Abstract
In the second part of this study, a systematic comparison was made between two ion fragmentation acquisition modes, namely data-independent acquisition (DIA) and DIA with ion mobility spectrometry (IMS) technology. These two approaches were applied to the analysis of 192 doping agents in urine. Group I included 102 compounds such as stimulants, diuretics, narcotics, and β2-agonists, while Group II contained 90 compounds included steroids, glucocorticoids, and hormone and metabolic modulators. Important method parameters were examined and compared, including the fragmentation, sensitivity, and assignment capability with the minimum occurrence of false positive hits.The results differed between Group I and II in number of detected fragments when exploring the MS/MS spectra. In Group I only 13%, while in the Group II 64% of the substances had a higher number of fragments in DIA-IMS mode vs. DIA. In terms of sensitivity, the performance of the two modes with and without activated IMS dimension was identical for about 50% of the doping agents. The sensitivity was higher without IMS, i.e. in simple DIA mode, for 20–40% of remaining doping agents. Despite this sensitivity reduction with IMS, 82% of compounds from both Groups met the minimum required performance level (MRPL) criteria of the World Anti-Doping Agency (WADA) when the DIA-IMS mode was applied.Automated data processing is important in routine doping analysis. Therefore, processing methods were optimized and evaluated for the prevalence of false peak assignments by analysing the target substances at different concentrations in urine samples. Overall, a significantly higher number of misidentified compounds was observed in Group II, with an almost 2-fold higher number of misidentifications in DIA compared to DIA-IMS. This result highlights the benefit of the IMS dimension to reduce the rate of false positive in screening analysis. The optimized UHPLC-IM-HRMS method was finally applied to the analysis of urine samples from administration studies including nine doping agents from both Groups. However, to limit the number of interferences from the biological matrix, an emphasis is needed on the adequate settings of the data processing method.
Highlights
The list of substances prohibited by the World Anti-Doping Agency (WADA) is updated yearly, rendering anti-doping analysis challenging in different aspects [1,2]
The use of ultra-high performance liquid chromatography (UHPLC) hyphenated to ion mobility-high resolution mass spectrometry (IM-high-resolution mass spectrometer (HRMS)) is a attractive alternative for ITP that could meet most of the current challenges in anti-doping analysis
In the first article of this series [3], a large database including retention times and traveling-wave collision cross section acquired on N2 (TWCCSN2) values for almost 200 target compounds across the various classes of substances on the prohibited list of WADA, including stimulants, narcotics, cannabinoids, diuretics, b2-agonists, b-blockers, anabolic agents, and hormone and metabolic modulators, was presented
Summary
The list of substances prohibited by the World Anti-Doping Agency (WADA) is updated yearly, rendering anti-doping analysis challenging in different aspects [1,2]. The use of ultra-high performance liquid chromatography (UHPLC) hyphenated to ion mobility-high resolution mass spectrometry (IM-HRMS) is a attractive alternative for ITP that could meet most of the current challenges in anti-doping analysis. In the first article of this series [3], a large database including retention times and traveling-wave collision cross section acquired on N2 (TWCCSN2) values for almost 200 target compounds across the various classes of substances on the prohibited list of WADA, including stimulants, narcotics, cannabinoids, diuretics, b2-agonists, b-blockers, anabolic agents, and hormone and metabolic modulators, was presented. The UHPLC-IM-HRMS method has demonstrated good performance and excellent stability in terms of intraday, interday, and interweek variability of retention times and TWCCSN2 values for all compounds at different concentrations in mixtures of standard solutions and in human urine samples. IM technology was demonstrated to be a very promising tool to filter the data and remove interferences for result interpretation from MS spectra [3]
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