RESUMO
The aim of this work was to validate the gas chromatography/combustion/isotope ratio mass spectrometry method in Havana Antidoping Laboratory and verify its implementation with a study of the Cuban population. The method was precise and accurate inside the linear working range; the limit of quantification and the uncertainty were compliant with TD2019IRMS. The study of the Cuban population showed no differences in δ13 C values between females and males. Only three values of Δδ13 C showed significant differences between sexes (PD-T, OHA-T, and 11-keto-Et-T). The values of δ13 C between -17.8 and -21.2 (upper and lower limits based on normal distribution) were consistent with other populations where C4 plant derivatives prevail in the diet.
Assuntos
Esteroides/urina , Detecção do Abuso de Substâncias/métodos , Adulto , Atletas , Isótopos de Carbono/urina , Cuba , Dopagem Esportivo , Feminino , Cromatografia Gasosa-Espectrometria de Massas/métodos , Humanos , Limite de Detecção , Masculino , Adulto JovemRESUMO
Carbon isotope ratio (CIR) confirmation is one of the most complex and delicate analyses in the doping control field, due to the nature of the molecules to be confirmed, normally present in urinary samples as a consequence of an endogenous production. The requirements for method validation established by the World Anti-Doping Agency (WADA) have been pushing the accredited laboratories to improve their methods. The choice of the method is always a cost benefit ratio involving a hard-working and time-consuming analysis and the guarantee of reporting of reliable results. This work presents the method fully validated by the Brazilian Doping Control Laboratory as part of the preparation for the Rio de Janeiro Summer Olympic and Paralympic Games 2016. Sample preparation encompassed solid-phase extraction, liquid-liquid extraction, enzymatic hydrolysis, acetylation, and purification by preparative high-performance liquid chromatography, and analyses were performed by gas chromatography/combustion/isotope ratio mass spectrometry. This proved to be a robust method to CIR confirmation in a big event, as demonstrated by the analysis of 179 samples during the Games 2016, from clearly negative results and adverse findings for testosterone (T) and related substances, boldenone and its metabolite, 19-norandrosterone and formestane. Two atypical findings were also reported for T and metabolites.
Assuntos
Isótopos de Carbono/urina , Dopagem Esportivo , Cromatografia Gasosa-Espectrometria de Massas/métodos , Congêneres da Testosterona/urina , Acetilação , Brasil , Cromatografia Líquida de Alta Pressão , Estranos/urina , Humanos , Extração Líquido-Líquido , Reprodutibilidade dos Testes , Extração em Fase Sólida , Esportes , Testosterona/análogos & derivados , Testosterona/urinaRESUMO
Measuring carbon isotope ratios (CIRs) of urinary analytes represents a cornerstone of doping control analysis and has been particularly optimized for the detection of the misuse of endogenous steroids. Isotope ratio mass spectrometry (IRMS) of appropriate quality, however, necessitates adequate purities of the investigated steroids, which requires extensive pre-analytical sample clean-up steps due to both the natural presence of the target analytes and the high complexity of the matrix. In order to accelerate the sample preparation and increase the automation of the process, the use of multidimensional gas chromatography (MDGC) prior to IRMS experiments, was investigated. A well-established instrumental configuration based on two independent GC ovens and one heart-cutting device was optimized. The first dimension (1D) separation was obtained by a non-polar column which assured high efficiency and good loading capacity, while the second dimension (2D), based on a mid-polar stationary phase, provided good selectivity. A flame ionization detector monitored the 1D, and the 2D was simultaneously recorded by isotope ratio and quadrupole mass spectrometry. The assembled MDGC set-up was applied for measuring testosterone, 5α- and 5ß-androstanediol, androsterone, and etiocholanolone as target compounds and pregnanediol as endogenous reference compound. The urine sample were pretreated by conventional sample preparation steps comprising solid-phase extraction, hydrolysis, and liquid-liquid extraction. The extract obtained was acetylated and different aliquots were injected into the MDGC system. Two high performance liquid chromatography steps, conventionally adopted prior to CIR measurements, were replaced by the MDGC approach. The obtained values were consistent with the conventional ones. Copyright © 2016 John Wiley & Sons, Ltd.
Assuntos
Anabolizantes/urina , Androsterona/urina , Isótopos de Carbono/urina , Cromatografia Líquida de Alta Pressão/métodos , Etiocolanolona/urina , Cromatografia Gasosa-Espectrometria de Massas/métodos , Esteroides/análise , Testosterona/urina , Anabolizantes/química , Androsterona/análise , Androsterona/química , Cromatografia Gasosa , Dopagem Esportivo , Etiocolanolona/análise , Etiocolanolona/química , Humanos , Espectrometria de Massas , Esteroides/química , Esteroides/metabolismo , Testosterona/análiseRESUMO
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.