Doping Control Laboratory of Athens, Olympic Athletic Centre of Athens "Spyros Louis" (OAKA), Kifissias 37, 15123 Maroussi, Greece; Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, 15771 Panepistimiopolis-Zographou, Athens, Greece.
J Chromatogr B Analyt Technol Biomed Life Sci. 2013 Dec 15;941:69-80. doi: 10.1016/j.jchromb.2013.10.008. Epub 2013 Oct 16.
In the present study a general screening protocol was developed to detect prohibited substances and metabolites for doping control purposes in equine sports. It was based on the establishment of a unified sample preparation and on the combined implementation of liquid and gas chromatographic MS analysis. The sample pretreatment began with two parallel procedures: enzymatic hydrolysis of sulfate and glucuronide conjugates, and methanolysis of the 17β-sulfate steroid conjugates. The extracts were treated for LC-TOF-MS, GC-HRMS and GC-MS assays. The majority of the prohibited substances were identified through a high mass accuracy technique, such as LC-TOF-MS, without prior derivatization. The sample preparation procedure included the formation of methylated and trimethylsilylated derivatives common in toxicological GC-MS libraries. The screening method was enhanced by post-run library searching using automated mass spectral deconvolution and identification system (AMDIS) combined with deconvolution reporting software (DRS). The current methodology is able to detect the presence of more than 350 target analytes in horse urine and may easily incorporate a lot of new substances without changes in chromatography. The full scan acquisition allows retrospective identification of prohibited substances in stored urine samples after reprocessing of the acquired data. Validation was performed for sixty representative compounds and included limit of detection, matrix interference - specificity, extraction recovery, precision, mass accuracy, matrix effect and carry over contamination. The suitability of the method was demonstrated with previously declared positive horse urine samples.
在本研究中，开发了一种通用筛选方案，用于检测赛马运动中的禁用物质和代谢物，以进行兴奋剂控制。它基于建立统一的样品制备方法，并结合液相色谱和气相色谱-质谱联用分析。样品预处理从两个平行程序开始：硫酸盐和葡萄糖醛酸缀合物的酶水解，以及 17β-硫酸酯类固醇缀合物的甲醇解。提取物经过 LC-TOF-MS、GC-HRMS 和 GC-MS 分析。大多数禁用物质通过高质量精度技术（如 LC-TOF-MS）进行鉴定，无需预先衍生化。样品制备程序包括形成毒理学 GC-MS 文库中常见的甲基化和三甲基硅烷基化衍生物。通过使用自动质谱解卷积和识别系统（AMDIS）与解卷积报告软件（DRS）结合进行后运行库搜索，增强了筛选方法。该方法能够检测马尿中超过 350 种目标分析物的存在，并且可以轻松地在不改变色谱法的情况下纳入许多新物质。全扫描采集允许在重新处理获取的数据后对存储的尿液样本进行禁止物质的回溯性鉴定。对 60 种代表性化合物进行了验证，包括检测限、基质干扰-特异性、提取回收率、精密度、质量精度、基质效应和残留污染。该方法的适用性通过先前宣布的阳性马尿样本得到了证明。
