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.

Characterisation of steroids in wooden crates of veal calves by accelerated solvent extraction (ASE) and ultra-high performance liquid chromatography coupled to triple quadrupole mass spectrometry (U-HPLC-QqQ-MS-MS).

Illegal steroid administration to enhance growth performance in veal calves has long been, and still is, a serious issue facing regulatory agencies. Over the last years, stating undisputable markers of illegal treatment has become complex because of the endogenous origin of several anabolic steroids. Knowledge on the origin of an analyte is therefore of paramount importance. The present study shows the presence of steroid analytes in wooden crates used for housing veal calves. For this purpose, an analytical procedure using accelerated solvent extraction (ASE(R)), solid-phase extraction (SPE) and ultra-high performance liquid chromatography coupled to triple quadrupole mass spectrometry (U-HPLC-MS-MS) is developed for the characterisation of androstadienedione (ADD), boldenone (bBol), androstenedione (AED), beta-testosterone (bT), alpha-testosterone (aT), progesterone (P) and 17alpha-hydroxy-progesterone (OH-P) in wood samples. In samples of wooden crates used for housing veal calves, ADD, AED, aT and P could be identified. Using the standard addition approach concentrations of these analytes were determined ranging from 20 +/- 4 ppb to 32 +/- 4 ppb for ADD, from 19 +/- 5 ppb to 44 +/- 17 ppb for AED, from 11 +/- 6 ppb to 30 +/- 2 ppb for aT and from 14 +/- 1 ppb to 42 +/- 27 ppb for P, depending on the sample type. As exposure of veal calves to steroid hormones in their housing facilities might complicate decision-making on illegal hormone administration, inequitable slaughter of animals remains possible. Therefore, complete prohibition of wooden calf accommodation should be considered.

Absence of an effect of dietary fibre or clinoptilolite on boar taint in entire male pigs fed practical diets.

This study aimed to evaluate the possibility of reducing boar taint in boars (Piétrain×Hybrid) by addition of different feed ingredients (raw potato starch (RPS) 10%, raw potato starch 10%+wheat bran 5% (RPS+WB), lupins 10%, inulin 5%, clinoptilolite 1%) to a standard diet over a period of 4-6 weeks before slaughter. Control boars (CBOAR) as well as barrows were fed the standard diet. Efficacy of the different feed ingredients was evaluated by different boar taint detection methods: hot iron method, consumer panel, expert panel and laboratory analysis. According to all detection methods, clear differences were noticeable between boars and barrows. No differences in boar taint incidence were found between the boars on the different dietary treatments as assessed by consumers, experts, hot iron method or the concentration of skatole in fat. A significant effect on indole level was found, but no further differentiation could be made. The concentration of backfat androstenone was significantly higher for the inulin and control boar group compared to the lupin group. In conclusion, none of the feeding strategies tested in this study reduced boar taint in boars at the given percentages.

Past, present and future of mass spectrometry in the analysis of residues of banned substances in meat-producing animals.

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.

Development and validation of a method for simultaneous analysis of the boar taint compounds indole, skatole and androstenone in pig fat using liquid chromatography-multiple mass spectrometry.

Boar taint in entire male pigs remains a problem for fresh pork production. Since castration of pigs will be prohibited in the future on animal welfare reasons, attempts are made to detect boar taint pre and post mortem. Post mortem techniques focus on simultaneous quantification of the three boar taint substances by one simple and reliable method. In this study a liquid chromatographic multiple mass spectrometric (LC-MS(n)) method has been developed and validated for the simultaneous determination of indole (2,3-benzopyrrole, ID), skatole (3-methylindole, SK) and androstenone (5alpha-androst-16-en-3-one, AEON) in pig fat samples. Sample preparation was kept as short as possible, since a single extraction method for structurally different indols and steroids was seeked after. Analytes were extracted from the fat matrix by methanol and clean-up consisted of freezing the extract in liquid nitrogen followed by a filtration step. The analytes were chromatographically separated on a Symmetry C(18) column. Recoveries for ID, SK and AEON, as calculated from fortified fat samples using 2-methylindole (2-MID) as internal standard, were 96, 91 and 104%, respectively. However, matrix interferences were encountered determining the androstenone levels in fat. Linearity, determined in fat samples, was in the range of 50-1600 microg kg(-1) for the indolic compounds and 125-2000 microg kg(-1) for the steroid AEON. The correlation coefficients (R(2)) of the calibration curves were 0.998 for ID, 0.997 for SK and 0.810 for AEON.

Metabolism of methenolone acetate in a veal calf.

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.

Testosterone metabolism in Neomysis integer following exposure to benzo(a)pyrene.

Cytochromes P450 (CYPs) are important enzymes involved in the regulation of hormone synthesis and in the detoxification and/or activation of xenobiotics. CYPs are found in virtually all organisms, from archae, and eubacteria to eukaryota. A number of endocrine disruptors are suspected of exerting their effects through disruption of normal CYP function. Consequently, alterations in steroid hormone metabolism through changes in CYP could provide an important tool to evaluate potential effects of endocrine disruptors. The aim of this study was to investigate the potential effects of the known CYP modulator, benzo(a)pyrene (BaP), on the testosterone metabolism in the invertebrate Neomysis integer (Crustacea; Mysidacea). N. integer were exposed for 96 h to 0.43, 2.39, 28.83, 339.00 and 1,682.86 microg BaP L(-1) and a solvent control, and subsequently their ability to metabolize testosterone was assessed. Identification and quantification of the produced phase I and phase II testosterone metabolites was performed using liquid chromatography coupled with multiple mass spectrometry (LC-MS2). Significant changes were observed in the overall ability of N. integer to metabolize testosterone when exposed to 2.39, 28.83, 339.00 and 1,682.86 microg BaP L(-1) as compared to the control animals.

Rapid and high-performance analysis of thyreostatic drug residues in urine using gas chromatography-mass spectrometry.

A more sensitive method was developed using the hyphenated technique of gas chromatography-mass spectrometry (GC-MS) supplementary to the official high-performance thin-layer chromatography (HPTLC) method. Even combined with less efficient extraction and clean-up methods, GC-MS is able to lower the detection limit to less than 50 ppb. The powerful technique of GC-MS-MS is tried out to reduce the detection limit even more, in combination with simplified extraction methods. This time-saving approach combined with the increase in sensitivity is of great importance for a routine technique.

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.

Gas chromatographic-tandem mass spectrometric analysis of clenbuterol residues in faeces.

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.

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.

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.

Graphical analysis of the formation function-II: the pseudo cross-over point.

In a further attempt to enhance the possibility of interpreting the formation function, a number of special shapes of the formation function are discussed, which give rise to a pseudo cross-over point. It is shown that under certain conditions a pseudo cross-over point can be found in the following cases: (1) two series of homonuclear complexes, (2) a mixture of a series of homonuclear and polymeric complexes, (3) a series of mononuclear complexes and two polynuclear complexes with nearly the same composition, (4) a system which gives a real cross-over point, and one or more polynuclear complexes, (5) a system of two polynuclear complexes. The conditions are mainly discussed in terms of the composition of the complexes. Calculated curves illustrate the different possibilities.

Graphical analysis of the formation function-I The real cross-over point.

From a mathematical treatment, it has been proved that a family of formation curves shows a real cross-over point in the following cases: (a) a mixture of mononuclear complexes BA, BA(2) ... BA(N) and polynuclear complexes of general formula (B(q)A(p))(n), provided that p/q < N; (b) a mixture of homonuclear complexes B(Q)A(p), where Q is constant, and mononuclear complexes shows at least one cross-over point if the maximum value for p is smaller than NQ and at least two cross-over points if NQ lies between the minimum value and the maximum value for p. Curves calculated for three different examples are in perfect agreement with the theory.

A potentiometric study of the complexes formed between Ni(II) and Zn(II) and 3-mercaptopropionic acid.

The composition and stability constants of the complexes formed between Ni(2+) and Zn(2+) and 3-mercaptopropionic acid (3-MPA) were studied by a potentiometric method at 25 degrees and in 0.5M KNO(3). For the system Zn(2+)/3-MPA. a mixture of the mononuclear complex BA(2) and the polynuclear complexes B(3)A(4). and B(4)A(6) was found (B means the metal ion and A the ligand). The system Ni(2+)/3-MPA can be represented by the complexes B(5)A(10), B(6)A(11) B(6)A(9) and B(6)A(12). In this series the last two complexes are predominant.

Gas chromatographic-mass spectrometric confirmation of 19-nortestosterone in the urine of untreated boars--effect of the administration of Laurabolin.

The presence of 17 beta-19-nortestosterone (nandrolone, NT, 17 beta-19-NT) and its epimer 17 alpha-19-nortestosterone (epiNT, 17 alpha-19-NT) was investigated in the urine of six untreated boars, obtained from experimental farms. The presence of 17 beta-19-nortestosterone was screened by RIA and HPTLC and confirmed by GC-MS analysis. Additionally, the two epimers (NT and epiNT) were investigated in the urine of a boar (two-year-old miniature male pig weighing 50 kg) before and after injection of 100 mg Laurabolin (nortestosterone laurate, Intervet N.V., Belgium). The isolation of the steroids was based on sample clean-up with solid phase extraction and subsequent high-performance liquid chromatography. Both gas chromatographic retention data and mass spectrometric data (selected ion monitoring and full spectrum) were used for detection and identification. The presence of 17 beta-19-nortestosterone in the urine of the boars that were not injected proves the endogenous production of the steroid. The absence of the 17 alpha-epimer in the urine of the injected boar suggests that 17 alpha-19-nortestosterone is not a major metabolite of 17 beta-19-nortestosterone.

A validated ultra-high performance liquid chromatography coupled to high resolution mass spectrometry analysis for the simultaneous quantification of the three known boar taint compounds.

Boar taint is an off-odour that can occur when meat or fat from entire male pigs is heated. Most of the currently available analytical methods are not capable of detecting the three known boar taint compounds (indole, skatole and androstenone) simultaneously, which renders their analysis often labour-intensive and time-consuming as separate analyses are required. In this study a validated U-HPLC-HR-Orbitrap-MS analysis method is described for the quantitative determination of the three boar taint compounds in fat. The sample pre-treatment involves a melting step followed by extraction with methanol and clean-up consisting of a freezing step and solid phase extraction (HLB cartridges). The analytes are then chromatographically separated and detected with an Exactive high-resolution mass spectrometer. Due to the absence of guidelines for the analysis of boar taint in fat, the Commission Decision 2002/657/EC [18] and ISO 17025 [19] guidelines were used as guideline for validation of the developed detection method. This resulted in limits of detection and limits of quantification between 2.5 and 7 µg kg(-1) and between 5 and 10 µg kg(-1) for the three compounds, respectively, which is far below the threshold values set at 100 µg L(-1) for indole, 200 µg L(-1) for skatole and 1000 µg L(-1) for androstenone in pig fat samples. The method obtained for the three compounds a repeatability (RSD) lower then 12.7% and a within-laboratory reproducibility (RSD) lower than 16.9%. The recovery of the three compounds ranged between 99 and 112 and an excellent linearity (R(2) ≥ 0.99) was found. In the future, this method may be extended with other compounds that turn out to be correlated with boar taint.

A review of analytical strategies for the detection of 'endogenous' steroid abuse in food production.

Detection of the abuse of synthetic steroids in food production is nowadays relatively straightforward using modern techniques such as gas or liquid chromatography coupled to mass spectrometry (GC-MS/MS or LC-MS/MS, respectively). However, proving the abuse of 'endogenous' (or naturally occurring) steroids is more difficult. Despite these difficulties, significant progress in this area has recently been made and a number of methods are now available. The aim of the current review was to systematically review the available analytical approaches, which include threshold concentrations, qualitative 'marker' metabolites, intact steroid esters, gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS), longitudinal testing and 'omics' biomarker profiling. The advantages/disadvantages of these methods are considered in detail, but the choice of which to adopt is dictated by a number of practical, political, and economic factors, which vary in different parts of the world. These include the steroid/species combination requiring analysis, the matrix tested, whether samples are collected from live or slaughtered animals, available analytical instrumentation, sample throughput/cost, and the relevant legal/regulatory frameworks. Furthermore, these approaches could be combined in a range of different parallel and/or sequential screening/confirmatory testing streams, with the final choice being determined by the aforementioned considerations. Despite these advances, more work is required to refine the different techniques and to respond to the ever increasing list of compounds classified as 'endogenous'. At this advanced stage, however, it is now more important than ever for scientists and regulators from across the world to communicate and collaborate in order to harmonize and streamline research efforts.

Predicting the likelihood of developing boar taint: early physical indicators in entire male pigs.

Three potential early-age predictors of which boars are likely to develop boar taint (testes volume, skin lesions and dirtiness) were measured on 102 boars every fortnight from 10 weeks of age until slaughter. These predictors were correlated with the level of boar taint according to the hot iron method and the concentrations of skatole and androstenone as determined by chemical analysis. The chance of no/low boar taint according to the hot iron method decreased with higher testes volume (weeks 22 and 24) and increased with skin lesion score (weeks 12, 16 and 18). For the concentrations of androstenone and skatole, the strongest correlation was found with testes volume in week 12. Skin lesions in week 16 were negatively correlated with skatole levels. Dirtiness was negatively correlated with skatole concentrations (week 18) but positively correlated with androstenone concentrations (weeks 20 and 22). Testes volume has the greatest potential for predicting the likelihood of developing boar taint.

Ultra-high performance liquid chromatography coupled to triple quadrupole mass spectrometry detection of naturally occurring thiouracil in urine of untreated livestock, domesticated animals and humans.

Thiouracil belongs to the xenobiotic thyreostats, which are growth-promoting agents illegally used in animal production. Recently it has been reported that thiouracil is suspected to have a natural origin. The European Union of Reference Laboratory guidance paper of 2007 acknowledged this by stating that thiouracil concentrations below 10 µg l⁻¹ might have a natural origin derived from the consumption of Brassicaceae. The present research aimed at endorsing this possible natural occurrence. Urine samples of animals (livestock and domesticated) with known and unknown clinical backgrounds were analysed for thiouracil with a newly developed ultra-high performance liquid chromatography coupled to a triple quadrupole mass spectrometric analysis method without derivatisation. In addition, a small-scale 9-day human experiment with Brassicaceae vegetables was performed to investigate if this natural prevalence could be extrapolated to the human population. The untreated animals had thiouracil concentrations below 10 µg l⁻¹ acknowledging the alleged natural occurrence of thiouracil. As for the humans, in 66.7% of the urine samples thiouracil was found above the CC(α) of 2.2 µg l⁻¹. However, the correlation with the Brassicaceae diet proved to be non-significant (p = 0.095). Nevertheless, these results clearly demonstrate the natural occurrence of thiouracil in urine of animals and humans. The exact origin of this natural thiouracil trace still needs to be identified.