Günter Gmeiner

Erythropoietin and Analogs

Erythropoietin (EPO), a glycoprotein hormone, stimulates the growth of red blood cells and as a consequence it increases tissue oxygenation. This performance enhancing effect is responsible for the ban of erythropioetin in sports since 1990. Especially its recombinant synthesis led to the abuse of this hormone, predominatly in endurance sports. The analytical differentiation of endogenously produced erythropoietin from its recombinant counterpart by using isoelectric focusing and double blotting is a milestone in the detection of doping with recombinant erythropoietin. However, various analogous of the initial recombinant products, not always easily detectable by the standard IEF-method, necessitate the development of analytical alternatives for the detection of EPO doping. The following chapter summarizes its mode of action, the various forms of recombinant erythropoietin, the main analytical procedures and strategies for the detection of EPO doping as well as a typical case report.

Carbon isotope ratio determination and investigation of seized testosterone preparations

In the present study, the content of a number of black market testosterone products collected in Austria has been analyzed. Additionally, (13) C/(12) C ratios were measured for testosterone in the products after cleavage of the testosterone ester. The aim was to determine whether some of these products had similar (13) C/(12) C ratios to those normally found for endogenous testosterone, which could prevent a positive isotopic ratio mass spectrometric (IRMS) finding in doping control. Moreover, it was investigated to what extent the preparations contained the masking agent epitestosterone, in order to lower the testosterone/epitestosterone (T/E) ratio in urinary steroid profiles. Out of 30 analyzed products, the declared ingredients differed from the actual content in 10 cases. Epitestosterone, however, could not be found in any of the products. The products displayed δ(13)C(VPDB) values between -23.6 and -29.4‰. For more than half of these products, the values were within a range reported for endogenous urinary steroids.

Use of dried blood spots in doping control analysis of anabolic steroid esters.

Dried blood spot (DBS) sampling, a technique for whole blood sampling on a piece of filter paper, has more than 50-years tradition, particularly in the diagnostic analysis of metabolic disorders in neonatal screening. Due to the minimal invasiveness, straightforwardness, robustness against manipulation and fastness DBS sampling recommends itself as an advantageous technique in doping control analysis. The present approach highlights the development of a screening assay for the analysis of eight anabolic steroid esters (nandrolone phenylpropionate, trenbolone enanthate, testosterone acetate, testosterone cypionate, testosterone isocaproate, testosterone phenylpropionate, testosterone decanoate and testosterone undecanoate) and nandrolone in DBS. The detection of the intact esters allows an unequivocal proof of the administration of conjugates of exogenous testosterone and its derivatives. Precise, specific and linear conditions were obtained by means of liquid chromatography high resolution/high accuracy mass spectrometry. Sensitivity in the low ppb range was accomplished by the preparation of the methyloxime derivatives of the target compounds. Labeled internal standards (d3-nandrolone, d3-nandrolone caproate and d3-nandrolone undecanoate) were applied to compensate for the broad range in chain length of the esters. The assay presented here outlines the application of DBS for the analysis of anabolic steroid esters in doping controls for the first time providing great potential to simplify the proof of exogenous administration of testosterone.

Screening of testosterone esters in human plasma

The detection of an intact ester of testosterone in plasma is leading towards unequivocal proof of the administration of exogenous testosterone. In the current study, a sensitive screening method for the detection of nine testosterone esters in human plasma was developed. By preparing oxime derivatives of intact testosterone esters, the sensitivity of the assay was increased. Furthermore, the method included liquid-liquid extraction (LLE) as sample clean-up, as well as online separation of the target analytes from the derivatization solution. The analysis was performed by liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS). The method developed herein is simple and rapid, and was validated according to World Anti-Doping Agency (WADA) guidelines.

Defending Dynepo detection

Doping analysis as the detection of prohibited substances in an athletes bodily specimen is not solely a scientific task; anti-doping scientists are challenged by the athletes experts and have to defend their results in front of arbitration panels such as the Court of Arbitration in Sports (CAS). Compliance with the internationally accepted standards issued by the World Anti-Doping Agency (WADA) is commonly the main aspect to prove and demonstrate. Taking the example of four cases of doping with Epoetin delta (Dynepo) in endurance disciplines like marathon, triathlon, and cycling, the challenges and experiences in court and the argumentation lines of the defence experts are discussed. In all cases, doping with Epoetin delta was detected by two methods: isoelectric focusing (IEF) and SDS-PAGE. Epoetin delta is known to be produced in a human fibrosoma cell line. The slightly more abundant bands alpha and beta result in a short detection window using IEF analysis alone. With the additional complementary information obtained by SDS-PAGE analysis, data interpretation and defence in court is facilitated.

Evaluation of longitudinal steroid profiles from male football players in UEFA competitions between 2008 and 2013

Testosterone and related compounds are the most recurrent doping substances. The steroid profile, consisting of the quantification of testosterone and its metabolites, has been described as the most significant biomarker to detect doping with pseudo-endogenous anabolic steroids. The steroidal module of the Athlete Biological Passport (ABP) was launched by the World Anti-Doping Agency (WADA) in 2014. To assess the value of introducing the module to its anti-doping programme, the Union of European Football Associations (UEFA) decided to analyze retrospectively the steroid profile data of 4195 urine samples, collected from 879 male football players and analyzed in 12 WADA-accredited laboratories between 2008 and mid-2013. This study focused on the evaluation of T/E ratios. The coefficient of variation (CV) and the adaptive model were the two statistical models used to study the longitudinal follow-up. A CV of 46% was determined to be the maximal natural intra-individual variation of the T/E when the sequence consisted of single data points analyzed in different laboratories. The adaptive model showed some profiles with an atypical T/E sequence and also enabled an estimate of the prevalence of external factors impacting the T/E sequences. Despite the limitations of this retrospective study, it clearly showed that the longitudinal and individual follow-up of the T/E biomarker of the players is a good tool for target testing in football. UEFA has therefore decided to implement the steroidal module of the ABP from the start of the next European football season in September 2015. Copyright

Synthesis and identification of hydroxylated metabolites of the anti‐estrogenic agent cyclofenil

The detection of metabolites of the anti-estrogenic substance cyclofenil, listed on the World Anti-Doping Agency (WADA) Prohibited List since 2004 is described. Target substances are hydroxylated metabolites, bearing an aliphatic hydroxyl group either in the 2-, 3- or 4-position of the aliphatic ring, in addition to the phenolic functions on the aromatic rings. Structural identification used NMR as well as high-resolution mass spectrometry after nano-electrospray ionisation (ESI). Unambiguous detection of all three synthesised cyclofenil metabolites M1-M3 was done using gas chromatography for separation and electron ionisation mass spectrometry for detection of the per-silylated compounds in comparison with a reference urine deriving from an excretion study within the WADA 2007 Educational Programme.

A facile and high yielding synthesis of 2,2,3,4,4-d5-androsterone-β-d-glucuronide—an internal standard in dope analysis

Abstract A facile six-step synthesis of 2,2,3,4,4-d5-androsterone-β- d -glucuronide (1) starting from epiandrosterone (2) in 63% yield is described and compared with several alternative synthetic pathways. Compound 1 can be used as an internal standard in screening procedures for anabolic steroids to monitor the hydrolysis step of the steroid glucuronides prior to gas chromatography–mass spectrometry (GC–MS) analysis. Thus, a time consuming solid-phase extraction step to remove possible hydrolysis inhibitors can be omitted.

Detection of testosterone esters in blood

Injections of synthetic esters of testosterone are among the most common forms of testosterone application. In doping control, the detection of an intact ester of testosterone in blood gives unequivocal proof of the administration of exogenous testosterone. The aim of the current project was to investigate the detection window for injected testosterone esters as a mixed substance preparation and as a single substance preparation in serum and plasma. Furthermore, the suitability of different types of blood collection devices was evaluated. Collection tubes with stabilizing additives, as well as non-stabilized serum separation tubes, were tested. A clinical study with six participants was carried out, comprising a single intramuscular injection of either 1000 mg testosterone undecanoate (Nebido(®)) or a mixture of 30 mg testosterone propionate, 60 mg testosterone phenylpropionate, 60 mg testosterone isocaproate, and 100 mg testosterone decanoate (Sustanon(®)). Blood was collected throughout a testing period of 60 days. The applied analytical method for blood analysis included liquid-liquid extraction and preparation of oxime derivatives, prior to TLX-sample clean-up and liquid chromatography-tandem mass spectrometry (LC-MS/MS) detection. All investigated testosterone esters could be detected in post-administration blood samples. The detection time depended on the type of ester administered. Furthermore, results from the study show that measured blood concentrations of especially short-chained testosterone esters are influenced by the type of blood collection device applied. The testosterone ester detection window, however, was comparable.

Murder by poisoning: successful analytical investigations of spectacular cases in Austria.

To prove murder by poisoning requires the application of analytical toxicology to detect the fatal substance and clear up the cause of death. Improvements in the development of mass spectrometry in combination with high-resolution chromatographic methods are steadily enhancing detection and identification power but making use of these advances relies on proper sample preparation as well as on knowledge about the chemical nature of the substances and their bio-transformation products. This review gives examples of case reports with successful analytical investigations of murder by poisoning in spectacular Austrian cases involving low molecular weight.