Mazzarino Monica, Pizzolato Francesca, Honesová Lenka, Tsivou Maria, Gmeiner Günter, Van Eenoo Peter
Doping Control Laboratory, Department of Diagnostic Sciences, Ghent University, Block B, Ottergemsesteenweg 460, BE-9000, Ghent, Belgium.
Doping Control Laboratory, Seibersdorf Labor GmbH, Campus Seibersdorf, 2444, Seibersdorf, Austria.
Anal Bioanal Chem. 2025 Feb;417(4):799-820. doi: 10.1007/s00216-024-05697-9. Epub 2025 Jan 3.
Dried urine spots have recently been proposed as an alternative matrix in the anti-doping field. Drying urine may open the opportunity to limit microbial and thermal degradation of the prohibited substances during transportation to the anti-doping laboratories without the need for refrigeration or freezing. In this study, a multi-targeted initial testing procedure was developed for the determination of 237 prohibited drugs/metabolites from 11 different classes in dried urine spots. The comparability between two different microsampling techniques (i.e., Whatman FTA DMPK-C cards and Mitra tips) was evaluated. The developed method was then used to evaluate the stability of the target compounds in urine for 7 days under different environmental conditions to simulate the transportation of the urine samples from the collection sites to anti-doping laboratories. Sample preparation consists of (i) extraction of the analytes from the collection device using a mixture of acetonitrile/methanol (1/1) for 30 min at 40 °C, (ii) enzymatic hydrolysis, and (iii) sample concentration by solid-phase extraction. Analysis was performed using liquid chromatography coupled to high-resolution mass spectrometry. The entire workflow was validated in terms of specificity (analytes were distinguishable from the matrix interferences), sensitivity (only with the Mitra tips the limits of detection comply with the World Anti-Doping Agency's requirements for the majority of the target compounds), carry-over (no signals in the negative urine injected after the positive urine), matrix effect (16-28% for Mitra tips and 22-35% for DMPK-C cards), and extraction yield (Mitra tips: 51-88%; DMPK-C cards: 40-76%). As proof of concept, authentic urine samples were analyzed: results obtained in dried urine were compared with those of fluid urine, providing good agreement. Stability studies showed that the target compounds were stable for the whole duration of the study (7 days) at -20 and 4 °C in both fluid and dried urine. At 50 °C or at 20-25 °C, several thiazide-based compounds were completely degraded to their degradation product in the first 24 h or after 3-4 days in fluid urine, whereas in dried urine the compounds were detectable for the entire duration of the study.
干尿斑最近被提议作为反兴奋剂领域的一种替代基质。尿液干燥可能提供机会，在无需冷藏或冷冻的情况下，限制违禁物质在运往反兴奋剂实验室途中的微生物和热降解。在本研究中，开发了一种多目标初始检测程序，用于测定干尿斑中11个不同类别中的237种违禁药物/代谢物。评估了两种不同微量采样技术（即Whatman FTA DMPK-C卡和Mitra吸头）之间的可比性。然后使用所开发的方法评估目标化合物在不同环境条件下在尿液中7天的稳定性，以模拟尿液样本从采集地点运往反兴奋剂实验室的过程。样品制备包括：(i) 使用乙腈/甲醇（1/1）混合物在40℃下从采集装置中提取分析物30分钟，(ii) 酶促水解，以及(iii) 通过固相萃取进行样品浓缩。使用液相色谱与高分辨率质谱联用进行分析。整个工作流程在特异性（分析物可与基质干扰区分开）、灵敏度（仅Mitra吸头的检测限符合世界反兴奋剂机构对大多数目标化合物的要求）、残留（阳性尿液后注入的阴性尿液中无信号）、基质效应（Mitra吸头为16 - 28%，DMPK-C卡为22 - 35%）和提取产率（Mitra吸头：51 - 88%；DMPK-C卡：40 - 76%）方面得到了验证。作为概念验证，对真实尿液样本进行了分析：将干尿斑中获得的结果与液态尿液的结果进行比较，结果吻合良好。稳定性研究表明，目标化合物在 - 20℃和4℃下，在液态和干尿斑中整个研究期间（7天）均保持稳定。在50℃或20 - 25℃下，几种噻嗪类化合物在液态尿液中在前24小时或3 - 4天后完全降解为其降解产物，而在干尿斑中整个研究期间均可检测到这些化合物。
