Abstract
In this series of two papers, 192 doping agents belonging to the classes of stimulants, narcotics, cannabinoids, diuretics, β2-agonists, β-blockers, anabolic agents, and hormone and metabolic modulators were investigated, with the aim to assess the benefits and limitations of ion mobility spectrometry (IMS) in combination with ultra-high performance liquid chromatography (UHPLC) and high resolution mass spectrometry (HRMS) in anti-doping analysis.In this first part, a generic UHPLC-IM-HRMS method was successfully developed to analyze these 192 doping agents in standard solutions and urine samples, and an exhaustive database including retention times, TWCCSN2 values, and m/z ratios was constructed. Urine samples were analyzed using either a simple “dilute and shoot” procedure or a supported liquid-liquid extraction (SLE) procedure, depending on the physicochemical properties of the compounds and sensitivity criteria established by the World Anti-Doping Agency (WADA) as the minimum required performance levels (MRPL). Then, the precision of the generic UHPLC-IM-HRMS method was assessed as intraday, interday as well as interweek variation of UHPLC retention times and TWCCSN2 values, for which RSD the values were always lower than 2% in urine samples. The possibility to filter MS data using IMS dimension was also investigated, and in average, the application of IMS filtration provided low energy MS spectra with 86% less interfering peaks in both standard and urine samples. Therefore, the filtered MS spectra allowed for an easier interpretation and a lower risk of false positive result interpretations. Finally, IMS also offers additional selectivity to the UHPLC-HRMS enabling to separate isobaric and isomeric substances. Among the selected set of 192 doping agents, there were 30 pairs of isobaric or isomeric compounds, and only two pairs could not be resolved under the developed conditions. This illustrates the potential of adding ion mobility to UHPLC-HRMS in anti-doping analyses.
Highlights
Anti-doping analysis is challenging domain of analyses in many aspects
In this series of two papers, 192 doping agents belonging to the classes of stimulants, narcotics, cannabinoids, diuretics, b2-agonists, b-blockers, anabolic agents, and hormone and metabolic modulators were investigated, with the aim to assess the benefits and limitations of ion mobility spectrometry (IMS) in combination with ultra-high performance liquid chromatography (UHPLC) and high resolution mass spectrometry (HRMS) in anti-doping analysis
(80e120%) were obtained for almost all compounds, with only two critical compounds, zilpaterol showing poor recovery and bolasterone metabolite being significantly affected by matrix effect, and another three compounds with recoveries ranging between 20 and 40%
Summary
Anti-doping analysis is challenging domain of analyses in many aspects. Each year, an updated version of the WADA Prohibited List [1] is released, with the inclusion of new substances and/or classes [2]. In recent years, doping laboratories have invested considerable resources to improve their methods, not just to improve the selectivity and to increase sample throughput, and to include the new target substances in compliance with the Prohibited List [3]. Good performance of the ITPs increases productivity, by limiting the number of time-consuming confirmatory analyses to a necessary minimum. For these reasons, method selectivity is a key aspect, to provide the certainty of identification for confirmatory procedures, and to improve the performance of the screening procedures, reducing time and costs of the overall analytical process [4,5]
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