有测试的运动药物检测样品中，完整的曲美他嗪是相关曲美他嗪相关分析物中丰度最高的。关于科隆兴奋剂检测实验室在 1999 年至 2013 年期间进行的曲美他嗪兴奋剂检测分析的回溯性数据挖掘显示，该药物的流行率相当高，尤其是在耐力和力量运动中，每年的检出率高达 39 次。
