Analytical Chemistry Research Group, Department of Physical and Analytical Chemistry, Faculty of Experimental Sciences, University of Jaén, Paraje Las Lagunillas, E-23071 Jaén, Spain.
Analytical Chemistry Research Group, Department of Physical and Analytical Chemistry, Faculty of Experimental Sciences, University of Jaén, Paraje Las Lagunillas, E-23071 Jaén, Spain.
Talanta. 2015 Mar;134:74-88. doi: 10.1016/j.talanta.2014.10.050. Epub 2014 Oct 31.
In this article, a screening method for the determination of 200 sport drugs in human urine has been developed using liquid-chromatography electrospray time-of-flight mass spectrometry (LC-TOFMS). The chromatographic separation of the targeted doping agents was carried out by fast liquid chromatography using a C18 column (4.6×50 mm) with 1.8 μm particle size. Accurate mass measurements of the selected ion (typically M+H and M-H) along with retention time matching was used for the screening and detection of the targeted species. The proposed methodology comprised also a simple sample treatment stage based on solid-phase extraction (SPE) with polymeric cartridges. The SPE method displayed satisfactory recoveries rates (between 70 and 120%) for the majority of the compounds at both concentration levels tested (2.5 and 25 μg L(-1)). The overall performance of the method was satisfactory with all 200 compounds fulfilling WADA minimum required performance levels (MRPLs), with limits of quantitation lower than 1 μg L(-1) for 80% of the compounds, and showing an appropriate linearity (r(2)>0.99) in most cases. Additionally, the ability of "in-source" collision induced dissociation (CID) for confirmatory purposes was examined using as criterion the presence of two high-resolution ions with relevant abundances for unambiguous confirmation. This stringent criterion was fulfilled for 75% of the species using in-source CID fragmentation. The use of an improved approach based on CID performed on a dedicated collision cell without precursor ion selection (using a Q-TOF) provided at least two ions in all cases with the exception of 2-aminoheptane. Finally, based on the use of diagnostic fragment ions, a workflow for the comprehensive screening and identification of non-targeted compounds (viz. compounds with no primary standards or retention time information available, such as metabolites) has been also examined using rat urine samples. The proposed screening method has proved to be effective for the analysis of targeted compounds, and also for the identification of metabolites, expanding easily the search for doping agents not only limited to specific banned parent compounds but also to derivate compounds with similar structure as well as metabolites.
本文开发了一种使用液相色谱-电喷雾飞行时间质谱（LC-TOFMS）测定人尿中 200 种运动药物的筛选方法。采用快速液相色谱法，使用粒径为 1.8μm 的 C18 柱（4.6×50mm）对靶向兴奋剂进行色谱分离。选择具有代表性的离子（通常为[M+H]+和[M-H]-）进行精确质量测量，并结合保留时间匹配，用于靶向物质的筛选和检测。该方法还包括基于聚合物小柱的固相萃取（SPE）的简单样品处理阶段。SPE 方法在两种测试浓度（2.5 和 25μg/L）下，对大多数化合物均显示出令人满意的回收率（70%至 120%之间）。该方法的整体性能令人满意，所有 200 种化合物均满足世界反兴奋剂机构（WADA）的最低性能要求（MRPL），80%的化合物的定量限低于 1μg/L，且大多数情况下表现出适当的线性（r2>0.99）。此外，还使用“源内”碰撞诱导解离（CID）来检查用于确证目的的能力，其标准是存在两个具有相关丰度的高分辨率离子，以进行明确确证。使用源内 CID 碎片时，75%的化合物符合这一严格标准。使用不进行前体离子选择（使用 Q-TOF）的专用碰撞池进行的改进 CID 方法在所有情况下都提供了至少两个离子，除了 2-氨基庚烷。最后，基于诊断碎片离子的使用，还使用大鼠尿液样本检查了一种用于非靶向化合物（即没有主要标准品或保留时间信息的化合物，如代谢物）综合筛选和鉴定的工作流程。所提出的筛选方法已被证明对靶向化合物的分析有效，也对代谢物的鉴定有效，不仅可以轻松扩展对禁用母体化合物的搜索，还可以扩展到结构相似的衍生化合物以及代谢物的搜索。
