You Youwen, Proctor Rachel M, Guo Kevin, Li Xiaoqing, Xue Evan, Guan Fuyu, Robinson Mary A
Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, New Bolton Center Campus, Kennett Square, PA 19348, USA.
Anal Methods. 2021 Apr 7;13(13):1565-1575. doi: 10.1039/d0ay02297g. Epub 2021 Mar 12.
High-resolution mass spectrometry (HRMS) is a very powerful technology for equine doping control analysis. The more recently developed hybrid type of Orbitrap-based HRMS instrument allows for both targeted and non-targeted screening analyses in a single liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) run. In the present study, an LC-HRMS/MS method was developed and validated to detect prohibited substances in equine sports. The substances were recovered from equine plasma by liquid-liquid extraction (LLE) using methyl tert-butyl ether and were separated on a C18 reversed-phase column using mobile phases of 5 mM ammonium formate and acetonitrile. A 7.5 min LC gradient was employed to elute substances and results indicated that the LC method generated sharp and symmetric chromatographic peaks. An in-house equine doping compound database and a spectral library were built to increase method specificity for substances of interest. Five criteria, i.e. accurate mass, retention time, isotope pattern, selected HRMS/MS fragment ions (compound database) and HRMS/MS spectra (spectral library), were employed for targeted screening. We utilized these criteria to validate targeted detection of 451 substances within our in-house equine doping compound database. By using all five criteria in screening, the false screening positive rate is significantly reduced. A screening strategy and a Microsoft Excel macro were developed to facilitate interpretation and reporting of results. As the simultaneous acquisition of the full scan HRMS data provides the opportunity for retrospective non-targeted analysis, our findings highlight the use of this novel methodology as a simple, rapid, and reliably reproducible strategy to meet the challenge of identifying an increasing number of doping substances that could potentially impact the integrity of the horse racing community.
高分辨率质谱（HRMS）是用于马类兴奋剂检测分析的一项非常强大的技术。最近开发的基于轨道阱的混合型HRMS仪器，能够在一次液相色谱 - 高分辨率串联质谱（LC - HRMS/MS）运行中同时进行靶向和非靶向筛查分析。在本研究中，开发并验证了一种LC - HRMS/MS方法，用于检测马类运动中的违禁物质。使用甲基叔丁基醚通过液 - 液萃取（LLE）从马血浆中回收这些物质，并在C18反相柱上使用5 mM甲酸铵和乙腈的流动相进行分离。采用7.5分钟的LC梯度洗脱物质，结果表明该LC方法产生了尖锐且对称的色谱峰。建立了一个内部马类兴奋剂化合物数据库和一个光谱库，以提高对感兴趣物质的方法特异性。采用准确质量、保留时间、同位素模式、选定的HRMS/MS碎片离子（化合物数据库）和HRMS/MS光谱（光谱库）这五个标准进行靶向筛查。我们利用这些标准验证了内部马类兴奋剂化合物数据库中451种物质的靶向检测。通过在筛查中使用所有五个标准，误筛阳性率显著降低。制定了一种筛查策略和一个Microsoft Excel宏，以方便结果的解释和报告。由于同时采集全扫描HRMS数据为回顾性非靶向分析提供了机会，我们的研究结果突出了这种新方法作为一种简单、快速且可靠可重复的策略的用途，以应对识别越来越多可能影响赛马界公正性的兴奋剂物质的挑战。
