Development and validation of a fast gas chromatography combustion isotope ratio mass spectrometry method for the detection of epiandrosterone sulfate in urine

In doping control, to confirm the exogenous origin of exogenously administered anabolic androgenic steroids (AAS), a gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) analysis is performed. Recently published work suggests that epiandrosterone sulfate (EpiAS) is a promising IRMS target compound for the detection of AAS, capable of prolonging the detection window. However, EpiAS is only excreted in urine in its sulfoconjugated form, while all other IRMS target compounds are excreted glucuronidated, meaning that EpiAS cannot be incorporated in the existing IRMS methods. A separate extensive sample preparation needs to be performed on this compound with a different hydrolysis and extraction procedure and a different liquid chromatography (LC) clean-up. The current work presents a new, fast, and easy to implement EpiAS IRMS method. The approach was based on the direct GC analysis of non-hydrolyzed EpiAS, making the solid phase extraction, hydrolysis, and acetylation step redundant. Sample preparation consisted of a simple liquid-liquid extraction, followed by LC fraction collection. A population study was performed to check compliance with the criteria drafted by the World Anti-Doping Agency (WADA). To verify the applicability of the developed approach, the method was applied to the samples of four administration studies (i.e. dehydroepiandrosterone (DHEA), testosterone gel (T gel), androstenedione (ADION), and intramuscular testosterone undecanoate. In contrast to previously published data, the strength of EpiAS as the target compound and the prolongation of the detection window in comparison with the conventional IRMS target compounds was less pronounced

GC-C-IRMS in routine doping control practice: 3 years of drug testing data, quality control and evolution of the method

In order to detect the misuse of endogenous anabolic steroids, doping control laboratories require methods that allow differentiation between endogenous steroids and their synthetic copies. Gas Chromatography Combustion Isotope Ratio Mass Spectrometry (GC-C-IRMS) is capable of measuring the carbon isotope ratio of urinary steroids and this allows differentiation between both. GC-C-IRMS and its application to doping control has evolved a lot during the last decade and so have the Word Anti-Doping Agency (WADA) technical documents that describe how GC-C-IRMS should be applied. Especially the WADA technical document of 2014 introduced a number of obligatory quality controls and a fixed methodology that should be used by all the doping control laboratories. This document imposed more uniform methods between the laboratories in order to decrease the inter laboratory standard deviation and acquire similar results for the analysis of the same urine samples. In this paper, three years of drug testing data of our GC-C-IRMS method in routine doping control practice is described, with an emphasis on the new WADA technical document and its implementation. Useful data for other doping control laboratories is presented focussing on general method setup, quality control and data collected from routine samples. The paper concentrates on how IRMS results shift or remain similar by switching to the 2014 WADA technical document and gives insight in a straightforward approach to calculate the measurement uncertainty

Confirmation of endogenous substance abuse in doping control by isotope ratio mass spectrometry

All doping control laboratories accredited by the World Anti-Doping Agency have been confronted with the task to develop analytical methods and establish criteria that allow endogenous substances to be distinguished from their synthetic copies. It has been known for some time that isotope ratio mass spectrometry (IRMS) is capable of meeting this challenge because endogenous and exogenous substances exhibit different isotope ratios. At this stage, the technique is primarily used for the differentiation between endogenous and exogenous steroids by comparison of the 13C/12C ratios. Endogenous reference compounds (ERC’s) are compounds of which the isotope ratio does not change after synthetic steroid administration. By comparison of the 13C/12C ratios of ERC’s with that of target compounds like testosterone and its metabolites, it is possible to differentiate between an exogenous or endogenous origin. Obviously, IRMS is not limited to steroids and 13C/12C measurements. The Technique can also be used for other endogenous substances that are known to be misused as doping, such as for example AICAR and adrenaline. Both compounds contain nitrogen, making 15N/14N ratio analysis a valuable option. Results of several new methods developed on IRMS will be discussed

Development of a sensitive GC-C-IRMS method for the analysis of androgens in doping control

All doping control laboratories accredited by the World Anti-Doping Agency (WADA) have been confronted with the task to develop analytical methods and establish criteria that allow endogenous steroids to be distinguished from their synthetic copies. It has been known for some time that gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) is capable of meeting this challenge by comparison of the 13C/12C ratios of the target compounds with those of endogenous reference compounds that are not affected by the administration of synthetic androgens. Testosterone and/or its main metabolites function as target compounds. Typical endogenous reference compounds include 5β-pregnanediol, 11-ketoetiocholanolone and 11-βhydroxyandrosterone. Synthetic copies are generally derived from stigmasterol and sitosterol; plant sterols obtained from soybean (Glycine max) which have a significantly different carbon isotope composition compared to endogenous steroids. As a consequence, the administration of synthetic analogs is detectable through a change in the carbon isotopic composition of testosterone and its metabolites. GC-C-IRMS however remains a very laborious and expensive technique because one can only determine the 13C/12C ratio of a pure compound. This means that a lot of purification steps have to be conducted and fractionation caused by one of these steps is unacceptable. On top of that, substantial amounts of urine are needed to meet the sensitivity requirements of the IRMS. Because the amount of received urine from an athlete is limited, doping control laboratories have to make their analysis as sensitive as possible so all the required doping tests can be executed. If less urine is consumed, then there is more available for additional tests. In this work we introduce a new type of injection which takes GC-C-IRMS to the next level. With the aid of a programmed temperature vaporizer we were able to increase the sensitivity of the IRMS with a factor of 10 and we drastically reduced the required amount of urine and the limit of detection. All this is achieved without having to change any of the IRMS detection parameters