Research projects

Code: 12A3JW 

Testosterone, an endogenous steroid banned by the World Anti-Doping Agency, is still one of the most widely used performance-enhancing drugs in the athletic community. Isotope ratio mass spectrometry (IRMS) is a commendatory technology used to detect urinary endogenous steroids by determination of the carbon isotope ratio (13C value). Recent investigation revealed that 13C labeled testosterone standard could be used to regulate the 13C value of testosterone products and lead to the false negative results in doping test. With increasing availability of the manipulated testosterone, significant efforts are needed by anti-doping authorities to develop methods to detect its abuse. 
The aim of this study is to establish method for the detection of testosterone manipulated with 13C labeled standards.  The key difference in structure between manipulated testosterone and non-manipulated testosterone is that certain amount of C-3 or/and C-4 are labeled by 13C in manipulated product which makes significant positive contribution to 13C value. In this study, chemical reactions will be explored to remove C-3 or C-4. For the manipulated testosterone, the 13C value of resultant of the reaction would be more negative than that of the reactant. By comparing the 13C values of reactant and resultant, the manipulated T could be detected. 

Main Findings: 

13C labeled standards can be used to control the δ13C value and produce manipulated testosterone (T) which cannot be detected by the current isotope ratio mass spectrometry (IRMS) method. The aim of this study is to establish a method for the detection of T manipulated with 13C labeled standards. A method was explored to remove the 13C labeled atom at C-3 in androsterone (Andro), the metabolite of T in urine, to produce the resultant (A-nor-5α-androstane-2,17-dione, ANAD). The difference in δ13C values between Andro and ANAD (∆δ13CAndro-ANAD, ‰) would change significantly in case manipulated T is abused. After administration of T manipulated with 13C labeled standards, urine samples were collected from volunteers and analyzed with the established method. Based on the population reference, the cut-off value of ∆δ13CAndro-ANAD for positive result was assessed. The developed method could be used to detect T manipulated with 3-13C labeled standards. The study results have been published in Analytica Chimica Acta (DOI: 10.1016/j.aca.2014.09.007).  

Code: 12A13OP 

Availability of reference materials is crucial in doping control since they are needed to confirm adverse analytical findings and to develop quantitative methods.  Most of the analytes included in doping control routine methods are phase I metabolites released to urine after enzymatic hydrolysis. However, the direct detection of phase II metabolites by LCMS/ MS is a common trend in doping control analysis. Therefore, the synthesis of phase II metabolites will become more important for the field in the next years. 
In previous WADA funded projects, our research group has identified several phase II metabolites which are useful for the detection of the misuse of several anabolic steroids. Among them, preliminary results indicate that 6β-hydroxyandrosterone glucuronide and 6βhydroxyetiocholanolone glucuronide can improve the detection of testosterone misuse. Several sulphate metabolites like epiboldenone sulphate or 17β-methyl-5β-androstane-3α,17α-diol 3αsulphate can help in the detection of boldenone and methyltestosterone misuse. The structure of these metabolites has been confirmed by their synthesis at small scale. This synthesis at small scale was necessary because none of the detected metabolites is commercially available. 
Larger amounts of the metabolites are needed for their complete characterization by NMR techniques. Besides, the availability of these larger amounts would also allow for the development of analytical approaches able to detect and confirm their presence in urine samples. 
Therefore, the goals of this project are (i) to synthesize at a larger scale several phase II metabolites previously identified as useful for the doping control field (6β-hydroxyandrosterone glucuronide, 6β-hydroxyetiocholanolone glucuronide, epiboldenone sulphate, 17α-methyl-5βandrostane-3α,17β-diol 3α-sulphate, 17β-methyl-5β-androstane-3α,17α-diol 3α-sulphate and 17βmethyl-5α-androstane-3α,17α-diol 3α-sulphate), (ii) to fully characterize them by well established techniques (NMR spectroscopy, mass spectrometry), (iii) to study their stability and (iv) to develop analytical approaches to quantify conjugated metabolites of testosterone (6βhydroxyandrosterone glucuronide, 6β-hydroxyetiocholanolone glucuronide). The synthetized standards will be distributed among WADA accredited laboratories. 

Main Findings: 

Availability of reference materials is crucial in doping control since they are needed to confirm adverse analytical findings and to develop quantitative methods. Most of the analytes included in doping control routine methods are phase I metabolites released to urine after enzymatic hydrolysis. However, the direct detection of phase II metabolites by LC-MS/MS is a common trend in doping control analysis. Therefore, the synthesis of phase II metabolites will become more important for the field in the next years.In previous WADA funded projects, our research group has identified several phase II metabolites which are useful for the detection of the misuse of several anabolic steroids. Among them, preliminary results indicate that 6β-hydroxyandrosterone glucuronide and 6β-hydroxyetiocholanolone glucuronide can improve the detection of testosterone misuse. Several sulfate metabolites like epiboldenone sulfate or 17β-methyl-5β-androstane- 3α,17α-diol 3α-sulfate can help in the detection of boldenone and methyltestosterone misuse. The structure of these metabolites has been confirmed by their synthesis at small scale since none of the detected metabolites is commercially available.

This project aimed to (i) synthesize selected phase II metabolites at large scale, (ii) characterize these metabolites (iii) develop analytical methodology for the quantitation of conjugated metabolites of testosterone by LC-MS/MS and (iv) distribute of the synthesized material among WADA accredited laboratories.

The main goals of this project have been reached. Firstly, we have synthesized at a larger scale 6β-hydroxyandrosterone glucuronide, 6β-hydroxyetiocholanolone glucuronide, epiboldenone sulfate, 17α-methyl-5βandrostane-3α,17β diol 3α-sulfate, 17β-methyl-5α-androstane-3α,17α-diol 3α-sulfate and 17βα-methyl-5α-androstane-3α,17α-diol 3α-sulfate. Additionally, we have fully characterized these metabolites by well established techniques like NMR spectroscopy and mass spectrometry. We have developed and validated an analytical method based on LC-MS/MS for the quantification of glucuronide metabolites of testosterone. Finally, we have distributed the synthesized sulfates among WADA accredited laboratories.

Code: 12D11VB 

No studies have examined the influence of factors such as exercise and dehydration on the pharmacokinetic profile of asthmatic drugs (beta2-agonists). Exercise may modify the metabolism and excretion of drugs, and sweat loss during exercise can result in dehydration, both affecting the urine concentration of the drug. Furthermore, most current pharmacokinetic studies of beta2-agonists have been performed in non-athlete European subjects, and are therefore not representative for neither athletes nor for the most of the human population not being Europeans. 
During the recent years WADA have changed the restriction towards beta2-agonists with the introduction of urinary thresholds instead of “therapeutic use exceptions” for the commonly used asthmatic drugs salbutamol, salmeterol and formoterol. A large number of studies have investigated the pharmacokinetic profile of these substances, providing support for the urinary threshold of 1000 ng/mL for salbutamol and of 30 ng/mL for formoterol. However, the background evidence for supporting these thresholds is to some degree still inadequate. Humans from various ethnical backgrounds respond different to drugs. 
The genetic variations and impact of exercise and fluid intake are of crucial relevance when it comes to determination of urinary thresholds on the prohibited list. Today, little are known of the pharmacokinetic and pharmacodynamic profile of asthmatic drugs in response to exercise or of genetic variations. It is not unreasonable to assume that therapeutic use of asthmatic medication could result in urine concentrations above the urinary threshold limit of salbutamol, and hence a false positive doping test. If this is the case, the current threshold on the prohibited list may be too low and should be changed. 
The purpose of this study is to examine the pharmacokinetic and pharmacodynamic profile of inhaled beta2-agonists in athletes’ blood and urine during exercise and dehydration, as well as the variations between ethnical groups.

Main Findings: 

The purpose of the research proposal was to investigate the influence of exercise and deshydration on the pharmacokinetics of therapeutic inhaled salbutamol and terbutaline in healthy well-trained men. In study I, we investigated pharmacokinetics of salbutamol after inhalation of the maximal WADA-permitted dose (8×200 μg) during three conditions: exercise (EX), exercise+dehydration (EXD) and rest (R). Exercise consisted of 75 min cycling at 60% of VO2max and a 20-km time-trial. Fluid intake was 2300, 270, and 1100 mL during EX, EXD, and R, respectively. The 2016 WADA decision limit (1200 ng/mL) for salbutamol was exceeded in 23, 31, and 10% of the urine samples during EX, EXD and R, respectively, when unadjusted for USG. When adjusted for USG, the corresponding percentages fell to 21, 15, and 8%. During EXD, mean urine concentrations of salbutamol exceeded (1325±599 ng/mL) the decision limit 4 h after administration when unadjusted for USG. Our data from study I demonstrated a high risk of AAFs in urine samples of salbutamol the first 6 hours after inhalation during EXD and EX with 28 and 20% samples being above the threshold limit for salbutamol, respectively. Adjustment of samples to a USG of 1.02 g/mL reduced the amount of false positive AFFs. Nine of 13 subjects had urine samples that exceeded the decision limit, hence being false positive. In conclusion, exercise and dehydration affect urine concentrations of salbutamol and increase the risk of Adverse Analytical Findings in samples collected after inhalation of that maximal permitted (1600 μg) for salbutamol. This should be taken into account when evaluating doping cases of salbutamol. 
In study II, we investigated pharmacokinetics of terbutaline after inhalation of 8×500 μg as a single dose during three conditions: Exercise in hot ambient conditions (30-35°C)(EXH), exercise in normal ambient conditions (20-25°C)(EX), and rest (20-25°C)(R). Exercise consisted of 130 min at various intensities. When unadjusted for USG, urine concentrations of terbutaline after 4 h were different in the order EXH>EX>R (P≤0.01). When unadjusted for USG, urine concentrations of terbutaline were 299±151 ng/mL higher (P≤0.001) after 4 h compared to adjusted concentrations in EXH. In conclusion, exercise in hot ambient conditions results in higher urine concentrations of terbutaline. This should be taken into account when evaluating doping cases of terbutaline. 
Our observations from study I & II clearly indicate that dehydration due to insufficient fluid intake or exercise in hot ambient conditions increases urine concentrations of beta2-agonists in urine samples that are unadjusted for USG. While adjustment of urine samples for USG also has some limitations, the present findings suggest that it may be relevant to consider USG of beta2-agonist urine samples in doping control. 

Code: 12A5CG

The number of peptide hormones, growth hormones and related substances which are required to be tested for are consistently increasing. The main catalyst for this fact is the marketing of products which have had limited data published in medical journals and are then produced and sold via internet websites. This makes them easily accessible and a concern for fair play in sport as well as the health of the athlete as the products may not have therapeutic use approval in humans and can be of poor quality.

In general the analysis of peptide hormones has been addressed one by one with many of the techniques requiring large volumes of urine and complicated extraction protocols. Recently several publications have multiplex methods to include a selection of peptides of interest to antidoping laboratories. These methods have involved either a direct analysis of urine samples for compounds expected at high concentrations or used antibodies for purification to enable lower detection limits.

Particular areas have been identified as playing an important role for the development of a multi-residue analysis technique; extraction/concentration including the use of two dimensional (2D) liquid chromatography, availability of standards and metabolites of peptide hormones. The extraction/concentration of the samples will be examined include solid phase extraction (cartridges/pipette tips), magnetic beads with antibodies, molecular weight cut-off filtration and 2D liquid chromatography. Many of the peptides which are sold over the internet are not available from suppliers of standard materials which makes it more difficult for laboratories to provide testing methodologies. This project includes making available standards suitable for laboratories to develop method with for testing procedures. An in vitro enzyme assay will also be setup with kidney microsomes to be applied to new peptide based drugs to determine the metabolites that are likely to be found in urine.

Main Findings

For over a decade the Australian Sports Drug Testing Laboratory (ASDTL) has been analyzing black market seizures for the Australian Border Force. Many different peptide hormone products have been identified and include Gonadotropin Releasing Hormone (GnRH) and analogues (eg. triptorelin), Growth Hormone Releasing Hormone (GHRH) and analogues (eg. CJC-1295), Growth Hormone Secretagogues (GHS) and mimetics (eg. ipamorelin) and Growth Hormone Releasing Peptides (eg. GHRP-6). Many of these substances continue to be confiscated at the border which means the risk of abuse by athletes in Australia, as well as other in countries around the world remains. The detection in bodily matrices is important, not only for ensuring fairness in sport but also ensuring the health and wellbeing of athletes.

As instrument sensitivity improved, publications were emerging outlining ‘dilute and shoot’ methods for small molecules. Given the difficulties encountered using SPE with the increasing list of peptides being analysed, a ‘dilute and shoot’ method was developed and validated where diluted samples were passed through Agilent Captiva ND Lipid plate to remove interferences. This methodology was superseded with the implementation of ionKey/MS systems and SPE sample preparation which can analyse approximately 50 small peptides, metabolites and peptide mimetics. Limits of detection for the majority of sample range between 0.1-0.5 ng/mL with recoveries between 20-99%. The workflow for the small peptide should also allow the addition of conjugated small molecules and the development will be included in future planned work.

The most significant impact of this project has been the production of reference material and the availability of these to WADA Laboratories. Some of the materials are available from commercial providers but the identification and production of new materials in particular metabolites can be considerably more responsive when initiated by a WADA laboratory than a commercial provider. The ASDTL now has available 60 parent and metabolites peptides.

Code: T11A03VB

Human chorionic gonadotropin (hCG) may be abused by male athletes in sports and 1s included in the banned substance list of the Wortd Anti-Doping Agency.The goal of this prej!;l.ct Is t9 develop an new analytical tool for the detection of hCG In eith!etes biological samples. In particular. we wm develop an isotopically-labeled version of hCG (PSAQ standard) to be used as qUi,,ritificaUon reference in mass spectrometry (MS)-based assays. ParUli:IJlarly, we w ii develop optimized protocols for expression, stable-Isotope-labelling and purification of hum?n choriogona9otropin (hCG).

hCG is an heterodfmer protein composed of an a chain (common to thyrotropin, lutropin and follitropin), and a ti chain. which confers its specific biplogical activity_ The a and (i subunM are non-covalently linked and carry several dtsulfide bonds (6 disulfide bonds per subunit). The 2 chains also harbour carbohydrate moieties. 2 N-glycosylations and 4 0-g!ycosylatlo s on the fi subunit and 2 N-glycosylatlp11s on the a subunit. For optimal assay accuracy and reliablUty. the PSAQ standard will be developed as an a? heterodimer. The PSAQ heterodimer will be purified and controlled for Isotope incorporation. MS-based analyses will also be perfomed to check the sequence, structure and post-translational modifications of the standard

Main findings

This program was a feasibility study aiming at developing expression, isotope-labelling and purification protocols to produce isotopically-labeled human choriogonadotropin PSAQ standard. Choriogonadotropin protein is a heterodimer composed of an alpha chain, which is also common to thyrotropin, lutropin, follitropin, and a beta chain, which confers its specific biological activity. The alpha and beta subunits are non-covalently linked and carry numerous disulfide bonds. The 2 chains also harbour carbohydrate moieties: 2 N-glycosylations and 4 O-glycosylations on the beta subunit and 2 Nglycosylations on the alpha subunit. The first step of evaluating the expression of a choriogonadotropin in human cell free expression system was failed. In a second step of the program, each subunit (alpha and beta) was cloned with its signal peptide into our dedicated mammalian expression vector. The DNA sequence coding for the poly-His purification tag was added at the C-terminal end of sequence coding for subunit alpha protein. Then both expression vectors coding for the alpha and beta chains were co-transfected in HEK 293 cells. The choriogonadotrophin heterodimer was successfully expressed and purified. The purity was estimated to be greater than 90% at this stage. The standard was treated with PNGase F which removes N-linked glycosylations. Alpha and beta subunits sequences were verified by LC-MS/MS analyses after reduction/alkylation and in-gel trypsin digestion. Sequences coverage was higher than 50% with 3 specific peptides detected for each subunit. Stable isotope incorporation was determined using MS data generated by LC-MS/MS analyses. This experiment confirmed that the isotope incorporation of arginine and lysine was greater than 98% with limited Arginine-to-Proline conversion.

Code: T11A02MT

Erythropoiesis-stimulating agents (ESAs) have been frequently detected in doping control samples in the past and are among the illicitly used drugs that are most often mentioned by confessing athletes. New ESA therapeutics represent new options for cheating athletes and new challenges for sports drug testing authorities as comprehensive detection assays are required. With the introduction of the recently approved EPO-mimetic drug Omontys (formerly referred to as Hematide and Peginesatide), a first peptidic ESA is principally available that necessitates complementary analytical methods as Omontys will not be detectable with conventional EPO analytical methodologies. First successful procedures for the analysis of Omontys from plasma, serum, and dried blood spots were recently reported; however, the most frequently available doping control samples are urine specimens. Since pharmacokinetic studies have demonstrated that a significant amount of Omontys is renally excreted, the transfer of the methodology to urine and the analysis of proof-of concept samples from human in-vivo studies are desirable.

In the present study, the completion of a detection assay for Omontys from urine samples by means of liquid chromatography-mass spectrometry is aimed. Pilot studies have demonstrated the capability of extracting, enzymatically hydrolysing and subsequently measuring a proteotypical peptide of Omontys from spiked urine specimens; in order to apply the methodology to authentic doping control samples, the method will be fully characterized and an administration study with the EPO-mimetic agent will be conducted with six human volunteers. The in-vivo derived samples will be measured with the developed assay to demonstrate its utility for the determination of the prohibited drug in authentic specimens.

Main findings

PEGylation of protein therapeutics by covalently attaching polyethylene glycol (PEG) polymers results in an increased molecular mass and, therefore, slower renal clearance, reduced proteolytic susceptibility, and decreased immunogenicity. Endogenously, PEGylated substances do not exist and, hence, the concept of targeting PEGylated compounds as an initial testing approach towards prohibited substances was considered. In this research project, different strategies including immunoaffinity purification & LC-MS/MS, commercial PEG-specific ELISAs, and SDS-PAGE in combination with iodine staining were tested to assess the capability of these approaches to allow for multiplexed detection of PEGylated proteins in doping control serum samples. In the course of the studies, ELISA kits and iodine staining were found to be of limited specificity. In addition, the diversity of PEGylations regarding size and composition resulted in limited recognition of PEGylated model compounds by anti-PEG antibodies used for immunoaffinity purification, suggesting that (to date) testing merely for the presence of PEG polymers in human serum does not facilitate sports drug testing approaches per se. However, Pegvisomant (i.e. PEGylated and sequence-modified human growth hormone) was successfully detected in serum samples with the employed methodologies, which demonstrates that in general mass spectrometric assays can be established if antibodies binding with appropriate efficiency to a broader spectrum of PEG conjugates are available.

Code: R11C01TF

We will analyze the effects of HGH and erythropoietin to search for genetic signatures of exposure to those doping agents. We have obtained results from several individual WADA-supported laboratories and will carry out extensive comparisons to identify genes whose expression is indicative of exposure to HGH or erythropoietin.

Main findings

These two related studies were designed to test the concept that exposure of humans to doping agents disturbs the normal expression of many of the 20,000 genes in human cells and whether those changes can be used as a rigorous proof or a “signature” of exposure to specific doping agents. These kinds of studies are made possible by modern genetic techniques that allow an estimate of the extent of expression of all human genes on a single square inch silicon chip. Our studies also were designed to determine if several studies carried out under different conditions in different laboratories could easily be analyzed even though they used slightly different techniques and methods of analysis. This method of analysis of multiple and slightly different sets of data from different studies is called “meta-analysis” and examples of meta-analysis have been successful in many other kinds of research. We therefore carried out meta-analysis of three independent studies of the effects on gene expression in blood samples from athletes of administration of human growth hormone (HGH) and three separate studies of the effects of erythropoietin (Epo) or hypoxia in the blood cells of human athletes and in mice.

As expected, we found that the genes that are expressed incorrectly after exposure to HGH and Epo are different from each other. However, we also discovered that the separate studies identified only a small number of genes that were disturbed in the same way in all three studies, leading to a conclusion that those genes are probably not specific “signatures” for exposure to HGH or Epo. We have concluded that these results could mean that these drugs do not cause significant changes in gene expression in blood cells. We prefer an explanation that the three separate HGH studies and the diverse Epo/hypoxia differed in many details that introduced too many slight differences in the timing of testing, dosages, methods of tissue preparation and methods of analysis that probably have hidden the gene expressions shared by the different studies. We are impressed that in other very recent studies supported by WADA in which one laboratory has carried out very careful studies of humans exposed to Epo, reproducible changes have been found that consistent with a genetic “signature” for exposure to Epo, demonstrating that extensive and well controlled single studies under some conditions can be an effective approach to identifying the genetic changes in blood samples or other tissues. We propose that a reasonable next step is to carry out a similar study with HGH exposure and compare results from such a study with those obtained in our meta-analysis. We are confident the overall concept of genetic signatures of doping is a correct and robust one and will add important new tools to the prevention and detection of doping.

Code: R11A01MT

The discovery and implementation of the long-term metabolite of metandienone, namely 17β-hydroxymethyl-17α-methyl-18-norandrost-1,4,13-trien-3-one to routine doping controls resulted in hundreds of adverse analytical findings for metandienone worldwide and impressively demonstrated that prolonged detection periods significantly increase the effectiveness of sports drug testing. For oxandrolone and other 17-methylated steroids, analogues to this metabolite have been described, but comprehensive characterization and pharmacokinetic data were still missing and dersirable. In this project, the synthesis of the two epimeric oxandrolone metabolites 17β-hydroxymethyl-17α-methyl-18-nor-2-oxa-5α–androsta-13-en-3-one and 17α-hydroxymethyl-17β-methyl-18-nor-2-oxa-5α–androsta-13-en-3-one using a fungus (Cunninghamella elegans) based protocol was conducted followed by full characterization of the obtained reference material by means of liquid chromatography-nuclear magnetic resonance spectroscopy and -high resolution/high accuracy mass spectrometry. To ensure a specific and sensitive detection in athlete’s urine samples, different analytical approaches were followed, such as LC-MS/MS (QqQ and Q-Orbitrap) and GC-MS/MS to detect and identify the new target analytes. The applied methods have demonstrated good specificity and no significant matrix interferences. Linearity (R2 > 0.99) was tested and precise results were obtained for the detection of the analytes (CV < 20%). Limits of detection (S/N) for confirmatory and screening analysis were estimated at one and two nanogram per milliliter of urine, respectively. The assay was applied to oxandrolone post-administration samples to obtain data on the excretion of the different oxandrolone metabolites. The studied specimens demonstrated significantly longer detection periods for the new oxandrolone metabolites compared to commonly targeted metabolites such as epioxandrolone or 18-nor-oxandrolone, presenting a promising approach to improve the fight against doping.

Main findings

According to the WADA statistics, anabolic androgenic steroids are still the most frequently detected substances in sports drug testing. Nevertheless, for some anabolic steroids as oxandrolone the number of positive findings is relativly low despite of very potent anabolic activity and only weak side effects making the steroid very attractive for cheating athletes. To improve sports drug testing and to develop comprehensive analytical strategies for an effective detection of an oxandrolone abuse, two isomeric oxandrolone metabolites Ox M1 and its epimer Ox M2 were successfully synthesized and fully characterized. Within an oxandrolone post administration study, this reference material was applied to obtain data on approximate detection windows. The first time, it has been demonstrated that Ox M1 and M2 provided significantly prolonged detection periods compared to commonly targeted metabolites such as epioxandrolone or 18-nor-oxandrolone. Based on these results, a very comprehensive, sensitive and reliable methodology based on LC-(ESI)-MS/MS and GC-MS/MS for screening and confirmatory analysis of the new long term metabolites Ox M1 and M2 was developed presenting a powerful tool in the fight against doping.

Code: 11A1JV 

When metabolites of nandrolone, boldenone or formestane are found at low concentrations in doping control tests, a further CIRMS-based analysis is compulsory in order to be sure that steroid misuse has occurred. For each analyte, the factors that could generate such findings are diffrent, but in all cases the urinary concentrations always range between 1 and 20 ng/mL. Due to the low concentration of the analytes and the small contribution of 13C to the analyte molecule, an important level of selectivity and sensitivity is necessary to characterize a 13C difference.  Therefore, the sample must be submitted to a long time-consuming sample preparation method. 
Nevertheless, to improve detection capabilities, some laboratories include a standalone clean-up step based on the use of high performance liquid chromatography (HPLC). The present project attempts to attain these objectives by means of on-line coupling of liquid chromatography and gas chromatography (LC-GC) using the TOTAD interface, developed and patented by the UCLM research group. This analytical methodology allows the complete LC fraction containing the analytes to be transferred to the GC and, moreover, provides a better signal/noise ratio because it provides cleaner extract. These aspects, will presumably improve sensitivity by one or two orders of magnitude. The project will focus on developing an analytical method in which the hydrolysed extract is derivatised (off-line) and analysed by LC-GC. Two different detectors will be used, leading to LC-GC-MS and/or LC-GC-C-IRMS coupling. Automation of the system by using an autosampler will also be attempted. The objective of the present project is to develop a sensitive, automatic, robust and reliable analytical method to be used for the analysis of the mentioned steroids in urine. The method should allow the detection limits required to be reached, thereby providing a powerful tool for the efficient control of the misuse of anabolic steroids. 

Main Findings: 

It has been developed a sensitive, automatic, robust and reliable method for analyzing steroid in a urine using on line coupled LC-GC-C-IRMS (liquid chromatography-gas chromatography-combustion isotope ratio mass spectrometry) with the TOTAD (Through iven Transfer Adsorption Desorption)  interface to discriminate between the endogenous or exogenous origin of the same, and by on-line coupled LC-GC-MS to confirm the purity and the identity of the steroid peaks. Urine samples spiked at 5 ng/mL or 10 ng/mL with the steroid to be analysed were used. Of each urine sample 20 mL was hydrolyzed, extracted, purified by a first RPLC step of un-derivatized steroids, derivatized to acetyl­steroids and, finally, submitted to on-line LC-GC with CIRMS or MS detection. In the coupled system a second RPLC cleaning of derivatized steroids is applied, and the l!...C fractions containing the analytes are completely transferred to GC-CIRMS or GC-MS. 
l!...C-GC-CIRMS methods were devefoped for the analysis of 19-Norandrosterone (19-NA) and for Bodenone (Bo) and its main metabolite (BoM). The method failed in the case of 19-NA due to an interfering compound. The derivatization of 
Formestane (F) did not work properly. so that the developed method is not applicable to F analysis. In the case of Bo and BoM the method was developed successfully. The volumes of the fractions transferred from LC to GC through a fraction collector were 1000 and 900 ftL, respectively. 138Cyp08 corrected values for Bo and BoM, as well as the /:J.8 in relation to the reference compound Pregnandiol (PD), clearly indicated the exogenous origin of the steroids. The Standard Deviations of the 138CVPoB values ranged from 0.85 to 1.11 for the entire method. A confirmatory on-line LC-GC-MS analytical method was developed for Bo and BoM. Relative Standard Deviations of the absolute peak areas were below 8%, except when Bo was spiked at 10 ng/L. Detection Limits were 0.5 ng/mL for Bo and 0.05 ng/mL for BoM in full scan. When 3 ions were selected, detection limits were 0.07 and 0.008 ng/mL respectively. 
For the first time, an analytical method involving the coupling of LC-GC with C­IRMS has been developed. The presented methods permit the origin of urinary Bo and BoM to be identified as endogenous or exogenous. The sensitivity is substantially improved compared with currently used methods, allowing the detection limits set by WADA to be attained.  The advantages of LC-CG coupling open uo the possibility of extending the methodology to other steroids and to other compounds currently analysed by GC. The high sensitivity achieved should permit the amount of the urine necessary for such analyses to be decreased. The LC-GC-C-IRMS method developed for the analysis of Boldenone has been published. Presumably, the LC-GC-MS method for the analysis of boldenone will be published shortly. 

Summary
The qualitative study draws conclusions from interviews with bodybuilders from England who practice doping behaviors and, within the framework of moral disengagement, identifies six ways in which an athlete justifies their to dope. The study provides a better understanding of the process of rationalizing doping and can help inform strategies to educate athletes and prevent moral disengagement.

Methodology

The study follows a cross-sectional research design. The researchers conducted interviews with sixty-four (64) bodybuilders from England who dope and, through deductive reasoning, identified ways in which moral disengagement can clarify the reasons for which an athlete decides to practice doping behaviors.

Results

The researchers identified six ways of justifying doping behaviours: 
• Moral justifications, in which the rationalization is based on downplaying the impact of doping; 
• Euphemistic labeling, i.e., speaking of drugs in such a way that makes them seem safer and less risky, e.g., referring to steroids as “juice”; 
• Advantageous comparison, in which an athlete declares doping reasonable because they are not smoking or drinking alcohol; 
• Displacement of responsibilities, i.e., “everyone else is doing it, so I have to”; 
• Diffusion of responsibility, i.e., “if everyone is doing it, it must be right”; 
• Distortion of consequences, in which an athlete deemphasizes the degree of harm caused by doping.

Significance for Clean Sport

The study examines moral disengagement and its role as a set of behaviors that athletes adopt to rationalize doping. The results provide a toolkit for identifying the reasons to dope within the proposed framework and help design strategies to counteract them. Research conducted by Boardley and Dewar can be used to inform educational approaches aimed at preventing moral disengagement. 

Doping in bodybuilders: a qualitative investigation of facilitative psychosocial processes

Moral disengagement and associated processes in performance-enhancing drug use: a national qualitative investigation