A comprehensive study on the performance of different retention mechanisms in sport drug testing by liquid chromatography tandem mass spectrometry.

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.