S.S. Sterk

Detection of anabolic androgenic steroid abuse in doping control using mammalian reporter gene bioassays

Anabolic androgenic steroids (AAS) are a class of steroid hormones related to the male hormone testosterone. They are frequently detected as drugs in sport doping control. Being similar to or derived from natural male hormones, AAS share the activation of the androgen receptor (AR) as common mechanism of action. The mammalian androgen responsive reporter gene assay (AR CALUX bioassay), measuring compounds interacting with the AR can be used for the analysis of AAS without the necessity of knowing their chemical structure beforehand, whereas current chemical-analytical approaches may have difficulty in detecting compounds with unknown structures, such as designer steroids. This study demonstrated that AAS prohibited in sports and potential designer AAS can be detected with this AR reporter gene assay, but that also additional steroid activities of AAS could be found using additional mammalian bioassays for other types of steroid hormones. Mixtures of AAS were found to behave additively in the AR reporter gene assay showing that it is possible to use this method for complex mixtures as are found in doping control samples, including mixtures that are a result of multi drug use. To test if mammalian reporter gene assays could be used for the detection of AAS in urine samples, background steroidal activities were measured. AAS-spiked urine samples, mimicking doping positive samples, showed significantly higher androgenic activities than unspiked samples. GC-MS analysis of endogenous androgens and AR reporter gene assay analysis of urine samples showed how a combined chemical-analytical and bioassay approach can be used to identify samples containing AAS. The results indicate that the AR reporter gene assay, in addition to chemical-analytical methods, can be a valuable tool for the analysis of AAS for doping control purposes.

Sex hormone-binding globulin regulation of androgen bioactivity in vivo: validation of the free hormone hypothesis

Sex hormone-binding globulin (SHBG) is the high-affinity binding protein for androgens and estrogens. According to the free hormone hypothesis, SHBG modulates the bioactivity of sex steroids by limiting their diffusion into target tissues. Still, the in vivo physiological role of circulating SHBG remains unclear, especially since mice and rats lack circulating SHBG post-natally. To test the free hormone hypothesis in vivo, we examined total and free sex steroid concentrations and bioactivity on target organs in mice expressing a human SHBG transgene. SHBG increased total androgen and estrogen concentrations via hypothalamic-pituitary feedback regulation and prolonged ligand half-life. Despite markedly raised total sex steroid concentrations, free testosterone was unaffected while sex steroid bioactivity on male and female reproductive organs was attenuated. This occurred via a ligand-dependent, genotype-independent mechanism according to in vitro seminal vesicle organ cultures. These results provide compelling support for the determination of free or bioavailable sex steroid concentrations in medicine, and clarify important comparative differences between translational mouse models and human endocrinology.

Nortestosterone: endogenous in urine of goats, sheep and mares?†

For a number of species it is known that nortestosterone, either the alpha- or beta-epimer, can be of endogenous origin. For goats and mares similar results have not yet been published. As a follow-up on the experiments with cattle, a large number of urine samples per animal were collected from pregnant goats, sheep and mares. These samples were analysed for the presence of alpha- and beta-nortestosterone and alpha-estradiol using GC-MS. The results show that in the goats and mares studied alpha-nortestosterone is present during pregnancy. In this study no alpha-nortestosterone could be demonstrated in sheep. From our study and recently published data, however, it is proven that alpha-nortestosterone can occur endogenously.

Confirmatory analysis of Trenbolone using accurate mass measurement with LC/TOF-MS.

The use of accurate mass measurement as a confirmation tool is examined on a TOF-MS and compared with confirmation using a triple quadrupole mass spectrometer (QqQ-MS). Confirmation of the identity of a substance using mass-spectrometric detection has been described. However, the use of accurate mass measurement for confirmatory analysis has not been taken into account. In this study, criteria for confirmation with accurate mass are proposed and feasibility is demonstrated. Mass accuracy better than 3ppm of the quasi-molecular ion and a fragment and their relative ratios determined with LC/TOF-MS are compared to the criteria of two transition ions and their ratio of LC/QqQ-MS. The results show that these criteria can be met for Trenbolone in samples of bovine urine and that single MS accurate mass measurement is comparable to nominal mass MS/MS for confirmation. The increase in popularity and availability of LC/TOF-MS instruments and the ease, of which exact masses can be measured, make it important to formulate criteria for this type of instrumentation. It is shown in this study that accurate mass measurement can be used for confirmatory analysis. However, more experiments need to be conducted to demonstrate the applicability of accurate mass measurement in general for residue analysis.

Sports doping: Emerging designer and therapeutic β2-agonists

Beta2-adrenergic agonists, or β2-agonists, are considered essential bronchodilator drugs in the treatment of bronchial asthma, both as symptom-relievers and, in combination with inhaled corticosteroids, as disease-controllers. The use of β2-agonists is prohibited in sports by the World Anti-Doping Agency (WADA) due to claimed anabolic effects, and also, is prohibited as growth promoters in cattle fattening in the European Union. This paper reviews the last seven-year (2006-2012) literature concerning the development of novel β2-agonists molecules either by modifying the molecule of known β2-agonists or by introducing moieties producing indole-, adamantyl- or phenyl urea derivatives. New emerging β2-agonists molecules for future therapeutic use are also presented, intending to emphasize their potential use for doping purposes or as growth promoters in the near future.

Production and stability testing of incurred reference materials for the anabolic steroid trenbolone in bovine urine

The production of stable reference materials with incurred residues of veterinary drugs is necessary for the validation of methods of analysis, including the determination of critical performance characteristics. A reference material for trenbolone in bovine urine was produced and the long-term stability was tested. From a pilot 16 week stability study on seven batches containing different additives it was concluded that the use of preservatives does not improve the stability of the residue. A final batch of reference material of 800 vials each containing 5 ml of urine with a target concentration of 5 micrograms l-1 was prepared. The homogeneity and long-term stability of the material were tested. The material was found to be homogeneous. Based on the results of a 52 week stability study it was concluded that the material is stable, using the current analytical methodology. For the development of reference materials, highly accurate and precise analytical methods are necessary. However, the current analytical methodology is not suitable for full evaluation and certification. Currently, a new LC-MS method is being developed. After validation of this method, the stability and homogeneity study will be repeated.

Bovine blood analysis for natural hormones : an overview of analytical strategies

Abstract The results of an inquiry, organized among a large number of European Community (EC) control laboratories, revealed that most EC countries have a control program for the natural hormones 17β-estradiol and 17β-testosterone which is based on the analysis of samples of plasma or serum by a (radio)immunoassay procedure. The diversity within these procedures, however, is large and good quality control programs are not available. As base for such a program a method based on gas chromatography—isotope dilution mass spectrometry was developed and validated for purposes of the simultaneous accurate quantification of low levels of 17β-estradiol and 17β-testosterone. Accuracy, repeatability and within-laboratory reproducibility of this method are adequate. However, to obtain a limit of determination that is low enough for accurate quantification at the EC-recommended action level of 0.04 μg 1−1 for 17β-estradiol the use of negative chemical ionisation is necessary.

Distinction of clenbuterol intake from drug or contaminated food of animal origin in a controlled administration trial – the potential of enantiomeric separation for doping control analysis

ABSTRACT The differentiation of clenbuterol abuse and unintentional ingestion from contaminated meat is crucial with respect to the valuation of an adverse analytical finding in human sports doping control. The proportion of the two enantiomers of clenbuterol may serve as potential discriminating parameter. For the determination of the individual enantiomers, specific methods were developed and validated for the different matrices under investigation based on chiral chromatography coupled to tandem mass spectrometry. Data are presented from the administration to humans of clenbuterol from a pharmaceutical preparation, and from cattle meat and liver containing residues. A shift in the proportion of the enantiomers in cattle meat is detected and this signature is also found in human urine after ingestion. Thus, an altered enantiomeric composition of clenbuterol may be used to substantiate athletes’ claims following adverse analytical findings in doping control. However, in meat, the enantiomeric composition was found to be highly variable. Species as well as tissue dependent variances need to be considered in interpreting enantiomer discrimination. Analysis of post administration urines from a controlled experiment comparing the administration of racemic clenbuterol from a registered pharmaceutical preparation and the administration of residue-containing meat and liver (nonracemic mixture) from treated animals is reported. Furthermore doping control samples from Mexican U17 World Championship 2011 of the Fédération Internationale de Football Association (FIFA), with adverse analytical findings for clenbuterol, were re-analysed. GRAPHICAL ABSTRACT

A view on the analytical design of future risk based residue control.

The current laboratory network system in support of residue monitoring programmes within the EU formally started in the early 1990s. Since then, it has undergone a gentle evolution incorporating new techniques and methods for quality assurance and, in parallel to the extension of the European Union itself, was further extended. However, a paradigm shift from production-based to risk-based control now is foreseen. This will have a serious impact on the type of methodologies used and subsequently on the specific roles of EU reference laboratories also. Here, we present our view on the changes that will inevitably take place in the years to come. Copyright

Hormone analysis in food products.

The administration of growth-promoting compounds, to food producing animals is banned within the European Union. We developed several methods whereby the cleanup is based on LLE and SPE and detection based on GC-MSMS or LC-MSMS to identify and confirm the identity of different growth promoting agents in several food products. This chapter describes methods to isolate and identify these growth promoting agents.