Simplifying and expanding analytical capabilities for various classes of doping agents by means of direct urine injection high performance liquid chromatography high resolution/high accuracy mass spectrometry

So far, in sports drug testing compounds of different classes are processed and measured using different screening procedures. The constantly increasing number of samples in doping analysis, as well as the large number of substances with doping related, pharmacological effects require the development of even more powerful assays than those already employed in sports drug testing, indispensably with reduced sample preparation procedures. The analysis of native urine samples after direct injection provides a promising analytical approach, which thereby possesses a broad applicability to many different compounds and their metabolites, without a time-consuming sample preparation. In this study, a novel multi-target approach based on liquid chromatography and high resolution/high accuracy mass spectrometry is presented to screen for more than 200 analytes of various classes of doping agents far below the required detection limits in sports drug testing. Here, classic groups of drugs as diuretics, stimulants, β₂-agonists, narcotics and anabolic androgenic Steroids as well as various newer target compounds like hypoxia-inducible factor (HIF) stabilizers, selective Androgen Receptor modulators (SARMs), selective Estrogen Receptor modulators (SERMs), plasma volume expanders and other doping related compounds, listed in the 2016 WADA prohibited list were implemented. As a main achievement, growth hormone releasing Peptides could be implemented, which chemically belong to the group of small Peptides (<2kDa) and are commonly determined by laborious and time-consuming stand-alone assays. The assay was fully validated for qualitative purposes considering the parameters specificity, robustness (rRT: <2%), intra- (CV: 1.7-18.4 %) and inter-day precision (CV: 2.3-18.3%) at three concentration levels, linearity (R²>0.99), limit of detection (0.1-25ng/mL; 3'OH-stanozolol glucuronide: 50pg/mL; dextran/HES: 10μg/mL) and matrix effects.