The number of substances nominally listed in the prohibited list of the World Anti-Doping Agency increases each year. Moreover, many of these substances do not have a single analytical target and must be monitored through different metabolites, artifacts, degradation products, or biomarkers. A new analytical method was developed and validated for the simultaneous analysis of peptides and organic molecules using a single sample preparation and LC-Q-HRMS detection. The simultaneous analysis of 450 target molecules was performed after cleanup on a mixed-mode solid-phase extraction cartridge, combined with untreated urine. The cleanup solvent and reconstitution solvent were the most important parameters for achieving a comprehensive sample preparation approach. A fast chromatographic run based on a multistep gradient was optimized under different flows; the detection of all substances without isomeric coelution was achieved in 11 minutes, and the chromatographic resolution was considered a critical parameter, even in high-resolution mass spectrometry detection. The mass spectrometer was set to operate by switching between positive and negative ionization mode for FULL-MS, all-ion fragmentation, and FULL-MS/MS . The suitable parameters for the curved linear trap (c-trap) conditions were determined and found to be the most important factors for the development of the method. Only FULL-MS/MS enables the detection of steroids and peptides at concentrations lower than the minimum required performance levels set by World Anti-Doping Agency (1 ng mL ). The combination of the maximum injection time of the ions into the c-trap, multiplexing experiments, and loop count under optimized conditions enabled the method to be applied to over 10 000 samples in only 2 months during the 2016 Rio Summer Olympic and Paralympic Games. The procedure details all aspects, from sample preparation to mass spectrometry detection. FULL-MS data acquisition is performed in positive and negative ion mode simultaneously and can be applied to untargeted approaches.

This paper summarises the results obtained from the doping control analyses performed during the Summer XXXI Olympic Games (August 3-21, 2016) and the XV Paralympic Games (September 7-18, 2016). The analyses of all doping control samples were performed at the Brazilian Doping Control Laboratory (LBCD), a World Anti-Doping Agency (WADA)-accredited laboratory located in Rio de Janeiro, Brazil. A new facility at Rio de Janeiro Federal University (UFRJ) was built and fully operated by over 700 professionals, including Brazilian and international scientists, administrative staff, and volunteers. For the Olympic Games, 4913 samples were analysed. In 29 specimens, the presence of a prohibited substance was confirmed, resulting in adverse analytical findings (AAFs). For the Paralympic Games, 1687 samples were analysed, 12 of which were reported as AAFs. For both events, 82.8% of the samples were urine, and 17.2% were blood samples. In total, more than 31 000 analytical procedures were conducted. New WADA technical documents were fully implemented; consequently, state-of-the-art analytical toxicology instrumentation and strategies were applied during the Games, including different types of mass spectrometry (MS) analysers, peptide, and protein detection strategies, endogenous steroid profile measurements, and blood analysis. This enormous investment yielded one of the largest Olympic legacies in Brazil and South America.

Zebrafish (Danio rerio) water tank (ZWT) approach was investigated as an alternative model for metabolism studies based on six different experiments with four model compounds. Sibutramine was applied for the multivariate optimization of ZWT conditions, also for the comparison of the metabolism among ZWT, humans and mice, beyond for the role of CYP2B6 in ZWT. After the optimization, 18 fish and 168 hours of experiments is the minimum requirement for a relevant panel of biotransformation products. A comparison among the species resulted in the observation of the same hydroxylated metabolites, with differences in metabolites concentration ratio. However, the ZWT allowed tuning of the conditions to obtain a specific metabolic profile, depending on the need. In addition, by utilizing CYP2B6 inhibition, a relevant ZWT pathway for the demethylation of drugs was determined. The stereospecificity of the ZWT metabolism was investigated using selegiline and no racemization or inversion transformations were observed. Moreover, the investigation of metabolism of cannabimimetics was performed using JWH-073 and the metabolites observed are the same described for humans, except for the hydroxylation at the indol group, which was explained by the absence of CYP2C9 orthologs in zebrafish. Finally, hexarelin was used as a model to evaluate studies by ZWT for drugs with low stability.As a result, hexarelin displays a very fast metabolization in ZWT conditions and all the metabolites described for human were observed in ZWT. Therefore, the appropriate conditions, merits, and relevant limitations to conduct ZWT experiments for the investigation of drug metabolism are described.

Glicocorticosteroides estão incluídos na lista de substâncias proibidas da Agência Mundial Antidoping como analitos que dispensam quantificação. Entretanto, abordagens semiquantitativas são necessárias antes de relatar um resultado analítico adverso para essa classe de substâncias. Cromatografia líquida acoplada à espectrometria de massa (LC-MS) tem sido empregada com excelente seletividade e especificidade na análise de xenobiontes em misturas complexas. Foram analisadas amostras contendo glicocorticosteroides exógenos, a partir de diferentes vias de administração, utilizando o método proposto. Amostras geradas após administração sistêmica (via oral) apresentaram níveis de prednisolona ou prednisona superiores a 30 ng mL -1 , limite mínimo de desempenho requerido usado como parâmetro de diagnóstico. Por outro lado, vias não-sistêmicas parecem não apresentar biodisponibilidade para gerar um caso adverso em análises de controle de dopagem. Considerando o número reduzido de estudos similares na literatura, mais estudos de excreção podem ser realizados usando o método relatado com o objetivo de fornecer mais informações sobre o perfil de excreção de glicocorticosteroides administrados por vias sistêmicas e não sistêmicas.Glucocorticosteroids are included in the World Anti-Doping Agency (WADA) prohibited list as non-threshold analytes. Nevertheless, at least semi-quantitative approaches are necessary before reporting an adverse analytical finding for this class of substances. The liquid chromatography coupled to mass spectrometry (LC-MS) has been used with excellent selectivity and specificity in xenobiotic analysis in complex matrices. Real samples containing exogenous glucocorticosteroids from different administration routes were analyzed using the method proposed. Samples from systemic administration (oral route) presented levels of prednisolone or prednisone higher than 30 ng mL -1 , the minimum requirement performance level used as diagnostic parameter. On the other hand, non-systemic routes seem to not have bioavailability to generate an adverse case in doping control analysis. Considering the low number of similar studies available in the literature, more urinary studies could be done using the method reported aiming to provide more information about the excretion profile of glucocorticosteroids administered by systemic and non-systemic routes.

The detection of 11 sympathomimetic alkylamines in urine was presented with a focus on human doping control is proposed using liquid chromatography tandem mass spectrometry (LC-QqQ) and high resolution mass spectrometry (LC-HRMS) as a screening tool after a dilute-and-shoot (DS) approach. For the LC-HRMS analyses, several compounds exhibited better limits of detection (L.O.D.) than the LC-QqQ. However, due to their small differences in structure, co-elution among the alkylamines was observed. Therefore, the chemical conversion of the alkylamines into an appropriate derivative for the confirmation analyses using gas chromatography-mass spectrometry (GC-MS) was evaluated. Five derivatization approaches were evaluated in an attempt to increase the analytical response and the confidence of the identification. The choice of the appropriated derivative for each alkylamine makes their spectra more easily interpretable, fulfills the WADA's rather strict identification criteria and enables the unequivocal identification of alkylamines in urine.

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