Thomas Piper

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.

Determination of 13C/12C ratios of urinary excreted boldenone and its main metabolite 5β-androst-1-en-17β-ol-3-one

Boldenone (androsta-1,4-dien-17beta-ol-3-one, Bo) is an anabolic steroid known to have been used in cattle breeding or equine sport as a doping agent for many years. Although not clinically approved for human application, Bo or its main metabolite 5beta-androst-1-en-17beta-ol-3-one (BM1) were detected in several doping control samples. For more than 15 years the possibility of endogenous Bo production in human beings has been discussed. This is a challenging issue for doping control laboratories as Bo belongs to the list of prohibited substances of the World Anti-Doping Agency and therefore the chance for false positive testing is significant. By GC/C/IRMS (gas chromatography/combustion/isotope ratio mass spectrometry) it should be possible to analyze the (13)C/(12)C ratio of either Bo or BM1 and to distinguish whether their source is endogenous or exogenous. Therefore a method was developed to determine the (13)C/(12)C ratios of Bo, BM1, pregnanediol, androsterone, etiocholanolone, and testosterone from a single urine specimen. The validity of the method was ensured by repeated processing of urine fortified with 2-50 ng/mL Bo and BM1. The specificity of the method was ensured by gas chromatography/mass spectrometry determinations. Out of 23 samples investigated throughout the last four years, 11 showed (13)C/(12)C ratios of Bo or BM1 inconsistent with an exogenous origin. Two of these samples were collected from the same athlete within a one-month interval, strongly indicating the chance of endogenous Bo production by this athlete.

Determination of 13C/12C ratios of endogenous urinary steroids excreted as sulpho conjugates

The application of a comprehensive gas chromatography/combustion/isotope ratio mass spectrometry-based method for the measurement of stable carbon isotopes of endogenous urinary steroids excreted as sulphates is presented. The key element in sample preparation is the consecutive cleanup with high-performance liquid chromatography of underivatized and acetylated steroids, which allows the isolation of seven analytes (pregn-5-ene-3β,17α,20α-triol, etiocholanolone, androsterone, epiandrosterone, dehydroepiandrosterone (DHEA), androst-5-ene-3β,17β-diol and androst-5-ene-3β,17α-diol) from a single urine specimen. These steroids are of particular importance to doping controls as they should enable the sensitive and retrospective detection of DHEA 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 the items, repeatability and reproducibility. The specificity was further demonstrated by gas chromatography/mass spectrometry 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 and urinary concentrations of all implemented steroids, a reference population of n = 67 subjects was measured to enable the calculation of reference limits for all relevant steroidal Δ values.The applicability of the developed method was tested by means of a DHEA excretion study. Despite the fact that orally ingested DHEA is preferentially converted into sulphated metabolites and that the renal clearance of sulphated steroids is slow, only the (13)C/(12)C ratio of EpiA demonstrated the potential to prolong the detection time for DHEA misuse.

Determination of the deuterium/hydrogen ratio of endogenous urinary steroids for doping control purposes

The development and application of a combined gas chromatography/thermal conversion/isotope ratio mass spectrometry (GC/TC/IRMS) method for D/H ratio determination of endogenous urinary steroids are presented. The key element in sample preparation was the consecutive cleanup with high-performance liquid chromatography of initially native and subsequently acetylated steroids. This strategy enabled sufficient cleanup off all target analytes for determination of their respective D/H values. Ten steroids (11beta-hydroxyandrosterone, 5alpha-androst-16-en-3alpha-ol, pregnanediol, androsterone, etiocholanolone, testosterone, epitestosterone, 5alpha-androstan-3alpha,17beta-diol, 5beta-androstan-3alpha,17beta-diol and dehydroepiandrosterone) were measured from a single urine specimen. Depending on the biological background, the determination limit for all steroids ranged from 10 to 15 ng/mL for a 20 mL specimen. The method was validated by application of linear mixing models on each steroid and covered repeatability and reproducibility. The specificity of the procedure was ensured by gas chromatography/mass spectrometry (GC/MS) analysis of the sample using equivalent chromatographic conditions to those employed in the GC/TC/IRMS measurement. Within the sample preparation, no isotopic fractionation was observed, and no amount-dependent shift of the D/H ratios during the measurement was noticed. Possible memory effects occurring during IRMS measurements were corrected by applying a simple rule of proportion. In order to determine the naturally occurring D/H ratios of all implemented steroids, a population of 18 male subjects was analyzed. Relevant mean Delta values among selected steroids were calculated which allowed us to study the metabolic pathways and production sites of all the implemented steroids with additional consideration of the corresponding (13)C/(12)C ratios.

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.

Combination of carbon isotope ratio with hydrogen isotope ratio determinations in sports drug testing

Carbon isotope ratio (CIR) analysis has been routinely and successfully applied to doping control analysis for many years to uncover the misuse of endogenous steroids such as testosterone. Over the years, several challenges and limitations of this approach became apparent, e.g., the influence of inadequate chromatographic separation on CIR values or the emergence of steroid preparations comprising identical CIRs as endogenous steroids. While the latter has been addressed recently by the implementation of hydrogen isotope ratios (HIR), an improved sample preparation for CIR avoiding co-eluting compounds is presented herein together with newly established reference values of those endogenous steroids being relevant for doping controls. From the fraction of glucuronidated steroids 5β-pregnane-3α,20α-diol, 5α-androst-16-en-3α-ol, 3α-Hydroxy-5β-androstane-11,17-dione, 3α-hydroxy-5α-androstan-17-one (ANDRO), 3α-hydroxy-5β-androstan-17-one (ETIO), 3β-hydroxy-androst-5-en-17-one (DHEA), 5α- and 5β-androstane-3α,17β-diol (5aDIOL and 5bDIOL), 17β-hydroxy-androst-4-en-3-one and 17α-hydroxy-androst-4-en-3-one were included. In addition, sulfate conjugates of ANDRO, ETIO, DHEA, 3β-hydroxy-5α-androstan-17-one plus 17α- and androst-5-ene-3β,17β-diol were considered and analyzed after acidic solvolysis. The results obtained for the reference population encompassing n = 67 males and females confirmed earlier findings regarding factors influencing endogenous CIR. Variations in sample preparation influenced CIR measurements especially for 5aDIOL and 5bDIOL, the most valuable steroidal analytes for the detection of testosterone misuse. Earlier investigations on the HIR of the same reference population enabled the evaluation of combined measurements of CIR and HIR and its usefulness regarding both steroid metabolism studies and doping control analysis. The combination of both stable isotopes would allow for lower reference limits providing the same statistical power and certainty to distinguish between the endo- or exogenous origin of a urinary steroid.

Degradation of urine samples and its influence on the 13C/12C ratios of excreted steroids

The degradation processes in deficiently stored urine samples are well investigated regarding steroid concentrations and diagnostic ratios, such as the quotient of testosterone divided by epitestosterone. In contrast, nothing is known about the influence on carbon isotope ratios (CIR) by inappropriate storage conditions. In general, it is assumed that degradation, i.e. deconjugation or dehydrogenation, does not change CIR and thus CIR can be used in cases where the steroid profile turns out to be invalid. Therefore, the CIR of urinary steroids was investigated in different urine samples during the course of degradation over a time period of six months. Several steroids excreted as glucuronides (androsterone (A), etiocholanolone (E), testosterone, pregnanediol (PD) and 5α- and 5β-androstane-3α,17β-diol) or sulfo-conjugated (A, E and androst-5-ene-3β,17β-diol (5EN17b)) were investigated together with their unconjugated correspondents (A, E, PD and 5EN17b) and the main dehydrogenation products (5α- and 5β-androstane-3,17-dion and androst-4-ene-3,17-dion). For this purpose, the exiting methods for CIR determination were extended and validated. In addition, the urinary concentrations of all investigated steroids were monitored. Particular attention was paid to dehydroepiandrosterone conjugated and unconjugated together with its degradation product 3α,5-cyclo-5α-androstan-6β-ol-17-one as here the strongest influence on CIR was expected.