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The administration of synthetic homologues of naturally occurring steroids can be demonstrated by measuring (13)C/(12)C isotopic ratios of their urinary metabolites. Gas chromatography-mass spectrometry/combustion/isotope ratio mass spectrometry (GC-MS/C/IRMS) was used in this study to appraise in a global approach isotopic deviations of two 17β-testosterone metabolites (17α-testosterone and etiocholanolone) and one 17β-estradiol metabolite (17α-estradiol) together with those of 5-androstene-3β,17α-diol as endogenous reference compound (ERC). Intermediate precisions of 0.35‰, 1.05‰, 0.35‰, and 0.21‰, respectively, were observed (n = 8). To assess the performance of the analytical method, a bull and a heifer were treated with 17β-testosterone propionate and 17β-estradiol-3-benzoate. The sensitivity of the method permitted the demonstration of 17β-estradiol treatment up to 24 days. For 17β-testosterone treatment, the detection windows were 3 days and 24 days for the bull and the heifer, respectively. The capability of GC-MS/C/IRMS to demonstrate natural steroid abuse for urinary steroids was eventually compared to those of mass spectrometry (LC-MS/MS) when measuring intact steroid esters in blood and hair.

Although substantial technical advances have been achieved during the past decades to extend and facilitate the analysis of growth promoters in cattle, the detection of abuse of synthetic analogs of naturally occurring hormones has remained a challenging issue. When it became clear that the exogenous origin of steroid hormones could be traced based on the (13)C/(12)C isotope ratio of the substances, GC/C/IRMS has been successfully implemented to this aim since the end of the past century. However, due to the costly character of the instrumental setup, the susceptibility of the equipment to errors and the complex and time consuming sample preparation, this method is up until now only applied by a limited number of laboratories. In this review, the general principles as well as the practical application of GC/C/IRMS to differentiate between endogenous steroids and exogenously synthesized homologous compounds in cattle will be discussed in detail, and will be placed next to other existing and to be developed methods based on isotope ratio mass spectrometry. Finally, the link will be made with the field of sports doping, where GC/C/IRMS has been established within the World Anti-Doping Agency (WADA) approved methods as the official technique to differentiate between exogenous and endogenous steroids over the past few years.

A method is described for the synthesis of α,α-dichloroaldehydes, free from monochloroaldehyde, by chlorination of aldehydes or alcohols in dimethylformamide at 40–90°C. Results are reported for: (yield %) propanal (90), butanal (78), pentanal (78), 3-methylbutanal (90), propanol (64), butanol (78), 3-methylbutanol (52), pentanol (66), 3,3-dimethylbutanol (86), hexanol (66), 2-phenylethanol (51), 3-phenylpropanol (54), 1,5-pentanediol [3,3-dichloro-2-hydroxytetrahydropyran (37), 2,3,3-trichlorotetrahydropyran (19)], 1,6-hexanediol [2,2,5,5-tetrachloro-1,6-hexanedial (63)]. Oxidative conversion (KMnO4, K2Cr2O7, H2O2-NaHCO3) into α,α-dichlorocarboxylic acids and methyl esters is discussed.

Reaction of dimethyl 2-bromo-2-methylpropylidene malonate with various nucleophiles mainly afforded dimethyl cyclopropane 1,1-dicarboxylates while treatment with amines gave substitution products.

Selective chlorination methods were elaborated for the conversion of cyclopentanone into 2, 2, 5, 5-tetrachloro-, 2, 2, 3, 5, 5-pentachloro- and 2, 2, 3, 3, 5, 5-hexachlorocyclopentanone. Dimethylformamide-hydrogen chloride mixtures served as catalysts for enolization and for dehydrochlorination to chlorinated cyclopentenones. The reactivity of the polychlorocyclopentanones was tested towards zinc (in acetic acid, methanol or acetone) and towards bases in protic solvents. A number of chlorinated cyclopentenones, substitution products and open chain compounds (Grob elimination) were obtained in good yields.

Heating of dialkyl 2-bromoalkylidenemalonates in boiling xylene resulted in lactonization with formation of 2-alkoxycarbonyl-2-buten-4-olides. Bromination of dimethyl 2-phenylpropylidenemalonate with NBS in CCI4 gave the corresponding butenolide. However, heating of the 2-bromocyclohexylidenemalonate only afforded the dehydrobromination product.

Efficient procedures for the synthesis of 3,3,5-trimethyl-1,2,4-cyclopentanetrione are described starting from the commercially available ketones 2,2,4-trimethylcyclopentanone, 2,4,4-trimethylcyclopentanone and 3,3,5-trimethylcyclohexanone. The methods involve polychlorination and dehydrochlorination in dimethylformamide followed by hydrolysis.

Reactions of allylic halides bearing two electron-withdrawing groups in γ-position with various nucleophiles (alkoxides, phenoxide, thiolates and cyanide) gave rise to substituted electrophilic cyclopropanes or ring-opening products, depending upon the reaction conditions, the nature of the nucleophiles and the activating groups and the substitution pattern of the allyl halides. A kinetic study revealed that the reaction involved a nucleophilic addition as the rate determining step followed by a fast ringclosure.

Regularly new anabolic steroids appear on the black market. In most cases these substances are marketed on websites or are confiscated during inspections. 1,(5alpha)-Androstene-17beta-ol-3-one, also known as 1-testosterone, is one of these substances presented to body-builders as a nutritional supplement or a pro-hormone. 1-Testosterone closely resembles the natural hormone testosterone except for a 1,2-double bound instead of a 4,5-double bound. 1-Androstene-3beta,17beta-diol is transformed into 1-testosterone after oral administration. 1-Testosterone, 1-androstene-3beta,17beta-diol and some other related 'new' anabolic steroids were studied with gas chromatography coupled to mass spectrometry (GC-MS) and Liquid chromatography coupled to tandem mass spectrometry (LC-MS2) methods. Similarities in spectra to known analytes, which may lead to pitfalls in the interpretation of the derivatised analytes, are discussed.

Currently, no analytical method is available to demonstrate progesterone administration in biological samples collected in rearing animals, and therefore, tracking the abuse of this popular growth promoter is arduous. In this study, a method is presented to reveal progesterone (PG) treatment on the basis of carbon isotope measurement of 5β-pregnane-3α, 20α-diol (BAA-PD), a major PG metabolite excreted in bovine urine, by gas chromatography–mass spectrometry/combustion/isotope ratio mass spectrometry (GC–MS/C/IRMS). 5-Androstene-3β,17α-diol (AEdiol) is used as endogenous reference compound. Intermediate precisions (n = 11) of 0.56‰ and 0.68‰ have been determined for AEdiol and BAA-PD, respectively. The analytical method was used for the very first time to successfully differentiate urine samples collected in treated and untreated animals.

The use of anabolic steroids has been banned in the European Union since 1981. In this study, the metabolism of the anabolic steroid methenolone acetate, was investigated in a male veal calf. After daily oral administration of methenolone acetate, three main metabolites were detected in both urine and faeces samples. Among these metabolites, alpha-methenolone was apparently the main one, but 1-methyl-5alpha-androstan-3,17-diol and 3alpha-hydroxy-1-methyl-5alpha-androstan-17-one were also observed. The parent compound was still detectable in faeces. As a consequence, abuse of methenolone acetate as growth promoter can be monitored by analysing urine and faeces samples. A few days after the last treatment, however, no metabolites were observed. Alpha-methenolone was detectable in urine until 5 days after the last treatment, but in faeces no metabolites were detectable after 3 days.

The Michael-induced ring closure (MIRC) of amines with 2-bromoalkylidenemalonates has been reinvestigated and the reaction products with primary amines have been identified as (2-iminoethyl)malonates and not 2-aminoalkylidenemalonates as previously reported. The (2-iminoethyl)malonates are formed by ring opening of the intermediate unstable 2-aminocyclopropane-1,1-di-carboxylates (β-ACCs) and were characterized spectroscopically and via chemical transformation.

4-Chloro-estr-4-en-17-ol-3-one, trivially named 19-norclostebol acetate or 4-chloro-19-nortestosterone acetate (NClTA), has been identified on the European black market in the late 1990s for possible use in breeding animals. After oral and subcutaneous administration of NClTA to bovine, urine samples were collected over a period of three weeks, and chemical structure of main excreted urinary metabolites was determined. After oral administration, the most abundant metabolites were mainly reduced as 4-chloro-19-norandrostan-3ξ-ol-17-one and 4-chloro-19-norandrostan-3ξ,17ξ-diol. They were identified until 1 week after administration. Following subcutaneous injection, 4-chloro-19-norandrostan-3ξ-ol-17-one was again of major abundance, but so were 4-chloro-19-norandrost-4-ene-3ξ,17ξ-diol and 4-chloro-19-norandrost-4-en-3ξ-ol-17-one. They were detected at least 3 weeks after administration. Whatever the route of administration, metabolites were found mainly glucurono-conjugated; the only exception was metabolite 4-chloro-19-norandrostan-3ξ-ol-17-one which was identified both in the sulpho- and glucurono-fractions.

Ion suppression, a matrix effect that affects quantitative mass spectrometry, is one of the main problems encountered in liquid chromatography/tandem mass spectrometry. Two different clean-up steps for the multi-residue analysis of beta-agonists in urine were evaluated with respect to minimisation of ion suppression, namely, a mixed-phase solid phase extraction (SPE) column, i.e., clean screen Dau (CSD), and a molecular imprinted polymer (MIP) SPE column. Ion suppression experiments revealed that CSD sample clean-up can lead to false negative results for some beta-agonists, and that clean-up using MIP columns is more selective for beta-agonists than the use of CSD columns.

Zilpaterol is a new powerful beta-agonist, which is officially registered for fattening purposes in cattle in Mexico and South Africa. Its chemical structure is different from the well-known beta-agonists. Therefore, the routinely used screening methods are not likely to be suited for the analysis of zilpaterol. Also gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry (LC-MS) methods need to be adapted to enable detection of zilpaterol. In this study, a LC-MS3 confirmatory method was developed for the simultaneous detection of zilpaterol and di-aromatic beta-agonists in urine samples. A LC-MS2 method was optimised for the detection of zilpaterol in faeces. To study the excretion profile in urine and faeces, a male veal calf was orally treated with daily doses of Zilmax ® during 2 weeks. Zilpaterol was mainly excreted via urine. © 2004 Elsevier B.V. All rights reserved.

Norchlorotestosterone acetate (NClTA) is an anabolic steroid which resembles chlorotestosterone acetate. It cannot yet be detected by routine methods used for anabolic steroids, because there is no knowledge of its metabolic pathway. The invertebrate Neomysis integer has been used as an alternative model to study the metabolism of NClTA. The experimental results indicated the presence of 4-norchloroandrost-4-ene-17-ol-3-one (NClT) and 4-norchloroandrost-4-ene-3,17-dione (NorClAD) as possible metabolites of NClTA. Subsequently NClTA and the synthesised metabolites NClT and NorClAD were incorporated into the routine multi-residue method for detection of anabolic steroids in kidney fat, urine, and faeces.

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. © 2002 Elsevier Science B.V. All rights reserved.

According to the European Commission Decision 2001/699/EC and 2001/705/EC certain fishery and aquaculture products, imported from China, Vietnam or Indonesia and intended for human consumption, must be subjected to a test in order to ensure the absence of chloramphenicol residues. For that reason an analytical method has been developed and validated based on ELISA for screening and gas chromatography–tandem mass spectrometry (GC–MS2) or liquid chromatography–tandem mass spectrometry (LC–MS2) for confirmation. The chloramphenicol ELISA was carried out directly on an aqueous extract of the shrimps or after an extraction with ethyl acetate. Confirmation of suspect samples was performed after extraction with ethyl acetate and defatting with n-hexane. The clean-up was based on solid phase extraction using C18 cartridges or reversed phase HPLC. After derivatisation with N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA), the final extracts were analysed by GC–MS2 in the negative ion chemical ionisation mode. Confirmation of chloramphenicol was also possible with LC–MS2 after the same clean-up. Both selective techniques made it possible to detect chloramphenicol residues at the 0.1 μg kg−1 level starting from 20 g of matrix for enzyme-linked immunosorbent assay (ELISA) with organic solvent extraction, or from 5 g of matrix for ELISA with aqueous extraction.

A rapid and easy-to-perform method for the screening and confirmation of estrogens, gestagens and androgens (EGAs) in kidney fat has been developed. In this investigation only 5 g of fat was needed. After extraction with acetonitrile the steroid phase was defatted using n -hexane. Followed by a saponification step, the sample extract was purified by solid phase extraction. After derivatisation as trimethylsilyl ether derivatives, the steroids in the final extract were analysed using gas chromatography-tandem mass spectrometry (GC-MS 2 ). With this method the Belgian National Minimum Required Performance Limits (National MRPLs) for EGAs could be fulfilled.

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. © 2002 Elsevier Science B.V. All rights reserved.

Every other year scientists working in the field of residue analysis participate the "International Symposium on Hormone and Veterinary Drug Residue Analysis" and "Euroresidue" conferences. In each symposium a lot of innovative information is presented. In order to obtain a retrieval system for this growing amount of information, a database dedicated to these conferences was created. The main principles of the database were explained during the Fourth Euroresidue Conference (2000, Veldhoven), but it will be extended from subsequent conferences. It is not the aim to compete with other databases but only to have an easy-to-operate database containing information on hormone and veterinary drug residue analysis. In this second edition, the abstracts of all the proceedings have been added. Moreover, the database may be downloaded up-to-date from a website. © 2002 Elsevier Science B.V. All rights reserved.

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. © 2002 Elsevier Science B.V. All rights reserved.

The use of identification points (IPs) is a new approach to set up quality criteria for the identification of organic residues and contaminants: a laboratory is allowed to use any molecular spectrometric technique or combination of techniques in order to earn a minimum number of points. The system of IPs balances the identification power of the different analytical techniques and has the advantage that new techniques can be introduced very easily. © 2001 Elsevier Science B.V. All rights reserved.

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.

The use of corticosteroids in combination with other hormonal substances has long been known to result in increased mass gain with bovines. Practice has demonstrated, however, that even the single use of a glucocorticoid may result in growth promoting effects. In addition to the popular dexamethasone, more recently other corticosteroids have also been misused for fattening purposes. The first part of this study deals with the detection of two of them, namely betamethasone and triamcinolone acetonide. Betamethasone was administered orally to a cow at a dose of 50 mg d-1 for 5 d, then later the same cow was injected intramuscularly with a dose of 50 mg of betamethasone dipropionate. Excretion in urine and faeces was followed with both HPLC-enzyme immunoassay and a previously described method based on negative chemical ionization mass spectrometry (NCI-MS) after oxidation. For the triamcinolone acetonide study a cow was treated with 50 mg d-1 of the drug during a 7 d period. Excretion in faeces was followed with GC-NCI-MS. As triamcinolone acetonide is resistant to the previously described oxidation procedure, however, a hydrolysis step had to be introduced prior to oxidation. In addition to this specific modification necessary for triamcinolone acetonide, in a subsequent part of this study the original oxidation procedure with pyridinium chlorochromate was re-investigated especially to shorten the procedure. With the introduction of potassium dichromate the reaction time could be decreased from 3 h to 10 min.

European legislation forbids the use of beta-agonists as growth-promoting substances in cattle raised for human consumption. However, the use of beta-agonists is allowed as a therapeutic treatment of tocolysis for female cattle during calving and of respiratory diseases and tocolysis for horses not raised for human consumption. A maximum residue limit (MRL) of 0.5 microgram kg-1 for clenbuterol in the liver of cattle and horses is proposed by law. Residues of beta-agonists in liver are identified with LC-MS-MS. Using ion trap technology, it was possible to identify each analyte without the need to resolve completely the chromatographic peaks. For each analyte, specific fragment ion spectra were obtained. The coeluting or incompletely resolved peaks were separated mass spectrometrically. For tulobuterol, bromobuterol and mabuterol, qualitative information was obtained. All beta-agonists could be detected up to a concentration of 0.1 microgram kg-1. For clenbuterol, a limited quantitative validation was performed. A working range was defined for which the method was applicable. Quantification was based on the integration of the response of the analytes in spiked blank liver samples. The mean recovery was 15%. The relative standard deviation (RSD) values at different concentrations were below the maximum allowed RSD. The limit of detection of clenbuterol was 0.11 microgram kg-1. The limit of quantification was 0.21 microgram kg-1. It was possible to quantify clenbuterol below one-half of the MRL. The advantage of this method is the ease of use of the mass spectrometric separation to qualify and quantify the presence of four beta-agonists in liver.

Qualitative methods are used for illegal drugs (e.g. hormones) which have a so-called zero tolerance (i.e. the presence of even a very small amount is not tolerated). In practice a limit of determination is used to prove the presence or absence of an analyte. Quantitative analysis is necessary especially for residues with a maximum residue limit (MRL). The method used in this case must have a limit of quantification (much) lower than the MRL. In any case, quality criteria must be fulfilled before quantification. In this investigation the pro's and con's of different strategies towards the use of qualitative and quantitative methods are discussed from the point of view and the daily experience of routine control in two field laboratories and one national reference laboratory.

Although the ability to differentiate between endogenous steroids and synthetic homologues on the basis of their (13)C/(12)C isotopic ratio has been known for over a decade, this technique has been scarcely implemented for food safety purposes. In this study, a method was developed using gas chromatography-mass spectrometry/combustion/isotope ratio mass spectrometry (GC-MS/C/IRMS) to demonstrate the abuse of 17β-estradiol in cattle, by comparison of the (13)C/(12)C ratios of the main metabolite 17α-estradiol and an endogenous reference compound (ERC), 5-androstene-3β,17α-diol, in bovine urine. The intermediate precisions were determined as 0.46 and 0.26‰ for 5-androstene-3β,17α-diol and 17α-estradiol, respectively. This is, to the authors' knowledge, the first reported use of GC-MS/C/IRMS for the analysis of steroid compounds for food safety issues.

This study represents the second part of an interlaboratory study intended to develop an official modular Community confirmatory method for the detection of beta-agonists in animal feed. Homogeneous pools of primary extracts were prepared by means of an extraction module based on the conclusions of a previous part of this work. The primary extracts were further processed by four laboratories each using a different clean-up scheme. The final extracts thus obtained were cross-distributed between the same laboratories and measured either by GCMS or HPLC. Two laboratories (B and D) applied separate clean-up schemes for clenbuterol and salbutamol. All clean-up schemes for clenbuterol were found to be compatible with all end-determination steps. In contrast, for salbutamol clean-up method D was found not to be compatible with the end-determination steps applied by laboratories B and C. The results of this study have clearly demonstrated that the clean-up methods for both clenbuterol and salbutamol applied by laboratory B yielded superior recoveries with an acceptable standard deviation. Therefore, in conclusion to this study, the participating laboratories recommend the clean-up schemes applied by laboratory B to serve as part of the official Community confirmatory method.

In all EU member states, the use in livestock farming of certain substances having a hormonal action is prohibited. Clenbuterol, the beta-adrenergic agonist, has some growth promoting characteristics. Screening for clenbuterol can be carried out by an immunoassay. Gas chromatography-mass spectrometry (GC-MS) is very valuable for confirmatory purposes. In full scan MS it is impossible to fulfil the EU criteria of four diagnostic ions with one single ionisation mode. Some alternative possibilities are: (1) the use of two different ionisation modes, (2) the use of different derivatization methods or (3) the use of tandem MS. Each derivatisation or ionisation mode on its own did not give a sufficient number of ions. By combining these different possibilities we were able to obtain four ions, fulfilling the EU criteria.

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.

The European Commission, Measurement and Testing Programme (BCR) has initiated a project to improve the methodology for analysis of beta-agonists in animal feeding stuffs. An intercomparison of methods for clenbuterol in animal feed is described. The study involved 13 European laboratories which analysed a blank feed and three feed samples with three different levels of clenbuterol contamination. The participants used a variety of extraction (organic or aqueous solvents), clean-up (liquid-liquid, silica and C-18 solid phase and immuno-affinity chromatography) and end-point detection (HPLC, GC-MS and TLC) steps. The purpose of this study was to identify and to quantify clenbuterol. The coefficient of variation from all the results for the low level (25 micrograms/kg) was 39%, for the intermediate level (100 micrograms/kg) 52% and for the high level (1000 micrograms/kg) 35%. The study showed that the initial extraction, the modular clean-up step and their compatibility to the HPLC and the GC-MS determination step were critical steps.

The detection of illegal growth promoters in cattle urine by conventional immunoassays, such as radioimmunoassay (RIA) and enzyme‐linked immunosorbent assay (ELISA) has been extensively described. ...

Multi‐analyte screening methods for β‐agonists in faeces and feeds based on enzyme immunoassay (EIA) were developed. Commercially available EIA kits were used, and the results presented. The extraction for both matrices was performed under acid conditions. The addition of an organic solvent to the extradant, resulting in higher recoveries, was only retained for feeds as the background absorption for faeces increased substantially. Clean‐up for faecal extracts was achieved in a one‐step extraction with isobutyl alcohol. Detection limits in faeces were slightly different for the two types of kits used. They ranged from 0.8ng g−1 for clenbuterol to 10ng g−1 for terbutaline. In feeds, corresponding detection limits for both compounds were 5 ng g−1 and 110 ng g−1. Confirmation of EIA positives was established by liquid chromatography with post‐column diazotation and gas chromatography‐mass spectrometry. In the screening of faeces over 1 year about 70% of the EIA positive samples were confirmed as positive.

International audience; Corticosteroids are hormonal substances widely used in human and veterinary medicine for their anti-inflammatory properties. Among the numerous existing artificial corticosteroids, dexamethasone remains the most commonly used, mainly throughout esterified forms such as acetate or phosphate. An experimental study was designed to assess its drug residue levels in urine and feces, as well as its fixation in bovine hair following a single administration of 0.15 mg/kg b.w. dexamethasone acetate and 0.12 mg/kg b.w. dexamethasone sodium phosphate. Different analytical methods based on GC-MS or LC-MS/MS were used for measuring dexamethasone and its esterified forms, which were implemented in 3 different European laboratories in the field that collaborated for this study. The obtained results confirmed the high and rapid urinary excretion rate of dexamethasone, with a maximal concentration (267 mu g/L) measured one day after administration and 98% elimination within 3 days. The concentrations obtained with the GC-NCI-MS procedure (using chemical oxidation as derivatization) were found significantly higher than the ones obtained with LC-ESI-MS/MS, indicating a possible contribution of dexamethasone phase I and/or II metabolites to the monitored signal. Fecal elimination was also found rapid (95% elimination within 3 days) with a maximum concentration level (28.5 mu g/kg) observed one day after administration. Detectable levels of dexamethasone in hair appeared on day 2 (11.5 mu g/kg), reached a maximum around one week, and could be identified until 22 days upon treatment, establishing the suitability of hair as a biological matrix for medium to long-term residue controls of dexamethasone. (C) 2010 Elsevier Inc. All rights reserved.

In the analysis of residues of anabolic agents the use of combined techniques, especially gas chromatography—mass spectrometry, is of increasing interest. When this method is used for the analysis of residues of anabolics it is very important to have a knowledge of the characteristics of the different types of apparatus on the market and to be sure that the results are not “apparatus-dependent”. In this study two mass-selective detectors, the Hewlett-Packard 5970 and the ion trap detector 800, were compared by injecting the same samples of derivatized anabolics (standard solutions) into the two systems.

A residue is a trace (microg kg(-1), ng kg(-1)) of a substance, present in a matrix. Banned substances, such as growth promoters, which are abused in animal fattening and where this article is focused on, may be divided into four major groups: thyreostats, anabolics or anabolic steroids, corticosteroids and beta-agonists or repartitioning agents. The combination of chromatographic techniques with mass spectrometry (GC-MS(n), LC-MS(n), etc.) plays a key role in the production of specific results in residue analysis. In this review, the past, present and future of mass spectrometry in this area are discussed in the light of the impact of these substances on human health and the reliable production of analytical results, ready for challenge in a court.

Cholesterol is a well-known component in fats of animal origin and it also is the precursor of natural hormones. Phytosterols appear in plants and only differ slightly in structure from cholesterol. An important difference however is the low absorption in the gut of phytosterols and their saturated derivatives, the phytostanols. As a result, there is time for all kind of reactions in faecal material inside and outside of the gut. Determination of the abuse of natural hormones may be based on gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Abuse of natural hormones changes the 13C/12C ratio of some metabolites during a relatively long time. The formation of (natural) hormones in the gut may interfere with this method. Designer drugs are mainly known from sports doping. In animal fattening, designer drugs may be used as well. Small changes in the structure of (natural) hormones may lead to a new group of substances asking for new strategies for their detection and the constatation of their abuse.

Since the 1970s, many analytical methods for the detection of illegal growth promoters, such as thyreostats, anabolics, beta-agonists and corticosteroids have been developed for a wide range of matrices of animal origin, including meat, fat, organ tissue, urine and faeces. The aim of this study was to develop an analytical method for the determination of ng L(-1) levels of estrogens, gestagens, androgens (EGAs) and corticosteroids in aqueous preparations (i.e. drinking water, drinking water supplements), commercially available on the 'black' market. For this, extraction was performed with Bakerbond C18 speedisk, a technique commonly used in environmental analysis. After fractionation, four fractions were collected using a methanol:water gradient program. Gas chromatography coupled to electron impact multiple mass spectrometry (GC-EI-MS2) screening for the EGAs was carried out on the derivatized extracts. For the detection of corticosteroids, gas chromatography coupled to negative chemical ionization mass spectrometry (GC-NCI-MS) was used after oxidation of the extracts. Confirmation was done by liquid chromatography coupled to electrospray ionization multiple mass spectrometry (LC-ESI-MS2). The combined use of GC and LC coupled to MS enabled the identification and quantification of anabolics and corticosteroids at the low ng L(-1) level. This study demonstrated the occurrence of both androgens and corticosteroids in different commercial aqueous samples.

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-MSn) and gas chromatography-multiple mass spectrometry (GC-MSn) 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).

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. © 2004 Taylor & Francis Ltd.

A new approach to the search for residues of unknown growth promoting agents such as anabolic steroids and beta-agonists in feed is presented. Following primary extraction and clean-up, samples are separated using gradient liquid chromatography (LC). The effluent is split towards two identical 96-well fraction collectors and an optional electrospray quadrupole time-of-flight mass spectrometry (QTOFMS) system for accurate mass measurement. One 96-well plate is used for a bioassay (enzyme-immuno assay, receptor assay) and will detect the bioactivity and position of the relevant peak in the chromatogram. The positive well in the second 96-well plate is used for identification by LC/QTOFMS/MS. The value of this LC/bioassay/QTOFMS/MS methodology is highlighted by the finding and structure elucidation of a new beta-agonist in a feed extract.

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.

In the European Union, the use of growth promoting substances such as thyreostats, anabolics (products with estrogenic, androgenic or gestagenic action) and beta-agonists in animal fattening is forbidden. Corticosteroids, such as dexamethasone, although considered catabolic substances, have been administered to food producing animals in order to achieve mass gains. For the analysis of injection sites and of suspect cocktails (found at the farm), a number of HPTLC and HPLC methods are used. However, in injection sites and also in cocktails found at the farm, sometimes many unknown substances are found. In this investigation, a multiple mass spectrometric (MSn) method was developed. The method is based on rapid extraction of the matrix with methanol and direct infusion of the extract into the interface of the mass spectrometer. Tables that summarise the masses of corticosteroids and their possible esters are presented.

Within several regional field laboratories and the national reference laboratory a harmonised methodology for the analysis of anabolic residues in faecal samples was developed. The method consists of a liquid–liquid and a solid-phase extraction step, followed by a high-performance liquid chromatography purification step. Using gas chromatography-mass spectrometry, currently illegally used anabolic steroids can be detected in faeces at the ppb level. Within this context acidification, followed by centrifugation under cooling, allows efficient, practical and rapid defatting of faecal samples. Furthermore, a combination of a silica and an aminopropyl solid-phase extraction column was found to give the best results as regards the sample purification process.

The objective of this intercomparison study was to evaluate the qualitative aspects and the interlaboratory performance of the method selected to be recommended as the official Community reference confirmatory method for the analysis of beta-agonists in animal feed. This method contains three possible options, i.e. a narrow range method for clenbuterol-type compounds based either on HPLC or on GCMS as the end-determination step and a broad range GCMS method for clenbuterol-type and salbutamol-type-beta-agonists. Three types of animal feed materials were provided: a series of blank materials and two series of materials contaminated with clenbuterol and salbutamol at a low and a high level, respectively. The results showed that the majority of the laboratories were able to identify blank, low and high level materials both for clenbuterol and salbutamol. For clenbuterol the narrow range GCMS method has been shown to be the most satisfactory. Although the participants had comments on the purity of the extracts obtained by means of the broad range method it was found appropriate as a multi-residue method which is able to measure simultaneously clenbuterol-type and salbutamol-type beta-agonists. A statistical evaluation of the quantitative measurement was also performed.

New projects of the European Commission, Measurement and Testing Programme (BCR) were set up in order to develop a modular sample preparation system for the determination of beta-agonists and animal feeds. Three phases are included: an extraction study, a clean-up study and finally a Second Intercomparison. This paper describes the extraction study in which four laboratories were involved. A total of 33 extraction conditions were tested regarding their yield on clenbuterol and salbutamol, their compatibility towards several clean-up and chromatographic end-methods and the influence of undesired coextractives. The conditions differed with respect to five factors: with or without organic solvent, temperature, pH, agitation and centrifugation. Their influence was examined via a ruggedness-test approach. A unique set-up allowed the combination of individual results in a complete factorial design. The addition of an organic solvent was found to be the most important factor. Interactions between factors were also studied. The best combinations of factors regarding the extraction are given. Finally limits for applicability and influence of organic solvents, pH and temperature were evaluated in a fifth laboratory towards enzyme immunoassay as detection method.

For several years, the misuse of dexamethasone and its esters in livestock production has been clearly demonstrated. The first part of the present study deals with the elaboration of a sensitive and specific method for the determination of residues of dexamethasone in excreta at the ppb level. Sample preparation for urine and faeces, including high-performance liquid chromatography (HPLC) fractionation, was carried out. The detection was based on established methodology employing negative chemical ionization–mass spectrometry (NCI–MS) after oxidation of the dexamethasone. In comparison with previous literature, the yield of oxidized dexamethasone was substantially improved and the oxidation procedure was made more simple and robust. In the second part of the study, the relationship between the dose of dexamethasone administered and the levels of the drug in excreta was investigated using this method, as was the ratio between drug levels in urine and faeces. Treatment was carried out for 7 d with an oral dose of 50 mg d–1, the maximum levels found in urine and faeces were 980 and 744 ppb, respectively. While the elimination via faeces responded much slower at the start and the end of treatment, the final part of both excretion profiles were very similar and a level of 1 ppb was reached in both matrices 9 d after the end of treatment. Gas chromatography–mass spectrometry (GC–MS) results obtained for the urine samples were compared with those obtained with direct enzyme immunoassay.

Since the late 1980s all of the laboratories involved in high-performance thin-layer chromatography (HPTLC) control of hormonal residues in kidney fat, have occasionally detect a green fluorescent spot with similar RF values and colour to those observed for methyltestosterone (MT). This spot (product) could lead to false positive results for MT and was thus named 'le faux méthyl' (the false methyl) by a french speaking colleague. All of the samples with a false methyl spot also contained a relatively high concentration of progesterone. Differentiation of this product from methyltestosterone can be performed in three ways: firstly, extra HPTLC on reversed-phased plates, secondly, extra purification of the extract with HPLC prior to HPTLC and thirdly, gas chromatography--mass spectrometry. This interference was identified as 20 beta-hydroxyprogesterone, a by-product of progesterone. The problem of the false methyl was not only linked with the TLC characteristics of MT but also to the progesterone used as standard. Some laboratories used an analytical-reagent grade standard and others used commercial progesterone powders as standards (e.g., obtained in crude form from pharmaceutical companies). The commercial-grade progesterones showed two spots in comparison with the analytical standard that showed just one spot. As the false methyl was observed not only in kidney fat and meat samples, but also in illegal hormone cocktails, it was concluded that we had detected a by-product of an illegally used 'natural progesterone'.