Ute Mareck

Factors influencing the steroid profile in doping control analysis.

Steroid profiling is one of the most versatile and informative screening tools for the detection of steroid abuse in sports drug testing. Concentrations and ratios of various endogenously produced steroidal hormones, their precursors and metabolites including testosterone (T), epitestosterone (E), dihydrotestosterone (DHT), androsterone (And), etiocholanolone (Etio), dehydroepiandrosterone (DHEA), 5alpha-androstane-3alpha,17beta-diol (Adiol), and 5beta-androstane-3alpha,17beta-diol (Bdiol) as well as androstenedione, 6alpha-OH-androstenedione, 5beta-androstane-3alpha,17alpha-diol (17-epi-Bdiol), 5alpha-androstane-3alpha,17alpha-diol (17-epi-Adiol), 3alpha,5-cyclo-5alpha-androstan-6beta-ol-17-one (3alpha,5-cyclo), 5alpha-androstanedione (Adion), and 5beta-androstanedione (Bdion) add up to a steroid profile that is highly sensitive to applications of endogenous as well as synthetic anabolic steroids, masking agents, and bacterial activity. Hence, the knowledge of factors that do influence the steroid profile pattern is a central aspect, and pharmaceutical (application of endogenous steroids and various pharmaceutical preparations), technical (hydrolysis, derivatization, matrix), and biological (bacterial activities, enzyme side activities) issues are reviewed.

Determination of 13C/12C ratios of endogenous urinary steroids : method validation, reference population and application to doping control purposes

The application of a comprehensive gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS)-based method for stable carbon isotopes of endogenous urinary steroids is presented. The key element in sample preparation is the consecutive cleanup with high-performance liquid chromatography (HPLC) of underivatized and acetylated steroids, which allows the isolation of ten analytes (11beta-hydroxyandrosterone, 5alpha-androst-16-en-3beta-ol, pregnanediol, androsterone, etiocholanolone, testosterone, epitestosterone, 5alpha-androstane-3alpha,17beta-diol, 5beta-androstane-3alpha,17beta-diol and dehydroepiandrosterone) from a single urine specimen. These steroids are of particular importance to doping controls as they enable the sensitive and retrospective detection of steroid abuse by athletes. Depending on the biological background, the determination limit for all steroids ranges from 5 to 10 ng/mL for a 10 mL specimen. The method is validated by means of linear mixing models for each steroid, which covers repeatability and reproducibility. Specificity was further demonstrated by gas chromatography/mass spectrometry (GC/MS) for each analyte, and no influence of the sample preparation or the quantity of analyte on carbon isotope ratios was observed. In order to determine naturally occurring (13)C/(12)C ratios of all implemented steroids, a reference population of n = 61 subjects was measured to enable the calculation of reference limits for all relevant steroidal Delta values.

Determination of the origin of urinary norandrosterone traces by gas chromatography combustion isotope ratio mass spectrometry

On the one hand, 19-norandrosterone (NA) is the most abundant metabolite of the synthetic anabolic steroid 19-nortestosterone and related prohormones. On the other hand, small amounts are biosynthesized by pregnant women and further evidence exists for physiological origin of this compound. The World Anti-Doping Agency (WADA) formerly introduced threshold concentrations of 2 or 5 ng of NA per ml of urine to discriminate 19-nortestosterone abuse from biosynthetic origin. Recent findings showed however, that formation of NA resulting in concentrations in the range of the threshold levels might be due to demethylation of androsterone in urine, and the WADA 2006 Prohibited List has defined NA as endogenous steroid. To elucidate the endogenous or exogenous origin of NA, (13)C/(12)C-analysis is the method of choice since synthetic 19-nortestosterone is derived from C(3)-plants by partial synthesis and shows delta(13)C(VPDB)-values of around -28 per thousand. Endogenous steroids are less depleted in (13)C due to a dietary mixture of C(3)- and C(4)-plants. An extensive cleanup based on two high performance liquid chromatography cleanup steps was applied to quality control and doping control samples, which contained NA in concentrations down to 2 ng per ml of urine. (13)C/(12)C-ratios of NA, androsterone and etiocholanolone were measured by gas chromatography/combustion/isotope ratio mass spectrometry. By comparing delta(13)C(VPDB)-values of androsterone as endogenous reference compound with NA, the origin of NA in doping control samples was determined as either endogenous or exogenous.

13C/12C ratios of endogenous urinary steroids investigated for doping control purposes.

In order to detect the misuse of endogenous anabolic steroids such as testosterone by athletes a total of n = 1734 suspicious urine samples were investigated by gas chromatography/combustion/isotope ratio mass spectrometry throughout the years 2005, 2006 and 2007. The (13)C/(12)C ratio of a target substance (androsterone, a testosterone metabolite) was compared to the (13)C/(12)C ratio of an endogenous reference compound (11beta-hydroxyandrosterone).N = 1340 samples were investigated due to elevated testosterone/epitestosterone ratios, with n = 87 (6.5%) exceptional findings regarding their isotopic ratios. An additional n = 164 samples were investigated because of elevated dehydroepiandrosterone concentrations, with n = 2 (1.2%) exceptional findings. The remainder were subjected to isotope ratio analysis because of elevated androsterone levels or because this was requested by sports federations.Significant differences between female and male samples were found for the (13)C/(12)C ratios of androsterone and 11beta-hydroxyandrosterone but not for samples taken in or out of competition.A further n = 645 samples originating from other World Anti-Doping Agency accredited laboratories, mainly throughout Europe as well as South America, South Africa and Southeast Asia, were investigated. The (13)C/(12)C ratios of the urinary steroids differ significantly for each geographical region, reflecting the dietary status of the individuals.The system stability over time has been tested by repeated injections of a standard solution and repeated processing of frozen stored blank urine. Despite a drift over time in absolute (13)C/(12)C ratios, no significant change in the difference of (13)C/(12)C (11beta-hydroxyandrosterone) minus (13)C/(12)C (androsterone) could be observed.

Doping-control Analysis of the 5α-reductase Inhibitor Finasteride: Determination of Its Influence on Urinary Steroid Profiles and Detection of Its Major Urinary Metabolite

5α-Reductase inhibitors such as finasteride are prohibited in sports according to the World Anti-Doping Agency. This class of drugs is used therapeutically to treat benign prostatic hyperplasia, as well as male baldness, by decreasing 5α-reductase activity. Accordingly, metabolic pathways of endogenous as well as synthetic steroids are influenced, which complicates the evaluation of steroid profiles in sports drug testing. The possibility of manipulating steroid excretion profiles and, presumably, to mask steroid abuse was investigated in 5 administration studies with use of finasteride at different doses, with and without coadministration of 19-norandrostenedione. The evaluation of urinary steroid profiles demonstrated the intense effect of finasteride on numerous crucial analytical parameters, in particular the production of 5α-steroids such as androsterone and 5α-androstane-3α,17β-diol, which was significantly reduced. In addition, the excretion of the main metabolite of norandrostenedione, norandrosterone, was significantly suppressed, by up to 84%, in elimination studies. For doping-control analysis the use of 5α-reductase inhibitors causes considerable problems because steroid profile parameters, which are commonly considered stable, are highly affected and complicate the detection of steroid abuse. In addition, the suppression of production and renal excretion of 5α-steroids such as 19-norandrosterone generated from anabolic agents such as 19-norandrostenedione may lead to false-negative doping-control results, because urine specimens are reported positive only when a threshold level of 2 ng/mL is exceeded. Finally, a method for the determination of the major urinary metabolite of finasteride (carboxy-finasteride) in routine doping-control screening with use of liquid chromatography-tandem mass spectrometry is described, allowing the detection of carboxy-finasteride for up to 94 hours in urine specimens collected after an oral administration of 5 mg of finasteride.

Determination of the prevalence of anabolic steroids, stimulants, and selected drugs subject to doping controls among elite sport students using analytical chemistry

Abstract Drug abuse by adolescents has been investigated in various surveys that reported correlations between age, gender, and activity. However, none of these studies included chemical analyses to help substantiate the statements of participants. In the present study, the urine specimens of 964 students (439 females, 525 males; mean age 22.1 years, s = 1.7), who applied to study sports sciences at university, were assessed for anabolic steroids, stimulants, and selected drugs prohibited in sports. In total, 11.2% of the urine specimens provided contained drugs covered by doping controls. The most frequently detected compound was the major metabolite of tetrahydrocannabinol (9.8%) followed by various stimulants related to amphetamine and cocaine (1.0%). Indications of anabolic steroid use were found in 0.4% of urine samples but originated from contraceptives containing norethisterone. The present study provided unambiguous data on the status quo of drug (ab)use by adolescents hoping for a career related to elite sport or sports sciences. No use of anabolic steroids was detected. However, evidence for stimulants and tetrahydrocannabinol administration was obtained, although not reported by any participant, which highlights the issue of under-reporting in surveys based solely on questionnaires.

Determination of salbutamol and salbutamol glucuronide in human urine by means of liquid chromatography-tandem mass spectrometry.

The determination of salbutamol and its glucuronide in human urine following the inhalative and oral administration of therapeutic doses of salbutamol preparations was performed by means of direct urine injection utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS) and employing d(3)-salbutamol and d(3)-salbutamol glucuronide as internal standards. Unconjugated salbutamol was detected in all administration study urine samples. Salbutamol concentrations following inhalation were commonly (99%) below 1000 ng/ml whereas values after oral administration frequently (48%) exceeded this threshold. While salbutamol glucuronide was not detected in urine samples collected after inhalation of the drug, 26 out of 82 specimens obtained after oral application contained salbutamol glucuronide with a peak value of 63 ng/ml. The percentage of salbutamol glucuronide compared to unconjugated salbutamol was less than 3%. Authentic doping control urine samples indicating screening results for salbutamol less than 1000 ng/ml, showed salbutamol glucuronide concentrations between 2 and 6 ng/ml, whereas adverse analytical findings resulting from salbutamol levels higher than 1000 ng/ml, had salbutamol glucuronide values between 8 and 15 ng/ml. The approach enabled the rapid determination of salbutamol and its glucuronic acid conjugate in human urine and represents an alternative to existing procedures since time-consuming hydrolysis or derivatization steps were omitted. Moreover, the excretion of salbutamol glucuronide in human urine following the administration of salbutamol was proven.

Temporal indication of cannabis use by means of THC glucuronide determination

According to the regulations of the World Anti-Doping Agency (WADA), the use of cannabinoids is forbidden in competition. In doping controls, the detection of cannabinoid misuse is based on the analysis of the non-psychoactive metabolite 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (carboxy-THC). The determination of values greater than 15 ng/mL in urine represents an adverse analytical finding; however, no accurate prediction of the time of application is possible as the half-life of carboxy-THC ranges between three and four days. Consequently the detection of carboxy-THC in doping control urine samples collected in competition might also result from cannabis use in out-of-competition periods. The analysis of the glucuronide of the pharmacologically active delta 9-tetrahydrocannabinol (THC-gluc) may represent a complementary indicator for the detection of cannabis misuse in competition.An assay for the determination of THC-gluc in human urine was established. The sample preparation consisted of liquid-liquid extraction of urine specimens, and extracts were analysed by liquid chromatography/tandem mass spectrometry (LC-MS/MS). Authentic doping-control urine samples as well as specimens obtained from a controlled smoking study were analysed and assay characteristics such as specificity, detection limit (0.1 ng/mL), precision (>90%), recovery ( approximately 80%), and extraction efficiency (90%) were determined.

Detection of dehydroepiandrosterone misuse by means of gas chromatography- combustion-isotope ratio mass spectrometry.

According to World Anti-Doping Agency (WADA) rules (WADA Technical Document-TD2004EAAS) urine samples containing dehydroepiandrosterone (DHEA) concentrations greater than 100 ng mL−1 shall be submitted to isotope ratio mass spectrometry (IRMS) analysis. The threshold concentration is based on the equivalent to the glucuronide, and the DHEA concentrations have to be adjusted for a specific gravity value of 1.020. In 2006, 11,012 doping control urine samples from national and international federations were analyzed in the Cologne doping control laboratory, 100 (0.9%) of them yielding concentrations of DHEA greater than 100 ng mL−1. Sixty-eight percent of the specimens showed specific gravity values higher than 1.020, 52% originated from soccer players, 95% were taken in competition, 85% were male urines, 99% of the IRMS results did not indicate an application of testosterone or related prohormones. Only one urine sample was reported as an adverse analytical finding having 319 ng mL−1 DHEA (screening result), more than 10,000 ng mL−1 androsterone and depleted carbon isotope ratio values for the testosterone metabolites androsterone and etiocholanolone. Statistical evaluation showed significantly different DHEA concentrations between specimens taken in- and out-of-competition, whereas females showed smaller DHEA values than males for both types of control. Also a strong influence of the DHEA excretion on different sport disciplines was detectable. The highest DHEA values were detected for game sports (soccer, basketball, handball, ice hockey), followed by boxing and wrestling. In 2007, 6622 doping control urine samples were analyzed for 3α,5-cyclo-5α-androstan-6β-ol-17-one (3α,5-cyclo), a DHEA metabolite which was described as a useful gas chromatography-mass spectrometry (GC-MS) screening marker for DHEA abuse. Nineteen urine specimens showed concentrations higher than the suggested threshold of 140 ng mL−1, six urine samples yielded additionally DHEA concentrations higher than 100 ng mL−1, none of them showing positive IRMS findings. These results should be taken into consideration in future discussions about threshold values for endogenous steroids in doping control.

Reporting and managing elevated testosterone/epitestosterone ratios—Novel aspects after five years' experience

The testosterone/epitestosterone (T/E) ratio was implemented as an indirect parameter for the detection of testosterone administration with an empirically established threshold value at T/E = 6. In 2005, the T/E reporting threshold was lowered from six to four. Between 2005 and 2009, 63 510 doping control urine samples were analyzed in the Cologne laboratory. A total of 1442 specimens (2.3%) showed a T/E > 4; 80 (5.5%) of which were tested positive by means of isotope ratio mass spectrometry (IRMS); and most of which (68) originated from strength sport disciplines. Specimens of high T/E ratio showed a much higher probability for being confirmed to contain exogenous testosterone using IRMS analysis than samples of low T/E values. Considering the small number of adverse analytical findings triggered by lowering the T/E reporting threshold (978 urine specimens with T/E ratios between 4 and 6 yielded only 4 (0.4%) positive IRMS findings) and the known limitations of the T/E ratio as discriminating parameter (UGT2B17 polymorphism), the currently mandatory approach shows only marginal overall efficiency. A more effective tool for the detection of the misuse of testosterone would be the implementation of individual reference ranges. Until athlete steroidal passports are available, it is suggested to exceed the threshold level for T/E from 4 to 6 and perform obligatory IRMS analysis for specimens showing T/E > 6. Further conditions triggering IRMS analysis could be suppressed luteinizing hormone (LH) values in males and disproportionate changes of relevant parameters in individual profiles evidently not resulting from ethanol consumption.