Testosterone Abuse


The instinct for man to compete against his peers dates back to the earliest times, and so does the temptation to gain a competitive advantage by unfair means. Today, this unfair advantage can come in many forms, but enhancing performance by chemical means is one of the most sophisticated and hardest to detect. With a responsibility to the public to ensure fair competition, as well as to maintain the health of the athletes themselves, authorities must use the highest performance cutting-edge technology detect and enforce the principle of clean competition.

Stable isotope analysis provides a unique tool for determining whether an athlete has used performance enhancing drugs or if they simply benefit from an natural capacity for high performance. The World Anti Doping Agency has declared that stable isotope analysis is a mandatory technique for sports testing, but similar principles can also be applied to the detection of steroid abuse in cattle.

Detecting synthetic steroids

The application of synthetic forms of endogenous anabolic androgenic steroids by an athlete is known to boost that athletes performance. This is unfair and so must be detected by means of a urinary analysis. GC/C/IRMS has been approved by the World Anti-Doping Agency (WADA) as the official method for detecting this doping procedure and is laid out in WADA Technical Document TD2016IRMS.


Gas Chromatography / Combustion / Isotope Ratio Mass Spectrometry requires the very highest sensitivity to be able to detect the smallest levels of steroid abuse whilst offering analytical robustness that ensures that the sample analysis is consistently successful, especially during major sporting events. Our AnthrovisION GC/C/IRMS system provides exceptional levels of performance for the most demanding situations.

IonOS Software

IONOS provides a number of powerful features for the anti-doping laboratory. All data processing is done within IonOS which means that all data processing can be traced back to the original instrument data. Our patented Peak Mapping algorithm is able to automatically detect isotope standards such as USADA-33 and USADA-34 and automatically apply an isotope calibration. Finally, our Calculated Columns functionality is able to automatically calculate Endogenous Reference Compound (ERC) and Target Compound (TC) combinations and flag them as lying outside the permissible ranges.

Sports doping publications using our instruments

Our customers use our instruments to do some amazing research in the sports doping application field. To show you how they perform their research and how they use our IRMS instruments, we have collected a range of peer-reviewed publications which cite our products. You can find the citations below and then follow the links to the publishing journal should you wish to download the publication.

If you would like to investigate our available citations in more detail, or email the citation list to yourself or your colleagues then take a look at our full citation database.

18 results:

A simplified and accurate method for the analysis of urinary metabolites of testosterone-related steroids using gas chromatography/combustion/isotope ratio mass spectrometry.
Rapid communications in mass spectrometry : RCM (2013)
Alexandre Ouellet, Nicolas LeBerre, Christiane Ayotte

RATIONALE: The analysis of urinary metabolites of testosterone-related steroids through the measurement of their carbon isotopic signature (δ(13) C) by gas chromatography/combustion/mass spectrometry (GC/C/IRMS) is a confirmation method employed in doping control analyses. Stringent analytical conditions are essential to an accurate and precise analysis as well as the proper selection of the metabolites, which forms the basis of the refined method presented in this paper. METHODS: In a simplified approach, following enzymatic hydrolysis and extraction from a relatively low volume of urine sample, a one-step high-performance liquid chromatography (HPLC) purification was developed for seven diagnostic urinary metabolites (TS) including testosterone itself, dehydroepiandrosterone, 5α- and 5β-androstanediol, epitestosterone, androsterone, etiocholanolone and two endogenous reference compounds (ERC), 5β-pregnanediol and 5α-androst-16-en-3β-ol. These steroids were pooled in three fractions and analyzed as such. With regards to the GC/C/IRMS analysis, a multi-level isotopic calibration using the 'identical treatment' principle was created. RESULTS: The proposed isotopic calibration yielded results for purified reference steroids with a precision ≤0.15 and accuracy of ≤0.30 ‰ (between-assay, n = 26). Compared to other common endogenous reference compounds, those selected in this study had δ(13) C values close to the target metabolites which, along with the proposed isotopic calibration, produced narrow reference intervals within ± 3‰ for most diagnostic TS-ERC pairs, in compliance with the requirements of the World Anti-Doping Agency. CONCLUSIONS: These carefully controlled analytical conditions are compatible with routine operations, affording accurate and precise results for the more diagnostically relevant metabolites such as testosterone itself and the 5α- and 5β-androstanediols. The values of the TS-ERC pairs measured in reference populations are described and the results from the routine testing of several hundreds of athletes' samples are discussed. Robust, this technique permitted the detection of adverse findings that would have been missed had these low level metabolites not been analyzed.

Effect of changes in the deuterium content of drinking water on the hydrogen isotope ratio of urinary steroids in the context of sports drug testing.
Analytical and bioanalytical chemistry (2013)
Thomas Piper, Karoline Degenhardt, Eugen Federherr, Andreas Thomas, Mario Thevis, Martial Saugy

The hydrogen isotope ratio (HIR) of body water and, therefore, of all endogenously synthesized compounds in humans, is mainly affected by the HIR of ingested drinking water. As a consequence, the entire organism and all of its synthesized substrates will reflect alterations in the isotope ratio of drinking water, which depends on the duration of exposure. To investigate the effect of this change on endogenous urinary steroids relevant to doping-control analysis the hydrogen isotope composition of potable water was suddenly enriched from -50 to 200 ‰ and maintained at this level for two weeks for two individuals. The steroids under investigation were 5β-pregnane-3α,20α-diol, 5α-androst-16-en-3α-ol, 3α-hydroxy-5α-androstan-17-one (ANDRO), 3α-hydroxy-5β-androstan-17-one (ETIO), 5α-androstane-3α,17β-diol, and 5β-androstane-3α,17β-diol (excreted as glucuronides) and ETIO, ANDRO and 3β-hydroxyandrost-5-en-17-one (excreted as sulfates). The HIR of body water was estimated by determination of the HIR of total native urine, to trace the induced changes. The hydrogen in steroids is partly derived from the total amount of body water and cholesterol-enrichment could be calculated by use of these data. Although the sum of changes in the isotopic composition of body water was 150 ‰, shifts of approximately 30 ‰ were observed for urinary steroids. Parallel enrichment in their HIR was observed for most of the steroids, and none of the differences between the HIR of individual steroids was elevated beyond recently established thresholds. This finding is important to sports drug testing because it supports the intended use of this novel and complementary methodology even in cases where athletes have drunk water of different HIR, a plausible and, presumably, inevitable scenario while traveling.

Two-dimensional gas chromatography with heart-cutting for isotope ratio mass spectrometry analysis of steroids in doping control.
Drug testing and analysis (2012)
a D Brailsford, I Gavrilović, R J Ansell, D a Cowan, a T Kicman

The accuracy and precision of gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) measurements are highly dependent on analyte purity. Reliable analysis of urinary steroids for doping control therefore requires extensive and time-consuming sample preparation (i.e. liquid chromatography fraction collection) prior to GC-C-IRMS analysis. The use of two-dimensional GC (GC-GC) with heart-cutting (Deans Switch) as a possible approach to reduce the sample purification required for IRMS analysis is described herein. The system uses a low thermal mass oven (LTM) incorporated into an existing GC-C-IRMS system. GC-GC allowed the use of a cyanopropyl/phenyl column in the first dimension to optimize the separation of underivatized steroids, while a phenyl-methylpolysiloxane column in the second dimension focuses the selectively cut analytes into narrower peaks for more sensitive and reliable MS analysis. In addition, to confirm analyte identity, eluent from the second GC was split, with 20 % entering a scanning MS, and 80 % flowing to the IRMS. As a proof concept, the developed method was then used to analyze a single spot urine (5 ml) from an individual receiving T therapy (2 × 50 mg sachets of Testogel(®)). The T delta value (-27.8 ‰, [T] = 38 ng/ml) was clearly distinct from 11-ketoetiocholanolone (-22.5 ‰) (used as an endogenous reference compound (ERC)), indicating T as being of exogenous origin. The simultaneous analysis by the scanning MS yielded a full scan mass spectrum of the same chromatographic peak, thus confirming the peak to be T.

Examination of the kinetic isotopic effect to the acetylation derivatization for the gas chromatographic-combustion-isotope ratio mass spectrometric doping control analysis of endogenous steroids.
Drug testing and analysis (2012)
Yiannis S Angelis, Maroula K Kioussi, Polyxeni Kiousi, J Thomas Brenna, Costas G Georgakopoulos

In gas chromatographic-combustion-isotope ratio mass spectrometry (GC-C-IRMS) doping control analysis, endogenous androgenic anabolic steroids and their metabolites are commonly acetylated using acetic anhydride reagent, thus incorporating exogenous carbon that contributes to the measured isotope ratio. Comparison of the endogenous δ(13)C of free, mono-, and di-acetylated steroids requires application of corrections, typically through straightforward use of the mass balance equation. Variability in kinetic isotope effects (KIE) due to steroid structures could cause fractionation of endogenous steroid carbon, resulting in inaccurate results. To test for possible KIE influence on δ(13)C, acetic anhydride of graded isotope ratio within the natural abundance range was used under normal derivatization conditions to test for linearity. In all cases, plots of measured steroid acetate δ(13)C versus acetic anhydride δ(13)C were linear and slopes were not significantly different. Regression analysis of the Δδ(13)C of enriched acetic anhydrides versus Δδ(13)C of derivatized steroids shows that KIE are similar in all cases. We conclude that δ(13)C calculated from the mass balance equation is independent of the δ(13)C of the acetic anhydride reagent, and that net KIE under normal derivatization conditions do not bias the final reported steroid δ(13)C.

Validation and Applications of Gas Chromatography-Combustion/isotope Ratio Mass Spectrometric Method to Control Misuse of Androgens in Humans
Mass Spectrometry Letters (2011)
Kang-Mi Mi Lee, Ho-Jun Kim, Eun-Sook Jeong, Hye-Hyun Yoo, Oh-Seung Kwon, Chang-Bae Jin, Dong-Hyun Kim, Jae-Ick Lee

The misuse of anabolic androgenic steroids is of particular concern in sports and society. Thus, it is of great importance to discriminate endogenous steroids such as testosterone or testosterone prohormones from their chemically identical synthetic copies. In this study, gas chromatography-combustion/isotope ratio mass spectrometric (GC−C/IRMS) method has been developed and validated for discriminating the origin of anabolic androgenic steroids. The method involves the solid-phase extraction, enzymatic hydrolysis with β-glucuronidase, HPLC-fractionation for the cleanup and analysis by GC−C/IRMS. The difference (∆13C) of urinary δ13C values between synthetic analogues and endogenous reference compounds (ERC) by GC−C/IRMS was used to elucidate the origin of steroids, and intra- and inter-day precision, specificity and isotope fractionation were evaluated. The present GC−C/IRMS method combined with HPLC cleanup was accurate and reproducible enough to be successfully applied to the test of urine sample from suspected anabolic steroid abusers.

External calibration in gas chromatography-combustion-isotope ratio mass spectrometry measurements of endogenous androgenic anabolic steroids in sports doping control.
Journal of chromatography. A (2011)
Maroula K Kioussi, Yiannis S Angelis, Adam T Cawley, Michalis Koupparis, Rymantas Kazlauskas, J Thomas Brenna, Costas G Georgakopoulos

An alternative calibration procedure for the gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) measurements of the World Antidoping Agency (WADA) Accredited Laboratories is presented. To alleviate the need for externally calibrated CO₂ gas for GC-C-IRMS analysis of urinary steroid metabolites, calibration using an external standard mixture solution of steroids with certified isotopic composition was investigated. The reference steroids of the calibration mixture and routine samples underwent identical instrumental processes. The calibration standards bracketed the entire range of the relevant δ¹³C values for the endogenous and exogenous steroids as well as their chromatographic retention times. The certified δ¹³C values of the reference calibrators were plotted in relation to measured m/z ¹³CO₂/¹²CO₂ (i.e. R(45/44)) mass spectrometric signals of each calibrator. δ¹³C values of the sample steroids were calculated from the least squares fit through the calibration curve. The effect of the external calibration on δ¹³C values, using the same calibration standards and set of urine samples but different brands of GC-C-IRMS instruments, was assessed by an interlaboratory study in the WADA Accredited Laboratories of Sydney, Australia and Athens, Greece. Relative correspondence between the laboratories for determination of androsterone, etiocholanolone, 5β-androstane-3α,17β-diacetate, and pregnanediacetate means were SD(δ¹³C)=0.12‰, 0.58‰, -0.34‰, and -0.40‰, respectively. These data demonstrate that accurate intralaboratory external calibration with certified steroids provided by United States Antidoping Agency (USADA) and without external CO₂ calibration is feasible and directly applicable to the WADA Accredited Laboratories for the harmonization of the GC-C-IRMS measurements.

Isotope ratio mass spectrometry analysis of the oxidation products of the main and minor metabolites of hydrocortisone and cortisone for antidoping controls.
Steroids (2009)
C Buisson, C Mongongu, C Frelat, M Jean-Baptiste, J de Ceaurriz

Metabolites of hydrocortisone (HC) and cortisone (C), namely tetrahydrocortisol (THF), tetrahydrocortisone (THE), allo-THF, allo-THE for the main metabolites and 11-hydroxyandrosterone, 11-hydoxyetiocholanolone, 11-ketoandrosterone, and 11-ketoetiocholanolone for the minor metabolites, as well as the two main metabolites of testosterone, androsterone and etiocholanolone, were separated from each other using HPLC fractionation of urine extracts. An isotopic ratio mass spectrometry (IRMS) analysis determined the absolute delta(13)C values of 5alpha-androstanetrione (5alpha-AT) and 5beta-androstanetrione (5beta-AT) as the oxidation products (ox-products) of the HC and C metabolites and as target compounds (TCs). We also performed IRMS analysis of 5alpha-androstanedione (5alpha-AD) and 5beta-androstanedione (5beta-AD) as the ox-products of etiocholanolone and androsterone and as endogenous reference compounds (ERCs). Urine samples came from two male volunteers treated with a single 10-mg oral dose and a single 100-mg intramuscular dose of HC hemisuccinate, a male volunteer treated with a single 25-mg oral dose of C acetate, and a control group of 30 drug-free athletes. The mean -3SD of delta(13)C depletion values from the controls were -1.46, -1.98, -1.78 and -2.42 for 5beta-AT-5beta-AD, 5alpha-AT-5beta-AD, 5beta-AT-5alpha-AD and 5alpha-AT-5alpha-AD, respectively, indicating -3 per thousand as a safe cut-off value for differentiating the pharmaceutical from the natural form. In the main metabolite fraction, delta(13)C depletion values peaked around -5 per thousand and -9 per thousand after oral and intramuscular administration of HC, respectively, and around -6 per thousand after oral administration of C. In comparison, less impressive results were obtained when IRMS analysis focused on the ox-products of the minor metabolites.

Development and application of stable carbon isotope analysis to the detection of cortisol administration in cattle
F Andre, B Le Bizec, E Bichon, F Kieken, N Cesbron, F Monteau, S Pre

The use of anabolic agents in food-producing animals has been prohibited within the EU since 1988. The control of the illegal use of natural steroid hormones in cattle is still an exciting analytical challenge as no definitive method and nonambiguous analytical criteria are available. We have used gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) to demonstrate the administration of cortisol to cattle. The method consisted of an efficient combination between OASIS HLB solid-phase extraction (SPE), oxidation, SiOH SPE and semi-preparative high-performance liquid chromatography (HPLC) for glucocorticoid purification. By comparison of the 13C/12C isotopic ratio of the oxidised product of cortisol, i.e. 5β-androstane-3,11,17-trione (5βAAT), with an endogenous reference compound (ERC), dehydroepiandrosterone (DHEA), the differentiation of cortisol metabolite origin, either endogenous or exogenous, has been achieved. After treatment of an animal, the δ13CVPDB values of 5βAAT reached −30 to −32‰, whereas the δ13CVPDB values of DHEA remained at −25‰. A significant difference in the δ13CVPDB values between DHEA and 5βAAT was measurable over a period of 3 days after a single administration of cortisol to the animal.

Analysis of exogenous nandrolone metabolite in horse urine by gas chromatography/combustion/carbon isotope ratio mass spectrometry.
Journal of pharmaceutical and biomedical analysis (2007)
Masayuki Yamada, Kenji Kinoshita, Masahiko Kurosawa, Koichi Saito, Hiroyuki Nakazawa

Nandrolone (17beta-hydroxy-4-estren-3-one, NAD) is an endogenous steroid hormone; thus, the detection of its metabolites is not conclusive of NAD doping in racehorses. NAD doping control in male horses is based on the threshold, namely, the concentration ratio of 5alpha-estran-3beta,17alpha-diol (ETA) to 5(10)-estren-3beta,17alpha-diol (ETE). The ETA/ETE ratio of 1/1 was determined based on statistical data of authentic horses in International Federation of Horseracing Authorities. To individuals with complex metabolic disorders, however, such a threshold might not be applicable. The aim of this study was to establish an analytical method that discriminates endogenous steroids from exogenous ones in horse urine after NAD administration using gas chromatography/combustion/carbon isotope ratio mass spectrometry (GC/C/IRMS). Urine was sampled from NAD-administered and authentic horses. Ten millilitres of urine was hydrolyzed and subjected to liquid-liquid extraction and solid phase extraction. The residue of the extracts purified by HPLC was derivatized by acetylation. As a result of measurement of the (13)C/(12)C ratio (delta(13)C) by GC/C/IRMS, the delta(13)C values of ETA for NAD-administered and authentic horses were -32.20+/-0.35 per thousand and -27.85+/-0.75 per thousand (n=60), respectively. The detection limit of ETA in this GC/C/IRMS analysis was approximately 25 ng/ml. This study indicates that the measurement of delta(13)C by GC/C/IRMS enables us to discriminate exogenous ETA derived from NAD administration from endogenous ETA, proving that GC/C/IRMS is a useful technique to complement the ETA/ETE ratio.

An overview of the doping control analysis during the Olympic Games of 2004 in Athens, Greece
Analytica Chimica Acta (2006)
M. Tsivou, N. Kioukia-Fougia, E. Lyris, Y. Aggelis, A. Fragkaki, X. Kiousi, Ph. Simitsek, H. Dimopoulou, I.-P. Leontiou, M. Stamou, M.-H. Spyridaki, C. Georgakopoulos

This study summarizes the results obtained from the doping control analysis during the period of the XXVIII summer Olympic Games (30 July–29 August 2004). The analysis of all doping control samples was performed at the Doping Control Laboratory (DCL)—the World Anti- Doping Agency (WADA) Accredited Laboratory of Athens. Three thousand six hundred and seventeen tests were conducted in total throughout the games. In 23 specimens the presence of a prohibited substance was confirmed. Sixteen of those were related to anabolic agents. The screened results were confirmed with various mass spectrometry analytical techniques, such as gas chromatography/high resolution mass spectrometry (GC/HRMS), gas chromatography/mass spectrometry (GC/MS), gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) and liquid chromatography/mass spectrometry (ion trap) (LC/MS). The results of the first time applied screening and confirmatory procedures for the detection of recombinant human growth hormone in serum were also presented. Besides, 107 therapeutic use exemptions (TUE) were verified for glucocorticosteroid and beta2-agonist use.