Vinícius F. Sardela

Development and validation of a ultra high performance liquid chromatography-tandem mass spectrometric method for the direct detection of formoterol in human urine.

Formoterol is a long acting β(2)-agonist and has proven to be a very effective bronchodilating agent. Hence it is frequently applied therapeutically for the treatment of asthma. Because β(2)-agonists might be misused in sports for the stimulatory effects and for growth-promoting action their use is restricted. Since January 2012, formoterol is prohibited in urinary concentrations higher than 30 ng/mL. The objective of this study was to develop and validate a simple and robust ultra high performance liquid chromatographic-tandem mass spectrometric (UHPLC-MS/MS) method for the direct quantification of formoterol in urine. Sample preparation was limited to an enzymatic hydrolysis step after which 2 μL was injected in the chromatographic system. Chromatography was performed on a C(8)-column using gradient conditions. The mobile phase consisted of water/methanol (H(2)O/MeOH) both containing 0.1% acetic acid (HOAc) and 1mM ammonium acetate (NH(4)OAc). Calibration curve were constructed between 15 and 60 ng/mL. Validation data showed bias of 1.3% and imprecision of 5.4% at the threshold. Ion suppression/enhancement never exceeded 7%. Calculating measurement uncertainty showed proof of applicability of the method. Stability of formoterol was also investigated at 56 °C (accelerated stability test) at pH 1.0/5.2/7.0 and 9.5. At the physiological pH values of 5.2 and 7.0, formoterol showed good stability. At pH 1.0 and 9.5 significant degradation was observed.

Analysis of sibutramine metabolites as N-trifluoroacetamide and O-trimethylsilyl derivatives by gas chromatography-mass spectrometry in urine.

A method for identifying the metabolites of sibutramine 1-(4(chlorophenyl)-N,N-dimethyl-alpha-(2-methylpropyl))cyclobutanemethanamine) in urine, utilizing a double derivatization strategy, with N-methyl-N-(trimethylsilyl)-trifluoroacetamide and N-methyl-bis-(trifluoroacetamide), in gas chromatography/mass spectrometry is proposed. This methodology results in mass spectra with at least three fragments in abundance superior to 20%, attending the World Anti-Doping Agency identification criteria for qualitative assays. The characterization of the derivatives was obtained through two ionization modes: Chemical Ionization and Electron Impact ionization, both in full scan mode. Sibutramine was administered to 5 (five) volunteers and the excretion profile followed for 92h. Routine analytical, hydroxy-cyclobutane-bis-nor-sibutramine which becomes the more abundant metabolite in the first 10h and hydroxy-isopropyl-bis-nor-sibutramine which becomes the most abundant after 40h, were proposed for doping monitoring.

Consequences of the formation of 3,4-dimethyl-5-phenyl-1,3-oxazolidine on the analysis of ephedrines in urine by gas chromatography and a new method for confirmation as N-trifluoroacetyl-O-t-butyldimethylsilyl ether derivatives.

The compound 3,4-dimethyl-5-phenyl-1,3-oxazolidine can appear as an artifact during the gas chromatographic analysis of ephedrines. Its presence is a risk for doping control and forensic analyses. An evaluation about the consequences of its formation showed the possibility of a false positive for ephedrine, a false negative for pseudophedrine and increased uncertainty in the quantitative approach. Misinterpretations can be avoided with the observation of fragments m/z 56 and 71 in the ephedrine mass spectrum during GC-MS analysis and also by the formation of N-TFA-O-TBDMS derivatives prior to GC analysis. These N-TFA-O-TBDMS derivatives lead to an increase in the number and mass of diagnostic ions, meet the identification criteria, and provide an improvement in chromatographic resolution, allowing the separation of the ephedrines.

Stimulant Doping Agents Used in Brazil: Prevalence, Detectability, Analytical Implications, and Challenges

This article presents the prevalence of stimulant doping among Brazilian athletes, the analytical approaches used, as well as a general evolution of the detectability of the stimulants being used. Results from the Brazilian accredited doping control laboratory are compared with the global statistics disclosed by the World Anti-Doping Agency. The high prevalence of stimulant doping in Brazil can be attributed to several reasons, including “self-administration,” a “body-shaping” culture, and the use of nutritional supplements.

Identification of sympathomimetic alkylamine agents in urine using liquid chromatography–mass spectrometry and comparison of derivatization methods for confirmation analyses by gas chromatography–mass spectrometry

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 WADAs rather strict identification criteria and enables the unequivocal identification of alkylamines in urine.

Systematic analysis of glycerol: colourimetric screening and gas chromatography–mass spectrometric confirmation

Glycerol is a naturally occurring polyol in the human body, essential for several metabolic processes. It is widely used in the food, pharmaceutical, and medical industries and in clinical practice as a plasma volume expander (PVE). Athletes, however, may use glycerol to mask the presence of forbidden substances or to enhance performance, inclusively through hyperhydration achieved by glycerol ingestion with added fluid. These practices are considered doping, and are prohibited by the World Anti-Doping Agency (WADA). Therefore, glycerol was introduced in the prohibited list. Doping through glycerol ingestion can readily be identified by detection of elevated glycerol concentrations in urine. In this paper, a protocol for the fast detection of glycerol in urine is proposed. It consists of a previous visual colourimetric screening, followed by a quantitative/qualitative confirmation analysis by mass spectrometry. The screening procedure involves a reaction in which polyhydric alcohols are oxidized by periodate to formic acid and formaldehyde, which is detected by the addition of a fuchsin solution. For the subsequent qualitative/quantitative confirmation analysis, a gas chromatography-mass spectrometry based approach with a non-deuterated internal standard and a drying step of only 10 min is proposed. The linear correlation was demonstrated within WADA´s threshold range. The calculated RSD were 2.1% for within-day precision and 2.8% for between-day precision. The uncertainty estimation was calculated, and a value of 2.7% was obtained. The procedure may also be used for the analysis of other polyols in urine, as for example the PVE mannitol.

Doping control analysis at the Rio 2016 Olympic and Paralympic Games

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. Copyright

Is zebrafish (Danio rerio) a tool for human‐like metabolism study?

One of the greatest challenges in anti-doping science is the large number of substances available and the difficulty in finding the best analytical targets to detect their misuse. Therefore, metabolism studies involving prohibited substances are fundamental. However, metabolism studies in humans could face an important ethical bottleneck, especially for non-approved substances. An emerging model for metabolism assessment is the zebrafish, due to its genetic similarities with humans. In the present study, the ability of adult zebrafish to produce metabolites of sibutramine and stanozolol, substances with a well-known metabolism that are widely used as doping agents in sports, was evaluated. They represent 2 of the most abused classes of doping agents, namely, stimulants and anabolic steroids. These are classes that have been receiving attention because of the upsurge of synthetic analogues, for which the side effects in humans have not been assessed. The samples collected from the zebrafish tank water were hydrolysed, extracted by solid-phase extraction, and analysed by liquid chromatography with high resolution mass spectrometry (LC-HRMS). Adult zebrafish could produce several sibutramine and stanozolol metabolites, including demethylated, hydroxylated, dehydroxylated, and reduced derivatives, all of which have already been detected in human urine. This study demonstrates that adult zebrafish can absorb, oxidise, and excrete several metabolites in a manner similar to humans. Therefore, adult zebrafish seem to be a very promising tool to study human-like metabolism when aiming to find analytical targets for doping control. Copyright

Urinary Excretion Profile of Luteinizing Hormone in Brazilian Athletes

Luteinizing hormone (LH) can be used by athletes as an alternative way to take illicit advantage of androgenic effects, since it stimulates the secretion of testosterone by the Leydig cells. As consequence, LH use is illegal for male athletes and the hormone is included in the World Anti-Doping Agency (WADA) prohibited list. Since LH is an endogenous substance, the strategy used to allow the detection of LH abuse was the establishment of a reference value. All analyses were developed by IMMULITE®, which was submitted to a complete validation performance with urine. The hormone showed to be stable at 4 °C and -20 °C for at least 40 days and for 10 days at room temperature and unstable after the second cycle of freezing and thawing. The reference population of male Brazilian athletes showed a cut-off value of 37.4 mIU mL-1. The results indicate the assay is suitable for application in doping control analysis.

Fast Ephedrine Quantification by Gas Chromatography Mass Spectrometry

Ephedrines are widely used in therapy. Because of their stimulant properties, these substances are relevant in different forensic fields. At present, the state of the art for ephedrines quantification relay based on a liquid chromatography mass spectrometry, mainly because of the dilute-andshoot approach. Notwithstanding, several gas chromatography based methods have already been described, all of them include cleanup steps, with the potential disadvantage of incurring errors and increasing the workload. In this paper, a straightforward method for ephedrine quantification based on gas chromatographic mass spectrometry, without cleanup and based on Doehlert matrix optimization is presented. Only 10 μL of a urine sample is necessary and for N-methylN-(trimethylsilyl)trifluoroacetamide/N-methyl-bis-trifluoracetamide derivatives, the intermediate precision was 2.77% for ephedrine, 9.20% for cathine, 8.29% for norephedrine and 4.27% for pseudoephedrine. The limit of detection was 20 ng mL for ephedrine, 30 ng mL for cathine and 40 ng mL for norephedrine and pseudoephedrine.