Implementation and Performance of the Gas Chromatography/Combustion/Isotope Ratio Mass Spectrometry-Based Method for the Confirmatory Analysis of Endogenous Anabolic Steroids during the Rio de Janeiro Olympic and Paralympic Games 2016

Abstract

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.

Application of online two‐dimensional high‐performance liquid chromatography as purification procedure to determine the origin of 19‐norandrosterone in urine by gas chromatography–combustion–isotope ratio mass spectrometry

“…Afterwards, the positive urine was fortified with blank urine to form a positive quality control (QCP) sample urine pool with concentration of 19‐NA around 10 ng/ml. The negative quality control (QCN) sample is prepared by using Fábio Azamor de Oliveira method 21 ; the concentration of 19‐NA is 15 ng/ml.…”

Annual banned‐substance review: Analytical approaches in human sports drug testing 2019/2020

“…While increasing sample throughput by optimizing sample preparation protocols and/or analytical turnaround times is not necessarily critical in research projects, fast reporting times for doping control analyses are desirable during major national and international sporting competitions. Consequently, options to enhance the laboratory's efficiency have been further explored regarding IRMS in particular and in that context, the GC/C/IRMS strategy employed at the Games of the XXXI Olympiad (2016) was recently discussed by de Oliveira et al 85 Putz et al presented an approach employing immunoaffinity purification of TCs and ERCs instead of frequently utilized HPLC fraction collection in order to expedite the overall analysis time 86 . A volume of 2–3 ml of urine was preconcentrated by SPE and retained unconjugated steroidal analytes were removed by subsequent LLE prior to an enzymatic hydrolysis of glucurono‐conjugated TCs and ERCs.…”