Transfer of flubendazole and tylosin from feed at cross-contamination levels to various poultry matrices.

Residues of veterinary drugs and feed additives used extensively in animal husbandry are sometimes found in edible matrices. In this study, broilers received experimental feed, containing either flubendazole or tylosin, at cross-contamination levels of 2.5%, 5%, and 10% of the therapeutic dose to determine the transfer ratio of these molecules from feed to poultry matrices. Breast and thigh muscle and liver samples were collected during treatment and depletion periods and then analyzed using liquid chromatography-tandem mass spectrometry. The parent molecule flubendazole and its 2 major metabolites were quantified. After 3 to 5 d, a plateau phase was reached, and a few days after withdrawal of the experimental feed, a depletion of residues was noted. Significant difference between both muscle types was noted for flubendazole. Strong metabolization of flubendazole in the liver was seen. For tylosin, no residue concentrations above the limit of quantification could be detected in muscle. None of the residue concentrations for either molecule exceeded the corresponding maximum residue limits.

Transfer of flubendazole and tylosin at cross contamination levels in the feed to egg matrices and distribution between egg yolk and egg white.

Chemical residues may be present in eggs from laying hens' exposure to drugs or contaminants. These residues may pose risks to human health. In this study, laying hens received experimental feed containing flubendazole or tylosin at cross contamination levels of 2.5, 5, and 10% of the therapeutic dose. Eggs were collected daily and analysis of the whole egg, egg white, and egg yolk was performed using liquid chromatography tandem mass spectrometry. Highest concentrations of the parent molecule flubendazole, as well as the hydrolyzed and the reduced metabolite, were detected in egg yolk. Residue concentrations of the parent molecule were higher compared with those of the metabolites in all egg matrices. No tylosin residue concentrations were detected above the limit of quantification for all concentration groups and in all egg matrices. Neither molecule exceeded the set maximum residue limits.

A general strategy to control antibody specificity against targets showing molecular and biological similarity: Salmonella case study.

The control of antibody specificity plays pivotal roles in key technological fields such as diagnostics and therapeutics. During the development of immunoassays (IAs) for the biosensing of pathogens in food matrices, we have found a way to rationalize and control the specificity of polyclonal antibodies (sera) for a complex analytical target (the Salmonella genus), in terms of number of analytes (Salmonella species) and potential cross-reactivity with similar analytes (other bacteria strains). Indeed, the biosensing of Salmonella required the development of sera and serum mixtures displaying homogeneous specificity for a large set of strains showing broad biochemical variety (54 Salmonella serovars tested in this study), which partially overlaps with the molecular features of other class of bacteria (like specific serogroups of E. coli). To achieve a trade-off between specificity harmonisation and maximization, we have developed a strategy based on the conversion of the specificity profiles of individual sera in to numerical descriptors, which allow predicting the capacity of serum mixtures to detect multiple bacteria strains. This approach does not imply laborious purification steps and results advantageous for process scaling-up, and may help in the customization of the specificity profiles of antibodies needed for diagnostic and therapeutic applications such as multi-analyte detection and recombinant antibody engineering, respectively.

Development of a strategy for the quantification of food allergens in several food products by mass spectrometry in a routine laboratory.

Worldwide, mass spectrometry is widely used to detect and quantify food allergens, especially in complex and processed food products. Yet, the absence of a regulatory framework for the developed methods has led to a lack of harmonization between laboratories. In this study, ten allergens were analyzed in eight food products by UHPLC-MS/MS, in order to establish criteria for the retention time, variation tolerance, the ion ratio deviation, and the signal-to-noise ratio for allergen detection. The set of criteria should help laboratories to compare results and avoid false positives and negatives. Furthermore, a strategy combining standard addition and labeled peptide correction was used to quantify milk, soy, peanut, and egg allergens in eight food products. This strategy is particularly interesting for routine laboratories, which receive hundreds of samples and cannot use an external calibration curve for each sample.

Discrimination between Synthetically Administered and Endogenous Thiouracil Based on Monitoring of Urine, Muscle, and Thyroid Tissue: An in Vivo Study in Young and Adult Bovines.

Thiouracil (TU), synthesized for its thyroid-regulating capacities and alternatively misused in livestock for its weight-gaining effects, is acknowledged to have an endogenous origin. Discrimination between low-level abuse and endogenous occurrence is challenging and unexplored in an experimental setting. Therefore, cows (n = 16) and calves (n = 18) were subjected to a rapeseed-supplemented diet or treated with synthetic TU. Significant higher urinary TU levels were recorded after TU administration (

Liquid chromatography coupled to tandem mass spectrometry for detecting ten allergens in complex and incurred foodstuffs.

Food allergy is a considerable heath problem, as undesirable contaminations by allergens during food production are still widespread and may be dangerous for human health. To protect the population, laboratories need to develop reliable analytical methods in order to detect allergens in various food products. Currently, a large majority of allergen-related food recalls concern bakery products. It is therefore essential to detect allergens in unprocessed and processed foodstuffs. In this study, we developed a method for detecting ten allergens in complex (chocolate, ice cream) and processed (cookie, sauce) foodstuffs, based on ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). Using a single protocol and considering a signal-to-noise ratio higher than 10 for the most abundant multiple reaction monitoring (MRM) transition, we were able to detect target allergens at 0.5mg/kg for milk proteins, 2.5mg/kg for peanut, hazelnut, pistachio, and cashew proteins, 3mg/kg for egg proteins, and 5mg/kg for soy, almond, walnut, and pecan proteins. The ability of the method to detect 10 allergens with a single protocol in complex and incurred food products makes it an attractive alternative to the ELISA method for routine laboratories.

Advances in ultra-high performance liquid chromatography coupled to tandem mass spectrometry for sensitive detection of several food allergens in complex and processed foodstuffs.

Sensitive detection of food allergens is affected by food processing and foodstuff complexity. It is therefore a challenge to detect cross-contamination in food production that could endanger an allergic customer's life. Here we used ultra-high performance liquid chromatography coupled to tandem mass spectrometry for simultaneous detection of traces of milk (casein, whey protein), egg (yolk, white), soybean, and peanut allergens in different complex and/or heat-processed foodstuffs. The method is based on a single protocol (extraction, trypsin digestion, and purification) applicable to the different tested foodstuffs: chocolate, ice cream, tomato sauce, and processed cookies. The determined limits of quantitation, expressed in total milk, egg, peanut, or soy proteins (and not soluble proteins) per kilogram of food, are: 0.5mg/kg for milk (detection of caseins), 5mg/kg for milk (detection of whey), 2.5mg/kg for peanut, 5mg/kg for soy, 3.4mg/kg for egg (detection of egg white), and 30.8mg/kg for egg (detection of egg yolk). The main advantage is the ability of the method to detect four major food allergens simultaneously in processed and complex matrices with very high sensitivity and specificity.

IGF -I plasma concentrations in non-treated horses and horses administered with methionyl equine somatotropin.

Insulin-like growth factor-I (IGF -I) is likely to be an indicator of somatotropin (ST) administration in the horse. To investigate the different ways ST administration may be detected, the following aspects of IGF -I concentrations in plasma were studied: (i) the daily variation; (ii) variation following a treadmill test; (iii) concentrations at rest and after exercise; and (iv) concentrations in plasma from two young horses and two adults treated with methionyl equine somatotropin (e ST). In the population of horses at rest, IGF -I mean concentration (SEM) was 261 (104) ng ml(-1). In post race samples, IGF -I mean concentration was 187 (100) ng ml(-1). All of these data indicate that exercise does not modify IGF -I concentration in plasma. The magnitude of the increase in IGF -I following administration of e ST differed according to the age of the horses. The critical value of 700 ng ml(-1)was exceeded for 1 day in adult horses and for at least 11 days in young horses. These results show that IGF -I has potential as an indirect marker of ST administration in horses.

The Production of Polyclonal Antibodies in Laboratory Animals. The Report and Recommendations of ECVAM Workshop 35.

This is the report of the thirty-fifth of a series of workshops organised by the European Centre for the Validation of Alternative Methods (ECVAM). ECVAM's main goal, as defined in 1993 by its Scientific Advisory Committee, is to promote the scientific and regulatory acceptance of alternative methods which are of importance to the biosciences and which reduce, refine or replace the use of laboratory animals. One of the first priorities set by ECVAM was the implementation of procedures which would enable it to become well informed about the state-of-the-art of non-animal test development and validation, and the potential for the possible incorporation of alternative tests into regulatory procedures. It was decided that this would be best achieved by the organisation of ECVAM workshops on specific topics, at which small groups of invited experts would review the current status of various types of in vitro tests and their potential uses, and make recommendations about the best ways forward (1). This joint ECVAM/FELASA (Federation of European Laboratory Animal Science Associations) workshop on The Immunisation of Laboratory Animals for the Production of Polyclonal Antibodies was held in Utrecht (The Netherlands), on 20-22 March 1998, under the co-chairmanship of Coenraad Hendriksen (RIVM, Bilthoven, The Netherlands) and Wim de Leeuw (Inspectorate for Health Protection, The Netherlands). The participants, all experts in the fields of immunology, laboratory animal science, or regulation, came from universities, industry and regulatory bodies. The aims of the workshop were: a) to discuss and evaluate current immunisation procedures for the production of polyclonal antibodies (including route of injection, animal species and adjuvant ); and b) to draft recommendations and guidelines to improve the immunisation procedures, with regard both to animal welfare and to the optimisation of immunisation protocols. This report summarises the outcome of the discussions and includes a number of recommendations and a set of draft guidelines (included in Appendix 1).

Preliminary study on the presence of prednisolone in porcine urine and liver - How to distinguish endogenous from therapeutically administered prednisolone.

In animal breeding in Europe, synthetic corticosteroids are not allowed as growth-promoting agents. However, prednisolone residues have recently been found in porcine urine samples collected at slaughterhouses. The aim of this work was therefore to look for prednisolone in porcine urine and liver, to determine if detected residues might be of endogenous origin, and to check the possible relation with stress. An analytical method developed in-house was validated, combining immunoaffinity-based purification and ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). This method was applied to urine and liver samples collected from sows experimentally treated either with prednisolone or tetracosactide hexaacetate (synthetic analogue of ACTH). Thanks to the performance of the analytical method, both cortisol and prednisolone were detected in all pig urine samples collected before or after administration of prednisolone or tetracosactide hexaacetate. High levels of prednisolone were found in porcine urine just after prednisolone administration, decreasing quickly to within the range detected in non-treated animals. In urine, the cortisol level varied depending on the time lapse between administration and sampling. On the other hand, prednisolone was detected also in liver samples of treated pigs. In this matrix, the cortisol level remained constant and prednisolone/cortisol level could be used to detect prednisolone administration at least 4 days after injection. In conclusion, the best indicator for detecting illicit prednisolone administration to pigs seems to be the prednisolone/cortisol ratio in liver samples. This preliminary work must be confirmed by a larger-scale study and metabolites should also be included.

Validation of methods for the detection and quantification of engineered nanoparticles in food.

The potential impact of nanomaterials on the environment and on human health has already triggered legislation requiring labelling of products containing nanoparticles. However, so far, no validated analytical methods for the implementation of this legislation exist. This paper outlines a generic approach for the validation of methods for detection and quantification of nanoparticles in food samples. It proposes validation of identity, selectivity, precision, working range, limit of detection and robustness, bearing in mind that each "result" must include information about the chemical identity, particle size and mass or particle number concentration. This has an impact on testing for selectivity and trueness, which also must take these aspects into consideration. Selectivity must not only be tested against matrix constituents and other nanoparticles, but it shall also be tested whether the methods apply equally well to particles of different suppliers. In trueness testing, information whether the particle size distribution has changed during analysis is required. Results are largely expected to follow normal distributions due to the expected high number of particles. An approach of estimating measurement uncertainties from the validation data is given.

Residues of sulfadiazine and doxycycline in broiler liver and muscle tissue due to cross-contamination of feed.

Veterinary drugs, such as antimicrobial compounds, are widely used in poultry and may lead to the presence of residues in matrices of animal origin, such as muscle and liver tissue. In this study, broilers received an experimental feed containing sulfadiazine or doxycycline at cross-contamination levels of 2.5, 5 and 10% of the therapeutic dose in feed. Breast and thigh muscle and liver samples were collected during treatment and depletion period and analysed via liquid chromatography-tandem mass spectrometry (LC-MS/MS). Concentrations reached a plateau phase 3-5 days after the start of experimental feeding. A rapid depletion of residues was noted after withdrawal of the experimental feed. No significant differences in measured concentrations were observed between the various muscle types. Residue concentrations for some experimental groups; the 10% group of sulfadiazine and the 5 and 10% group of doxycycline, however, exceeded their corresponding maximum residue limits (MRLs).

Transfer of the coccidiostats monensin and lasalocid from feed at cross-contamination levels to whole egg, egg white and egg yolk.

Recent legislation has addressed the unavoidable carry-over of coccidiostats and histomonostats in feed, which may lead to the presence of residues of these compounds in eggs. In this study, laying hens received cross-contaminated feed at a ratio of 2.5%, 5% and 10% of the therapeutic dose of monensin and lasalocid for broilers. The eggs were collected during the treatment and depletion period and were analysed using liquid chromatography-tandem mass spectrometry. The different egg matrices were separated and analysed during the plateau phase. High lasalocid concentrations, which exceeded the maximum residue level, and low monensin concentrations were found in whole egg. Plateau levels were reached at days 7-9 for lasalocid and at days 3-5 for monensin. For lasalocid, the highest concentrations were measured in egg yolk; residue concentrations in egg white were very low.

Residues of sulfadiazine and doxycycline in egg matrices due to cross-contamination in the feed of laying hens and the possible correlation with physicochemical, pharmacokinetic and physiological parameters.

In the poultry industry, the widespread use of veterinary drugs such as antimicrobial compounds may lead to the presence of residues in whole eggs, egg white and egg yolk. During this study, laying hens received experimental feed containing sulfadiazine or doxycycline at cross-contamination levels of 2.5%, 5% and 10% of the therapeutic concentration. Since the therapeutic dose is 250 mg kg(-1) for both substances, cross-contamination concentrations in the feed of 6.25, 12.5 and 25 mg kg(-1) were expected. Whole egg, egg white and egg yolk samples were collected during the treatment and depletion period and were analysed via liquid chromatography-tandem mass spectrometry. For both drugs, a plateau phase was reached within 3-5 days and residue concentrations were detected in all egg matrices. For the 10% cross-contamination group, residual sulfadiazine concentrations of 208, 299 and 60 µg kg(-1) and residual doxycycline concentrations of 455, 332, 206 µg kg(-1) were detected in whole egg, egg white and egg yolk on day 13 of the treatment period, respectively. Both sulfadiazine and doxycycline had higher concentrations in egg white than in egg yolk, but the egg white-egg yolk ratio was higher for sulfadiazine than for doxycycline. As neither drug is allowed in Belgium for use in laying hens, residues may pose food safety concerns.

Acute and long-term cardiomyopathy and delayed neurotoxicity after accidental lasalocid poisoning in horses.

BACKGROUND: Horses are extremely susceptible to ionophore intoxication. Although numerous reports are available regarding monensin, little is known about lasalocid toxicity. OBJECTIVES: To describe accidental lasalocid poisoning on a farm in Belgium. ANIMALS: Eighty-one horses, of which 14 demonstrated clinical signs from day 0-21 after being fed a new concentrate batch. One horse died on day 20 and another on day 27. METHODS: The most severe cases (n = 7), admitted to the clinic on day 29-46, underwent cardiac examination and blood biochemical analysis, including determination of plasma cardiac troponin I (cTnI) at admission and during follow-up. On day 57-70, cardiac examination, cTnI determination or both were undertaken on 72 remaining horses. RESULTS: Short-term effects of lasalocid intoxication included inappetance, lethargy, sweating, and muscular weakness. All 7 horses admitted to the clinic demonstrated signs of myocardial degeneration such as increased cTnI, dysrhythmia and reduced myocardial contractility. Four horses developed ataxia on day 40-50. Five horses died or were euthanized on day 30-370, 2 horses recovered fully and returned to previous athletic use. None of the 72 remaining horses exhibited clinical signs between day 57-70, but 34 had dysrhythmia and 13 had increased cTnI concentrations. After a period of rest, all horses returned to their previous work. Lasalocid was detected in hepatic tissue of 2 necropsied horses. CONCLUSIONS AND CLINICAL IMPORTANCE: Lasalocid intoxication induced myocardial and neurological damage. Although uncommon, this should be included as differential diagnosis for unexplained inappetance, signs of depression, cardiomyopathy, and ataxia in horses.

Detection and identification of plasma progesterone metabolites in the female Florida manatee (Trichechus manatus latirostris) using GC/MS/MS.

Florida manatees (Trichechus manatus latirostris) have relatively low peripheral concentrations of progesterone (P4). The objective of this study was to determine if these relatively low P4 concentrations are associated with a high ratio of progestin metabolites and to document metabolite concentrations from individual blood samples obtained from manatees during diestrus or pregnancy. Metabolites known to exist in elephants-terrestrial manatee relatives-were targeted. These included 5alpha-reduced progestins (5alpha-pregnane-3,20-dione [5alpha-DHP] and 3alpha-hydroxy-5alpha-pregnan-20-one [5alpha-P3-OH]) and 17alpha-hydroxyprogesterone (17alpha-OHP), which occurs in Asian elephants. An additional, inactive metabolite, 20alpha-hydroxyprogesterone (20alpha-OHP), indicative of P4 overproduction, was also targeted. Progesterone itself was the predominant progestin detected in pregnant and nonpregnant manatee plasma (n = 10) using gas chromatography-mass spectrometry with tandem quadrupole detectors (GC/MS/MS). Progesterone concentrations in pregnant females varied from early (moderate to high) through mid and late (low) pregnancy. Progesterone concentrations ranged from low to high in nonpregnant, nonlactating females. The most commonly detected metabolite was 5alpha-P3-OH (n = 7), which occurred in pregnant (lower limit of detection [LLOD] to high) and nonpregnant (trace to high) females. The 5alpha-DHP metabolite was also detected in pregnant (LLOD to moderate) and nonpregnant (low) females. The 17alpha-OHP metabolite was not detected in any tested female. The 20alpha-OHP metabolite was detected in one nonpregnant, nonlactating, captive female (LLOD). Metabolites were most prevalent during early pregnancy, concurrent with maximum P4 concentrations. Based on their concentrations in peripheral circulation, we inferred that these metabolites may have, opposite to elephants, a limited physiologic role during luteal, pregnant, and nonpregnant phases in the manatee.

Screening for the coccidiostats halofuginone and nicarbazin in egg and chicken muscle: development of an ELISA.

Nicarbazin and halofuginone have been widely used as coccidiostats for the prevention and treatment of coccidiosis in poultry. It has been shown that accidental cross-contamination of feed can lead to residues of these compounds in eggs and/or muscle. This paper describes a direct competitive assay for detecting halofuginone and nicarbazin, developed as qualitative screening assay. In an optimized competitive ELISA, antibodies showed 50% binding inhibition at approximately 0.08 ng ml(-1) for halofuginone and 2.5 ng ml(-1) for dinitrocarbanilide (marker residue for nicarbazin). Extraction from the matrix was carried out with acetonitrile followed by a wash with hexane. The assay's detection capability (CCbeta) for halofuginone was < 0.5 microg kg(-1) in egg and < 1 microg kg(-1) in muscle. For dinitrocarbanilide, the CCbeta was estimated at < 3 microg kg(-1) in egg and < 10 microg kg(-1) in chicken muscle.

Incidence of residues of nine anticoccidials in eggs.

A survey of the presence of residues of anticoccidials was performed. Three hundred and twenty egg samples, purchased in eight different European countries, were analysed for the presence of nine different compounds: dimetridazole, diclazuril, halofuginone, robenidine, nicarbazin, narasin, salinomycin, lasalocid and monensin. Analyses were performed by LC-MS/MS. Of the samples analysed, 114 (35.6%) contained one or more of the nine anticoccidials in concentrations ranging from 0.1 to 63 microg kg-1. Salinomycin and lasalocid account for more than 60% of all positive samples. Almost 90% of all positive samples contained less than 2 microg kg-1. Results were put into perspective of the farming method and country of origin.

Multi-allergen screening immunoassay for the detection of protein markers of peanut and four tree nuts in chocolate.

A multiresidue enzyme immunoassay was developed to check for the presence of markers of peanut, hazelnut, almond, cashew and Brazil nuts in a single run. The assay was designed under the competitive indirect format and adapted for screening purposes applied to chocolate samples. The limit of detection for this assay was below 1 microg g-1 protein for each allergenic food. In most cases, the high specificity of the antibodies used allowed the identification of each particular allergenic food with no possible confusion. This assay was proven to be useful as part of an analytical procedure involving the identification of the unknown allergenic food among peanut and other tree nuts in recalled samples before the application of a quantitative technique to determine the level of cross-contamination.

[Synthetic anabolic androgens in the fecal material of cattle: radioimmunological determination after purification by high performance liquid chromatography]. / Androgènes anabolisants de synthèse dans les matières fécales des bovins: dosage radio-immunologique après purification par chromatographie liquide à haute performance.

In answer to the mandatory control of the illegal use of anabolizing agents in meat-producing animals imposed by the EEC in farms, a method of analysis of faeces involving high performance liquid chromatography (HPLC) and radioimmunoassay (RIA) has been described. Four HPLC fractions were collected and submitted to corresponding RIA: 17 beta- and 17 alpha-trenbolone, 17 beta-nortestosterone, 17 alpha-nortestosterone and 17 alpha-methyltestosterone. The mean extraction and purification yield was estimated at 44 +/- 7% using tritiated 17 alpha-methyltestosterone as internal standard. Detection limits of the 3 hormones were estimated at 0.2-0.3 ng/g of faeces. About 50 samples can be analysed per week by this method.