Graham J. Trout

Carbon isotope ratio (δ13C) values of urinary steroids for doping control in sport

The detection of steroids originating from synthetic precursors in relation to their chemically identical natural analogues has proven to be a significant challenge for doping control laboratories accredited by the World Anti-Doping Agency (WADA). Endogenous steroid abuse may be confirmed by utilising the atomic specificity of gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) that enables the precise measurement of differences in stable isotope ratios that arise as a result of fractionation patterns inherent in the source of steroids. A comprehensive carbon isotope ratio (delta(13)C) profiling study (n=1262) of urinary ketosteroids is reported that demonstrates the inter-individual variation that can be expected from factors such as diet, ethnicity, gender and age within and between different populations (13 countries). This delta(13)C distribution is shown by principal component analysis (PCA) to provide a statistical comparison to delta(13)C values observed following administration of testosterone enanthate. A limited collection of steroid diol data (n=100; consisting of three countries) is also presented with comparison to delta(13)C values of excreted testosterone to validate criteria for WADA accredited laboratories to prove doping offences.

The detection of androstenedione abuse in sport: a mass spectrometry strategy to identify the 4-hydroxyandrostenedione metabolite.

Studies have shown that the administration of androstenedione (ADIONE) significantly increases the urinary ratio of testosterone glucuronide to epitestosterone glucuronide (T/E) - measured by gas chromatography/mass spectrometry (GC/MS) - in subjects with a normal ( approximately 1) or naturally high (>1) initial values. However, the urinary T/E ratio has been shown not to increase in subjects with naturally low (<1) initial values. Such cases then rely on the detection of C(6)-hydroxylated metabolites shown to be indicative of ADIONE administration. While these markers may be measured in the routine GC/MS steroid profile, their relatively low urinary excretion limits the use of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) to specifically confirm ADIONE administration based on depleted (13)C content. A mass spectrometry strategy was used in this study to identify metabolites of ADIONE with the potential to provide compound-specific detection. C(4)-hydroxylation was subsequently shown to be a major metabolic pathway following ADIONE administration, thereby resulting in urinary excretion of 4-hydroxyandrostenedione (4OH-ADIONE). Complementary analysis of 4OH-ADIONE by GC/MS and GC/C/IRMS was used to confirm ADIONE administration.

Strategies for rhepo Detection in Sport

This article examines available strategies for the detection of recombinant erythropoietin (rhEPO) abuse in sport. RhEPO was quickly recognized as an effective but hazardous performance-enhancing agent. In the absence of a valid procedure to detect rhEPO doping, at-competition health checks were introduced, which excluded athletes from competition when their hemoglobin or hematocrit values exceeded an arbitrary limit. This limited the danger to athletes, but did nothing to eliminate the use of rhEPO.Through the last decade, both direct and indirect methods for detecting rhEPO were investigated. No single indirect marker was found that satisfactorily demonstrated rhEPO use. A combination of blood and urine tests together formed the procedure and strategy approved by the International Olympic Committee (IOC) for detecting rhEPO use at the Sydney Olympics.However strategies for testing for EPO are as important as the developed laboratory analytical procedures. The use of extensive out-of-competition testing and analysis within the IOC accredited laboratory system is critical to any testing program. At-competition blood tests have merit as true health checks and will also be needed to detect acutely useful agents such as hemoglobin-based oxygen carriers. However the persistence of the “health check” rationale for on-site at-competition rhEPO testing has led to much wasted testing effort, as rhEPO use by athletes will rarely occur near to or at the time of the competition for fear of detection. Thus, direct testing methods (such as the rhEPO urine test) especially will fail due to the completed metabolism and elimination of administered rhEPO before the test, unless the international sporting federations use the information gathered to assist in targeted out-of-competition testing. This article discusses the limitations of testing at competition and proposed strategies for dealing with various phases of EPO doping in detail, concluding that no one single currently used strategy will detect all users of rhEPO.The development of strategies to diagnose rhEPO abuse may serve as a model to detect other biological agents.

Effects of recombinant human LH and hCG on serum and urine LH and androgens in men

Context  The administration of gonadotrophins is prohibited in sport but the effect in men of recently available recombinant hCG and LH on serum and urine concentrations of gonadotrophins and androgens has not been systematically evaluated in the antidoping context.

Screening for testosterone abuse in male athletes using the measurement of urinary LH, a revision of the paradigm.

The primary screening method for the detection of doping by athletes using synthetic versions of endogenous steroids such as testosterone relies on measurement of the ratio of testosterone (T) to epitestosterone (E) in urine. In 2005 the World Anti-Doping Agency (WADA) lowered the T/E value at which samples undergo further investigation from six to four. This has resulted in a large increase in the number of athletes with naturally elevated T/E ratios undergoing investigation without a corresponding increase in the number of proven doping offences involving testosterone.Our objective was to develop a new simple screening protocol that can, with high probability, not only distinguish athletes whose natural T/E values exceed four from those whose T/E values have been elevated by testosterone doping but also detect those athletes with naturally low T/E values that do not exceed four despite being administered testosterone.Testosterone (250 mg Sustanon) was administered weekly to a group of 47 young adult males for five weeks in a double-blind placebo controlled study and urine samples collected. The samples were analysed for steroid concentrations using GC/MS and for luteinizing hormone (LH) by immunoassay.The elevation of T/E that occurred in all subjects was accompanied by a significant reduction in urinary LH concentrations to levels that are rare in normal subjects.The appropriate measurement of urinary LH, with the measurement of T/E values, can markedly improve the efficiency of detection of doping with testosterone by male athletes, particularly those who have low natural T/E ratios.

Developments in Sports Drug Testing

AAustralian Sports Drug Testing Laboratory (ASDTL), Australian Government Analytical Laboratories, Pymble 2073, Australia. BNow at Research and Development, Australian Government Analytical Laboratories, Pymble 2073, Australia. CSchool of Chemistry, University of Sydney, Sydney 2006, Australia. DDepartment of Health Sciences, University of Technology, Sydney 2007, Australia. E Author to whom correspondence should be addressed (e-mail: graham.trout@agal.gov.au).