Thyreostatic drugs, stability in bovine and porcine urine.

Thyreostatic drugs, illegally administrated to livestock for fattening purposes, are banned in the European Union since 1981. For monitoring their illegal use, sensitive and specific analytical methods are required. In this context, the knowledge of the stability in a matrix is of primary importance. This study aimed at evaluating the effects of preservation, number of freeze-thaw cycles, and matrix-related variables on the stability of thyreostatic drugs in the urine of livestock. Finally, the developed conservation approach was applied on incurred urine samples, which displayed traces of the thyreostat thiouracil below the recommended concentration of 10 µg L(-1). The stability study confirmed the negative influence of preservation (8 h) at room temperature and at -70 °C, decreases in concentration of more than 78.0% were observed for all thyreostats, except for 1-methyl-2-mercaptoimidazole and 2-mercaptobenzimidazole. Additionally, investigation of matrix-related variables indicated significant impacts of the presence of copper (p = 0.001) and the pH (p = 0.002). Next, an optimised pre-treatment (pH 1 and 0.1 M ethylenediaminetetraacetic acid disodium salt dehydrate) significantly differing from the original conservation approach (p < 0.05) was developed, which proved capable of delaying the decrease in concentration and improved the detection in time for both spiked as well as incurred urine samples. In the future, it seems highly advisable to apply the developed pre-treatment on incurred urines upon sampling, before thyreostat analysis. Additionally, it is recommendable to limit preservation of urine samples at room temperature, but also in the freezer prior to thyreostat analysis.

Multiclass screening in urine by comprehensive two-dimensional liquid chromatography time of flight mass spectrometry for residues of sulphonamides, beta-agonists and steroids.

Nowadays routine residue monitoring involves the analysis of many compounds from different classes, mainly in urine. In the past two decades, developments heavily focused on the use of mass spectrometers (MS) and faster and more sensitive MS detectors have reached the market. However, chromatographic separation (CS) was rather ignored and the cognate developments in CS were not in line. As a result, residue analysis did not improve to the extent anticipated. CS by LC x LC is a promising technique and will enable a further increase in the range of compounds and compound classes that can be detected in a single run. In the present study, a self-built LC x LC system, using a 10 port valve, was connected to a single quadrupole MS with electrospray interface. Standards containing a mixture of sulphonamides, ß-agonists and (steroid) hormones, 53 compounds, in total, were analysed. Results demonstrated that these compounds were well separated and could be detected at low levels in urine, i.e. limit of detection (LOD) from 1 µg L-1 for most ß-agonists to 10 µg L-1 for some sulphonamides and most hormones. To enhance the sensitivity, optimisation was performed on an advanced commercial LC x LC system connected to a full scan accurate MS. This ultimately resulted in a fast high throughput untargeted method, including a simple sample clean-up in a 96-well format, for the analysis of urine samples.

Applicability of an innovative steroid-profiling method to determine synthetic growth promoter abuse in cattle.

A robust LC-MS/MS method was developed to quantify a large number of phase I and phase II steroids in urine. The decision limit is for most compounds lower than 1ngml-1 with a measurement uncertainty smaller than 30%. The method is fully validated and was applied to assess the influence of administered synthetic steroids and beta-agonists on the steroidogenesis. From three animal experiments, clenbuterol, diethylstilbestrol and stanozolol, the steroid profiles in urine of bovine animals were compared before and after treatment. It was demonstrated that the steroid profiles were altered due to these treatments. A predictive multivariate model was built to identify deviations from normal population steroid profiles. The abuse of synthetic steroids can be detected in urine samples from bovine animals using this model. The samples from the animal experiments were randomly analysed using this method and predictive model. It was shown that these samples were predicted correctly in the exogenous steroids group.

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.

Sports doping: emerging designer and therapeutic ß2-agonists.

Beta2-adrenergic agonists, or ß2-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 ß2-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 ß2-agonists molecules either by modifying the molecule of known ß2-agonists or by introducing moieties producing indole-, adamantyl- or phenyl urea derivatives. New emerging ß2-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.

Endogenous steroid profiling by gas chromatography-tandem mass spectrometry and multivariate statistics for the detection of natural hormone abuse in cattle.

For years it has been suspected that natural hormones are illegally used as growth promoters in cattle in the European Union. Unfortunately there is a lack of methods and criteria that can be used to detect the abuse of natural hormones and distinguish treated from non-treated animals. Pattern recognition of steroid profiles is a promising approach for tracing/detecting the abuse of natural hormones administered to cattle. Traditionally steroids are analysed in urine as free steroid after deconjugation of the glucuronide (and sulphate) conjugates. The disadvantage of this deconjugation is that valuable information about the steroid profile in the sample is lost. In this study we develop a method to analyse steroids at very low concentration levels (ng l(-1)) for the free steroid, glucuronide and sulphate conjugates in urine samples. This method was used to determine concentrations of natural (pro)hormones in a large population (n = 620) of samples from male and female bovine animals and from bovine animals treated with testosterone-cypionate, estradiol-benzoate, dihydroepiandrosterone and pregnenolone. The data acquired were used to build a statistical model applying the multivariate technique 'Soft Independent Modeling of Class Analogy' (SIMCA). It is demonstrated that by using this model the results of the urine analysis can indicate which animal may have had illegal treatment with natural (pro)hormones.

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.

Multi-residue screening of a minimum package of anabolic steroids in urine with GC-MS.

The method comprises the screening of two groups of anabolic compounds, the stilbenes and several steroids. All compounds, inclusive their metabolites when possible, for which gas chromatography-mass spectrometry (GC-MS) currently is the preferred analytical technique, are included. Two different derivatives are prepared. One group, including the stilbenes, is detected as HFB derivative (Method 1), the second group is detected as TMS derivative (Method 2). The method is used to perform a qualitative and semi-quantitative analysis of a minimum package of anabolic steroids to be included in National Residue Control Plans based on Council Directive 96/23 and complies with the current Minimum Required Performance Limits. The method has been validated according to Commission Decision 2002/657/EC. The CCalpha and CCbeta values are based on the detection of the most abundant ion. Results of validation experiments are presented. The method is flexible and due to the non-specific sample clean-up more and new anabolic compounds can be easily added in order to new monitoring requirements.

Development of a method which discriminates between endogenous and exogenous beta-boldenone.

One potential explanation for the presence of beta-boldenone in calf urine is contamination of the sample with feces containing beta-boldenone. It has been demonstrated that after oral and intramuscular administration of beta-boldenone esters, several metabolites are formed and excreted in urine. One of the (minor) metabolites is 6beta-hydroxy-17alpha-boldenone. This paper describes an analytical method that can discriminate between unconjugated boldenone, its glucuronide- and sulphate-conjugates, 6beta-hydroxy-17alpha/beta-boldenone and coprostanol, a marker for fecal contamination. The method was applied to all samples suspected to contain boldenone within the Dutch National Residue Control Plan. Approximately 10,000 samples of urine were screened (LC-MS) in 2004-2005 by VWA-East, one of the official Dutch control laboratories, from which 261 samples were suspected to contain boldenone. These samples were all analyzed for their conjugation state, 6beta-hydroxy-17alpha/beta-boldenone and for the presence of coprostanol. Alfa-boldenone, the major metabolite in bovine urine after boldenone-ester administration, was found in a large number of these samples. The presence of alpha-boldenone was proven also to be a result of fecal contamination. None of the samples tested contained residues of the metabolite 6beta-hydroxy-17alpha/beta-boldenone. Not finding this metabolite indicates that the origin of alpha-boldenone-conjugates is endogenous. The results confirm that the presence of unconjugated beta-boldenone and alpha-boldenone conjugates next to alpha-boldenone are no indicators for illegal administration of boldenone-esters. No indications were obtained that conjugated beta-boldenone can be of endogenous origin.

Phytosterol consumption and the anabolic steroid boldenone in humans: a hypothesis piloted.

The presence of the anabolic steroid boldenone in animals has become a research topic as its occurrence is proposed to be a marker for illegal hormone administration. However, boldenone can also be formed from beta-sitosterol, a phytosterol present in animal feed, as well as from endogenous sources. The observations in animals together with the increased consumption of phytosterol-enriched foods in the Western population led the authors to the hypothesis that consumption of phytosterol-enriched foods might possibly lead to increased boldenone levels in humans. The authors performed a pilot study among female volunteers (n = 10) to investigate whether boldenone concentrations in urine were detectable after consumption of 25 g day(-1) of phytosterol-enriched margarines for 1 week. Urine samples were collected at days 0, 3 or 4, and 7. Urine of a sitosterolemia (a rare autosomal recessively inherited lipid metabolic disorder) patient was collected as a positive control case. No traces of boldenone were detected in either the volunteers or in the patient. In conclusion, there is no evidence of formation of boldenone in women after consumption of the recommended amount of phytosterol-enriched margarines.

Determination of resorcylic acid lactones in biological samples by GC-MS. Discrimination between illegal use and contamination with fusarium toxins.

An EU project, FAIR5-CT-1997-3443, has been undertaken to distinguish illegal use of zeranol from consumption of food contaminated with Fusarium spp. toxin. One of the tasks was development of screening and confirmatory methods of analysis. This paper describes a new method based on two-step clean-up and GC-MS analysis. The first clean-up step is matrix-dependant; the second is applicable to both urine and meat. The MS is operated in negative chemical ionisation mode. The method is quantitative for zeranol and taleranol, alpha- and beta-zearalenol, and zearalenone and qualitative for zearalanone. Validation was performed according to the latest EU performance criteria (Commission Decision 2002/657). For analysis of urine CC(alpha) and CC(beta) for the method (microg L(-1)) were 0.06-0.11 for zeranol, 0.07-0.12 for taleranol, 0.07-0.11 for alpha-zearalenol, 0.21-0.36 for beta-zearalenol, 0.35-0.60 for zearalenone, and 0.19-0.33 zearalanone. Within-laboratory reproducibility was 16.2, 11.2, 31.9, 30.1, 26.6, and 54.2% for zeranol, taleranol, alpha-zearalenol, beta-zearalenol, zearalenone, and zearalanone, respectively. It was found that all the compounds are stable in urine at -20 degrees C for at least a year. Part of the validation program was organisation of a small proficiency study (ringtest) and a correlation study with an LC-MS-MS method developed by the Veterinary Science Division (VSD; Belfast, UK-NI). This study showed there was good correlation between results from both laboratories. The method can be used for quantitative analysis discriminating illegal use of zeranol from consumption of zearalenone-contaminated food.

Endogenous occurrence of some anabolic steroids in swine matrices.

Following findings of 17beta-19-nortestosterone (150-200 microg 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.

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.

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 assay for zeranol, taleranol and the Fusarium spp. toxins in bovine urine using liquid chromatography-tandem mass spectrometry.

A method is described for the quantitative determination of the veterinary drug zeranol, its epimer taleranol and the mycotoxins zearalenone, alpha-zearalenol and beta-zearalenol in bovine urine. The method is based on liquid chromatography coupled to negative-ion electrospray mass spectrometry-mass spectrometry of urine extracts prepared by solid-phase extraction with C(18) columns. Two transition ions at m/z 277 and 91 are monitored for zeranol and taleranol along with the transition ion at m/z 281 for their respective deuterated (d(4)) internal standards. Similarly, two transitions are monitored for each of the three mycotoxins along with a transition ion for each of their corresponding internal standards. The method has been validated according to the new European Union criteria for analysis of veterinary drug residues, and is suitable for monitoring urine samples taken under National Surveillance Schemes. The method has been validated at 1, 1.5 and 2 ng ml(-1) for zeranol and taleranol and at 5, 10 and 15 ng ml(-1) for each of the three mycotoxins. Correlation between the described method and a routine method, based on gas chromatography-mass spectrometry, was assessed using a range of naturally incurred samples.

Interlaboratory ring test of time-resolved fluoroimmunoassays for zeranol and alpha-zearalenol and comparison with zeranol test kits.

Many zeranol immunoassay test kits cross-react with toxins formed by naturally occurring Fusarium spp. fungi, leading to false-positive screening results. This paper describes the evaluation and application of recently published, dry reagent time-resolved fluoroimmunoassays (TR-FIA) for zeranol and the toxin alpha-zearalenol. A ring test of bovine urine fortified with zeranol and/or alpha-zearalenol in four European Union National Reference Laboratories demonstrated that the TR-FIA tests were accurate and robust. The alpha-zearalenol TR-FIA satisfactorily quantified alpha-zearalenol in urine fortified at 10-30 ng ml(-1). The specificity-enhanced zeranol TR-FIA accurately quantified zeranol in the range 2-5 ng ml(-1) and gave no false-positive results in blank urine, even in the presence of 30 ng ml(-1) alpha-zearalenol. Zeranol TR-FIA specificity was demonstrated further by analysing incurred zeranol-free urine samples containing natural Fusarium spp. toxins. The TR-FIA yielded no false-positive results in the presence of up to 22 ng ml(-1) toxins. The performance of four commercially available zeranol immunoassay test kits was more variable. Three kits produced many false-positive results. One kit produced only one potential false-positive using a protocol that was longer than that of the TR-FIA. These TR-FIAs will be valuable tools to develop inspection criteria to distinguish illegal zeranol abuse from contamination arising from in vivo metabolism of Fusarium spp. toxins.

Prevalence of zeranol, taleranol and Fusarium spp. toxins in urine: implications for the control of zeranol abuse in the European Union.

There is currently little information concerning the prevalence of zeranol and taleranol in animal urine following metabolism of the naturally occurring Fusarium spp. toxins. An epidemiological study is described which involves four European Union control laboratories in which 8008 urine samples were screened for the presence of zeranol using a time-resolved fluoroimmunoassay (TR-FIA). Of these samples, 93.6% screened negative for zeranol. All samples testing positive for zeranol were then analysed with a confirmatory method. Based on the confirmatory results, the TR-FIA-positive samples were then categorized as false-positive, true-positive or 'equivocal' (zeranol/taleranol and the Fusarium spp. toxins detected). The true-positive samples represented only 0.05% of the total number of samples (n = 4). After statistical analysis, 170 of 174 equivocal samples proved to belong to a 'normal' population in which the amount of zeranol/taleranol could be related to the total amount of Fusarium spp. toxins through a linear regression with a 99% prediction interval. This suggested that the presence of zeranol in these samples might be due to in vivo metabolism of the Fusarium spp. toxins. The presence of zeranol in the four remaining 'outliers' might be attributable to zeranol abuse rather than to natural contamination. The results are of interest for control laboratories as they might provide an analytical tool to help distinguish between abuse and natural contamination in zeranol testing.

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.

Multi-laboratory study of the analysis and kinetics of stanozolol and its metabolites in treated calves.

The European Union banned the use of anabolic steroids for cattle fattening in 1988. Analytical techniques able to detect trace amounts of the parent drugs and their metabolites are mandatory for the control of abuse. Stanozolol (Stan) is an anabolic steroid that is often found in injection sites and cocktails. However, it has never been detected in tissues (kidney fat, meat) or excreta (urine, faeces) taken during regulatory inspection. The difference between the structure of Stan and the other steroids (a pyrazole ring fused to the androstane ring system) is probably the cause of this phenomenon. In the multi-laboratory study described here, veal calves were treated with intramuscular doses of Stan. In the excreta of these calves the presence, absence and/or concentration of Stan and of its major metabolites 16 beta-hydroxystanozolol and 3'-hydroxystanozolol were determined. For the determination of these analytes the different laboratories used different extraction and clean-up procedures and also evaluated different analytical techniques such as GC-MS (negative chemical ionization) and LC-MS-MS. The aim of this investigation was to explore which analyte should be validated for veterinary inspection purposes.