Henrique Marcelo Gualberto Pereira

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.

Drug testing data from the 2007 Pan American Games: δ 13C values of urinary androsterone, etiocholanolone and androstanediols determined by GC/C/IRMS

The main purpose of this article is to show the application of the CG/C/IRMS in real time during competition in the steroid confirmation analysis. For this reason, this paper summarizes the results obtained from the doping control analysis during the period of the 2007 Pan American Games held in Rio de Janeiro, Brazil. Approximately 5600 athletes from 42 different countries competed in the games. Testing was performed in accordance to World Anti-Doping Agency (WADA) technical note for prohibited substances. This paper reports data where abnormal urinary steroid profiles, have been found with the screening procedures. One 8 mL urine sample was used for the analysis of five steroid metabolites with two separate analyses by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Urine samples were submitted to GC/C/IRMS for confirmation analysis to determine the (13)C/(12)C ratio of selected steroids. Fifty-seven urine samples were analyzed by GC/C/IRMS and the delta(13)C values ( per thousand) of androsterone, etiocholanolone, 5beta-androstane-3alpha, 17beta-diol (5beta-diol), 5alpha-androstane-3alpha, 17beta-diol (5alpha-diol) and 5beta-pregnane-3alpha, 20alpha-diol (5beta-pdiol), the endogenous reference compound are presented. One urine sample with a testosterone/epitestosterone (T/E) ratio of 4.7 was confirmed to be positive of doping by GC/C/IRMS analysis. The delta values of 5beta-diol and 5alpha-diol were 3.8 and 10.8, respectively, compared to the endogenous reference compound 5beta-pdiol, which exceeded the WADA limit of 3 per thousand. The results obtained by CG/C/IRMS confirmation analyses, in suspicious samples, were conclusive in deciding whether or not a doping steroid violation had occurred.

Controle de dopagem de anabolizantes: o perfil esteroidal e suas regulações

The concept of steroid profile is discussed in this paper. The main metabolic routes are presented. The importance of evaluating steroid profiles is demonstrated, with special attention to clinical medicine and sports. Parameters used in the literature for doping control of endogenous steroids are briefly evaluated, as well as the factors responsible for alterations in the normal steroid profile. Special focus is turned to the latter approach.

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.

Analysis of exemestane and 17β‐hydroxyexemestane in human urine by gas chromatography/mass spectrometry: development and validation of a method using MO‐TMS derivatives

Trimethylsilylation of anabolic agents and their metabolites is frequently achieved by using the derivatization mixture N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA)/NH(4)I/2-mercaptoethanol. Nevertheless, artifacts were formed when this mixture was employed in the monitoring of exemestane and its main metabolite 17β-hydroxyexemestane prior to GC-MS analysis. These artifacts were identified as the N-methyltrifluoroacetamide (MTFA) and trimethylsiloxyethylmercapto products of the respective trimethylsilyl (TMS) derivatives. Furthermore, artifact formation was evaluated taking the structure (1,4-diene-3-keto-6-exomethylene) of the compounds into account. Although these artifacts are relevant for investigations regarding the derivatization process and may be of interest in many fields, they are detrimental to cope with the requirements of the World Anti-Doping Agency (WADA) in terms of the limits of detection (LODs) required. To overcome this issue, a method using an alternative derivatization was proposed: formation of methyloxime-TMS derivatives through double derivatization using O-methylhydroxylamine/pyridine and MSTFA/TMS imidazole after enzymatic hydrolysis and liquid-liquid extraction. Samples from an excretion study after administration of exemestane to healthy volunteers were analyzed by the proposed method and detection of both exemestane and its main metabolite was possible. This method showed excellent results for both analytes meeting the LODs required for antiestrogenic agents (50  ng/mL) established by WADA. The method was validated for the main metabolite, it was robust and cost-effective for qualitative and quantitative purposes, with LOD and LOQ of 10  ng/mL and 25  ng/mL, respectively.

Improvements in steroid screening in doping control with special emphasis to GC-MS analytical conditions and method validation

A procedure is described for the simultaneous determination of androgenic substances including steroids and b2-agonists. The method involves analysis of hydrolized urinary anabolic compounds using liquid-liquid extraction, with subsequent conversion to trimethylsilylether derivatives for the analysis by GC-MS. Pulse split injection 1/10 of the TMS derivatives at 280 °C into the capillary column, initially maintained at 140 °C then programmed to 180 °C at 40 °C min-1, followed by 3 oC min-1 to 230 oC and then 40 oC min-1 to 300 oC, resulted in good resolution and peak shape for all compounds. The detection limits of most of the steroids were 1 ng mL-1 except for formebolone and trenbolone (25 ng mL-1). When applied to selected urine samples with evidence of bacterial degradation and metabolites from usual medications/vitamins, the method allowed rapid screening for androgens and other substances monitored in routine. The resolution was adequate to evaluate the endogenous steroid profile relevant to doping control and medical applications.

DNA Typing: An Accessory Evidence in Doping Control

A clear positive case for anabolic steroids doping was confounded by alleged urine tampering during doping control procedures. Review of the chain of custody showed no flaws, but nevertheless the athlete was adamant that the urine sample should be analyzed for DNA in order to support her contention that she was not the donor of the sample. The results obtained showed that the urine sample that scored positive for steroids contained nuclear DNA that could not be matched to the DNA obtained from the athletes blood. On the other hand, the same urine sample contained mitochondrial DNA whose nucleotide sequences spanning the hyper variable regions HV1 and HV2 proved to be identical to those determined in mitochondrial DNA amplified from the athletes blood. The occurrence of an extraneous genotype is compatible with exogenous nuclear DNA admixture to the athletes urine. Alternatively, taking in consideration the mitochondrial DNA, we could not exclude that a sibling or a maternal relative of the athlete could have acted as a donor of the urine utilized for doping control and DNA analysis. Both situations point to possible tampering of the urine by the athlete. Adjudication at CAS maintained previous national and international federation decision that there was no proof of a chain of custody flaw to justify the athletes allegation of urine substitution after collection.

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.