K De Wasch

Recent developments in the use and abuse of growth promoters

During the last few years, control within the European Union (EU) for illegal growth promoters in cattle and pigs revealed only a limited number of positives. Analysis of illegal preparations, however, showed that steroids, often (esters of) natural hormones, and -agonists are still used. Corticosteroids, controlled to a much lesser extent, seem to have become the most important group, while even thyreostats remain. Alarming information was obtained from specific investigations in which a large variety of products were found, some of which had never been reported to be misused in the field of growth promotion. For -agonists and quinoxaline compounds, analogues of known compounds are synthesised. Other compounds are readily available as they are registered as growth promoters in some countries outside Europe or are allowed for specific veterinary purposes. Some classes of veterinary drugs are misused for their secondary pharmacological effects, e.g. benzodiazepines as feed intake enhancers and non-steroidal anti-inflammatory drugs (NSAIDs) as pale meat-making agents. Several non-traditional substances are suspected to be used in the field of breeding animals. This is the case for growth hormones (GHs) and all substances acting over this anabolic compound, as for instance, orally GH secretagogue. Moreover, ecdysteroids, which according to old Russian studies, have anabolic activity, are actually very easy to purchase on the Internet. Recent findings in different classes of growth promoters are discussed in detail.

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.

Determination of mercaptobenzimidazol and other thyreostat residues in thyroid tissue and meat using high-performance liquid chromatography-mass spectrometry.

This paper describes a method for extraction of tapazol, thiouracil, methylthiouracil, propylthiouracil and mercaptobenzimidazol (MBI) from thyroid tissue. The solid-phase extraction procedure is optimized to obtain the maximum results for the main thyreostats including MBI. Different combinations of sample application, column conditioning and wash steps were tested. The analytes were extracted from the matrix with methanol. After solid-phase extraction they were derivatised with 7-chloro-4-nitrobenzo-2-furazan. Determination is carried out using liquid chromatography-electrospray mass spectrometry. The identification of the analytes was performed according to the final revision of the EU criteria (93/256/EC decision). The detection capability was 20 microg kg(-1) for all mentioned thyreostats.

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.

Evaluation and establishing the performance of different screening tests for tetracycline residues in animal tissues

Four methods intended for screening muscle tissue for residues belonging to the tetracycline group were compared using artificially contaminated as well as incurred samples. Two agar diffusion methods were studied: one with Bacillus subtilis as a test strain, the second with Bacillus cereus. Two variants of each method were compared: thin plates for analysis of intact or minced meat, and thick plates for analysis of meat fluid. The thin plate variants could not be evaluated with artificially contaminated samples because it was impossible to prepare homogeneously spiked, undiluted meat. The thick plates were suited for doxycycline and chlortetracycline, but they did not detect oxytetracycline or tetracycline in spiked meat fluid. The results of these tests done on incurred meat were very good for doxycycline and satisfying or just failing for oxytetracycline, while the best detection capability was obtained when intact frozen meat was examined on thin plates seeded with B. cereus. Two commercially available screening tests were also evaluated. The Premi® test, an inhibitor test with Bacillus stearothermophilus as a test strain and an indicator for growth, was not suited for detection of tetracyclines up to the maximum residue limit. Tetrasensor®, a receptor test specific for tetracyclines, proved a quick and simple test able to detect meat samples artificially contaminated with tetracycline, oxytetracycline, doxycycline or chlortetracycline, as well as meat incurred with oxytetracycline or doxycycline.

Determination of 16β-hydroxystanozolol in urine and faeces by liquid chromatography–multiple mass spectrometry

This paper describes the optimisation of the detection of stanozolol and its major metabolite 16beta-hydroxystanozolol in faeces and urine from cattle. Faeces are extracted directly with diisopropyl ether. Urine is first submitted to an enzymatic hydrolysis and then extracted over a modified diatomaceous earth column (Chem-Elut) with a mixture of diisopropyl ether-isooctane. In a final step an acidic back extraction is performed. For the LC-MS-MS detection two approaches are discussed. In a first approach the final extract is detected without derivatization, while the second approach makes use of a derivatization step for 16beta-hydroxystanozolol. While the MS-MS spectrum without derivatization exhibits extensive fragmentation, the spectrum of the derivative shows two abundant diagnostic ions with much more reproducible ion ratios. The derivatization method and the method without derivatization enable the detection of 16beta-hydroxystanozolol up to 0.03 microg l(-1) in urine and 0.07 microg kg(-1) in faeces. Until now there is no literature available for the detection of 16beta-hydroxystanozolol in faeces and urine at the ppt level.

Differentiation between dexamethasone and betamethasone in a mixture using multiple mass spectrometry

The objective of this study was to provide LC and GC-multiple mass spectrometry (MSn) data in positive and negative ion modes to prove the distinction between dexamethasone and betamethasone in a mixture of both components. Using GC-MS, the differentiation was based on a difference in the ratio of the ion traces of the two chromatographic peaks of the alpha and beta epimer with m/z 310 and 330. A minimum of 15% dexamethasone should be present in a mixture of both to detect it as present with a probability of 95%. In the same way betamethasone can be detected from 15% on. Because of the very similar structures of the dexamethasone and betamethasone epimers, no reversed-phase (RP) separations have been reported. Normal-phase separations have been reported in other studies. However because of the compatibility of RP mobile phases in the coupling with MS, the latter was the method of choice. In LC-MSn positive ion mode the product ion 355 was plotted against the sum of 337 and 319. With this combination dexamethasone and betamethasone could be discriminated in a mixture of 20 to 80% of each combination of analytes. In negative ion mode only two product ions were formed from the fragmentation of the acetate adduct, [M-H]- and [M-H-CH2O]-. The intensity of the fragment 391 ([M-H]-) was determined in the discrimination of the two epimers.

Confirmation of residues of thyreostatic drugs in thyroid glands by multiple mass spectrometry after thin-layer chromatographic screening

A method is described for the confirmation of high-performance thin layer chromatography (HPTLC) suspect results of residues of thyreostatic drugs in thyroid tissue. The method is based on the infusion of the remainder of the extract used for HPTLC via the electrospray interface into a mass spectrometer operating in the multiple stage mass spectrometry (MSn) mode. The clean-up of the samples was performed with a selective extraction procedure, based on a specific complex formation of the drugs with mercury ions, bound in an affinity column. The thyreostatic drugs were derivatised with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole.

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.

Comparison of the possibilities of gas chromatography-mass spectrometry and tandem mass spectrometry systems for the analysis of anabolics in biological material

Chromatographic techniques such as GC-MS play a most important role in modern multi-residue analysis of anabolic steroids. The major difference between GC-MS apparatus from different manufacturers is the way of detection and recording. Most apparatus use selected-ion monitoring (SIM) for the determination of low concentrations. Systems based on ion trap technology record in full-scan to even picogram concentrations using a computer algorithm to compare the most important peaks of the mass spectrum of the unknown to those of the standard. In this investigation the possibilities of ion trap GC-MS and the recently released GCQ MS and MS2 for the analysis of anabolics in biological material are compared.