Sofie Poelmans

Presence and metabolism of the anabolic steroid boldenone in various animal species : a review

The review summarizes current knowledge on the possible illegal use of the anabolic steroid boldenone. The presence of boldenone and metabolites in different animal species and the possibility of the occurrence of endogenous boldenone and metabolites is assessed, as are the methods of analysis used for detection. Different laboratories in the European Union have examined the occurrence of boldenone and its metabolites. The results were discussed at different meetings of a European Commission DG-SANCO Working Party and summarized in an expert report. The situation of the different laboratories at this time is also covered herein. The overall conclusion of the Working Party was that there was a necessity for further research to distinguish between naturally occurring and illegally used boldenone forms. The confirmation of the presence of boldenone metabolites (free and conjugated forms) in certain matrices of animals is proposed as a marker for the illegal treatment with boldenone.

Analytical possibilities for the detection of stanozolol and its metabolites

In sports doping, as well in man as in horseracing, stanozolol (Stan) was abused and became the subject of metabolism research. Also in veterinary practice, stanozolol became an important misused anabolic steroid. Like most other anabolic steroids, stanozolol has poor gas chromatographic behavior. It is difficult to detect in urine, because of low urinary excretion and renal clearance. This is due to the rapid metabolization, leading to low concentration levels of the parent compound found in urine. Therefore, most research studies have focused on the detection of its urinary metabolites. For the identification of the metabolites, different methods of extraction and detection are described in the literature. These are reviewed in this article. Most authors use a hydrolysis to free the phase II metabolites. Extraction procedures vary from solid-phase extraction (SPE), liquid-liquid (L-L) extraction to immunoaffinity chromatography (IAC). For the final detection, the use of gas chromatography (GC)-mass spectrometry (MS) can be compared with liquid chromatography (LC)-MSn. Different metabolites are identified depending on the administration of stanozolol in the animal experiment (oral or intramuscular). Analyses for these analytes in other matrices are also briefly discussed.

Testosterone and energy metabolism in the estuarine mysid Neomysis integer (Crustacea: Mysidacea) following exposure to endocrine disruptors

A diverse set of reference compounds suspected of having an endocrine-disrupting mode of action (i.e., testosterone, flutamide, ethinylestradiol, precocene, nonylphenol, fenoxycarb, and methoprene) were tested for acute toxicity to the estuarine mysid Neomysis integer (Crustacea: Mysidacea). Neomysis integer was very sensitive to all tested compounds, with 96-h median lethal concentrations in a narrow range between 0.32 and 1.95 mg/L. The pesticides methoprene and fenoxycarb, both synthetic insect juvenile hormone analogs, were most toxic to N. integer. In addition, the short-term sublethal effects of methoprene and nonylphenol (an estrogen agonist) on the energy and steroid metabolism of N. integer were evaluated. Both compounds significantly affected energy and testosterone metabolism of N. integer at concentrations below acute toxicity levels. Energy consumption in methoprene- and nonylphenol-exposed mysids was significantly induced at 100 microg/L, resulting in a lower cellular energy allocation in these animals. Testosterone phase I metabolism was affected at 10 microg/L, whereas glycosylation was the most important phase II pathway affected in mysids exposed to 100 microg/L of both compounds. Methoprene exposure resulted in a concentration-dependent increase in the metabolic androgenization ratio. Mysids exposed to nonylphenol at 10 microg/L had a significantly higher metabolic androgenization ratio. The present study indicates that energy and testosterone metabolism of mysids, as endpoints, are able to detect endocrine-disruptive activity of chemicals after short-term exposure to environmentally realistic levels of endocrine disruptors.

Endogenous occurrence of some anabolic steroids in swine matrices.

Following findings of 17β-19-nortestosterone (150–200 µg kg−1) in pigs of unspecified gender imported into the European Union, a study to determine steroid and hormone levels in swine from six age/gender categories (uncastrated ‘old’ boars, cryptorchids, one intersex, barrows, gilts and sows) was initiated. Indeed, for some hormones there has been a discussion about their being endo- or exogenous. Tissue and urine samples from swine from each of the six categories were obtained in Belgium, France, the Netherlands and the USA. Samples were analysed in three laboratories. Quantitation was obtained for norandrostenedione, 19-nortestosterone and boldenone. The results give a well-documented overview of the status of the presence of these hormones in swine. The data illustrate that uncastrated ‘old’ boars produce the highest percentage of ‘positive’ matrices, followed by the cryptorchids. Concentrations in the matrices of the barrows and the gilts are lower. Also, sow matrices contain low amounts of nor-steroids. Furthermore, urine samples from an intersex pig contains a higher concentration of nortestosterone than sows and can therefore be suspected for illegal use of these hormones. Veterinarians taking samples in pig farms for the analysis of hormones need to be aware of the presence and concentrations of these substances in the different categories.

Alternative to vertebrate animal experiments in the study of metabolism of illegal growth promotors and veterinary drugs

Abstract The continuous production of new illegal veterinary drugs and related products requires residue laboratories to initiate research into developing fast and accurate extraction and detection methods for the identification (and/or quantification) of the major analyte or metabolites of these compounds. In practice, animal experiments are carried out in which vertebrate animals (bovine, porcine, …) are treated orally or intramuscularly with the illegal compound. Different matrices (urine, faeces, blood) are collected over 2 or 3 weeks until the animal is sacrificed. Edible matrices (meat, liver, kidney,…) are collected. Because of the complexity of the animal experiment and the method development, a lot of valuable time and money is consumed. Recent studies have shown that some of these vertebrate experiments can be replaced by invertebrates metabolism studies. Vertebrate-type steroids such as testosterone have been used as substrates to study enzyme systems (cytP450) for the oxidative metabolism in invertebrates. Results from these studies provide information on the degree of similarity to the enzyme systems in vertebrates. These findings are of great importance to the research of illegally used substances but also to the downscaling of vertebrate animal experiments and their considerable cost factors. The invertebrate Neomysis integer (Crustacea, Mysidacea) has been used as an alternative model for the partial replacement of vertebrate animals in metabolism studies with illegal growth promotors and veterinary drugs. The principle of this assay and some examples are described.

Identification of "unknown analytes" in injection sites: a semi-quantitative interpretation

The analytical approach to the detection of residues of legally used veterinary medicinal products (VMPs) is similar to the approach of forbidden substances. The only difference lies in the quantitative component of the method. Since there is an evolution towards a different strategy in the screening for VMPs in matrices of slaughtered animals, a new approach was developed for determining the residues. The aim of this research was to create an efficient screening approach for determining the identity and/or quantity of legally and illegally used VMPs present at high concentrations in injection sites. The determination of these ‘unknown’ VMPs is combined with a fast report to the customer. Examples are given of the identification of phenylbutazone, penicillin G benzathine and florfenicol. For quantitative purposes, using a mini-validation procedure, concentrations far above the maximum residue limit (MRL) of the identified VMP can be reported. A quantitative validation normally consists of determining the required validation parameters at three levels: 1/2 MRL, MRL, 2 MRL. For highly concentrated injection sites, an alternative approach is proposed. The alternative validation consists of a comparison of the analyte concentration in the sample with the spike at the MRL and 10 times the MRL concentration.

Androstadienetrione, a boldenone-like component, detected in cattle faeces with GC-MSn and LC-MSn

Boldenone (1,4-androstadiene-17-ol-3-one, Bol) has been the subject of a heated debate because of ongoing confusion about its endogenous or exogenous origin when detected in one of its forms in faecal or urine samples from cattle. An expert report was recently written on the presence and metabolism of Bol in various animal species. Androstadienedione (ADD) is a direct precursor of 17β-boldenone (βBol). It is a 3,17-dione; ßBol is a 17-ol-3-one. Not much is published on 1,4-androstadiene-3,17-diol, which is a 3,17-diol (ADL). If animals were exposed for a longer period to one of these analytes, a metabolic pathway would be initiated to eliminate these compounds. Similar to recent testosterone metabolism studies in the aquatic invertebrate Neomysis integer, ADD, ßBol and ADL could also be eliminated as hydroxymetabolites after exposure. The presence of 11-keto-steroids or 11-hydroxy-metabolites in faecal samples can interfere with a confirmation method by gas chromatography-negative chemical ionization mass spectrometry (GC-NCI-MS), after oxidation of corticosteroids with a double bond in the A-ring (e.g. prednisolone or its metabolite prednisone). The presence of androstadienetrione (ADT) in faecal samples of cattle has never been reported. The origin of its presence can be explained through different pathways, which are presented in this paper.

Biological and Chemical Approaches for the Detection and Identification of Illegal Estrogens in Water-based Solutions

The continuous introduction of new products used as growth promoters in animal husbandry, for sports doping and as products for body-building requires residue laboratories to initiate research on developing a strategy for the identification of ’unknown’ components. In this study, a strategy is presented for elucidating the identity, the structure and the possible effects of illegal estrogenic compounds in an unidentified water-based solution. To obtain complete information on the composition and activity of the unidentified product, a multidisciplinary approach was needed. A case-study is described with a ’solution X’ found during a raid. First, in vivo techniques (animal trials with mice, anatomical and histological research) were combined with in vitro techniques (the yeast estrogenic screen (YES)). In a later stage of the investigation, HPLC-fractionation, liquid chromatography–multiple mass spectrometry (LC-MS n and gas chromatography-multiple mass spectrometry (GC-MS n were used. Finally, the identity of ’solution X’ was confirmed in a very low concentration range (10 ng/L estrone and 400 ng/l ethinyloestradiol).