Department of Analytical Chemistry, Institute of Fine Chemistry and Nanochemistry, Marie Curie Building (Annex), Campus of Rabanales, University of Córdoba, 14071 Córdoba, Spain.
Department of Analytical Chemistry, Institute of Fine Chemistry and Nanochemistry, Marie Curie Building (Annex), Campus of Rabanales, University of Córdoba, 14071 Córdoba, Spain.
J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Jul 15;1178:122821. doi: 10.1016/j.jchromb.2021.122821. Epub 2021 Jun 3.
Anti-doping substances listed by the World Anti-Doping Agency (WADA) include hundreds of compounds of very different physico-chemical properties. Anti-doping control laboratories need to screen all these substances in the so-called Initial Testing Procedures (ITPs) what is very challenging from an analytical point of view. ITPs are mostly based on reversed-phase (RP) liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using C18 columns, which feature poor retention and peak tailing for polar and basic compounds, respectively. While studies on this field dealing with the comparison of stationary phases are focused on certain chemical classes, this research provides a wide multi-target approach. For this purpose, a representative group of 93 anti-doping agents (log P from -2.4 to 9.2) included in ten different classes of prohibited substances was selected. A comprehensive study on the performance of six columns and four eluents on different separation parameters (retention factors, asymmetry factors, co-elutions, total run times) and matrix effects (signal enhancement or suppression) was performed for LC-MS/MS-based ITPs. Columns working in both RP [C18, C8, phenyl hexyl (PH), pentafluorophenyl (PFP) and mixed-mode hydrophilic/RP (HILIC-RP)) and hydrophilic (HILIC)] modes were investigated. Eluents contained methanol or acetonitrile as organic modifiers, with or without the addition of ammonium acetate. The best column-mobile phase binomial for ITPs was PFP using water-methanol (0.1% formic acid) as eluent, while HILIC was the best option for highly polar non-aromatic anti-doping agents, which were poorly addressed by PFP. Excellent good peak shapes and relative acceptable matrix interferences were obtained for HILIC-RP, which was tested for the first time for the analysis of anti-doping agents, although the number of compounds eluting too fast was too high. On the whole, the alkyl phase C18 showed the worst performance and although C8 and PH were better, their performance did not surpass that of PFP. Possible retention mechanisms underlying separation in the different stationary phases were discussed. This research provides valuable information to anti-doping control labs for improving LC-MS/MS-based ITPs and it proposes PFP as a suitable alternative to the already established C18.
世界反兴奋剂机构（WADA）列出的禁用物质包括数百种具有不同理化性质的化合物。反兴奋剂检测实验室需要在所谓的初始检测程序（ITP）中对所有这些物质进行筛选，这从分析角度来看是非常具有挑战性的。ITP 主要基于反相（RP）液相色谱与串联质谱（LC-MS/MS）联用，使用 C18 柱，极性和碱性化合物的保留和峰拖尾分别较差。虽然涉及固定相比较的研究主要集中在某些化学类别上，但本研究提供了一种广泛的多目标方法。为此，选择了代表一组 93 种兴奋剂（log P 从-2.4 到 9.2）的物质，这些物质包含在十种不同类别的禁用物质中。在基于 LC-MS/MS 的 ITP 中，对六种色谱柱和四种洗脱液在不同分离参数（保留因子、不对称因子、共洗脱、总运行时间）和基质效应（信号增强或抑制）方面的性能进行了全面研究。研究了在反相（RP）[C18、C8、苯基己基（PH）、五氟苯基（PFP）和混合模式亲水/RP（HILIC-RP）]和亲水（HILIC）模式下工作的色谱柱。洗脱液包含甲醇或乙腈作为有机改性剂，有或没有添加乙酸铵。对于 ITP，最佳的色谱柱-流动相组合是使用水-甲醇（0.1%甲酸）作为洗脱液的 PFP，而 HILIC 是高度极性非芳香性兴奋剂的最佳选择，PFP 对其的分析效果较差。首次用于兴奋剂分析的 HILIC-RP 获得了极好的峰形和相对可接受的基质干扰，尽管洗脱速度过快的化合物数量过多。总的来说，烷基相 C18 的性能最差，尽管 C8 和 PH 更好，但它们的性能并未超过 PFP。讨论了不同固定相分离的可能保留机制。本研究为反兴奋剂检测实验室提供了有价值的信息，以改进基于 LC-MS/MS 的 ITP，并提出 PFP 是 C18 的一种合适替代品。
