Quantitative risk model to estimate the level of antimicrobial residues that can be transferred to soil via manure, due to oral treatments of pigs.

Veterinary antimicrobials can spread via manure onto agricultural fields, representing an emission of these products or their active metabolites into the environment. This causes concerns regarding the role of antimicrobial residues in the development, selection and spread of resistance. Aiming to approach this issue quantitatively, first a literature review was performed on the bioavailability and extent of in vivo biotransformation of twelve antimicrobials commonly used in pigs orally, and on the level of their persistence in manure. This information was then used in a model estimating the level of each of these administered antimicrobials that is present in manure at the end of common storage durations in pits and, thus, readily applied onto soil. From the studied antimicrobials, the highest level of residues in stored manure was estimated for doxycycline (55% of the initial amount of doxycycline administered orally to pigs after six months of manure storage), as a combining result of its high use in pigs, low bioavailability and high stability in manure. Other antimicrobials (e.g. amoxicillin) are readily degraded and therefore pose less threat. The results of this study highlight the importance of rational antimicrobial use and of further research on pharmacokinetics of antimicrobials and their degraded products in different environmental compartments, to efficiently control the spread of residues and/or resistance genes from manure to these matrices.

Selection and transfer of an IncI1-tet(A) plasmid of Escherichia coli in an ex vivo model of the porcine caecum at doxycycline concentrations caused by crosscontaminated feed.

AIMS: The aim of this study was to investigate the effect of subtherapeutic intestinal doxycycline (DOX) concentrations (4 and 1 mg l-1 ), caused by cross-contamination of feed, on the enrichment of a DOX-resistant commensal Escherichia coli and its resistance plasmid in an ex vivo model of the porcine caecum. METHODS AND RESULTS: A DOX-resistant, tet(A)-carrying, porcine commensal E. coli strain (EC 682) was cultivated for 6 days in the porcine caecum model under different conditions (0, 1 and 4 mg l-1 DOX). EC 682, other coliforms and anaerobic bacteria were enumerated daily. A selection of isolated DOX-resistant coliforms (n = 454) was characterized by rep-PCR clustering, PCR assays (Inc1 and tet(A)) and micro broth dilution susceptibility tests (Sensititre). Both 1 and 4 mg l-1 DOX-enriched medium had a significantly higher selective effect on EC 682 and other resistant coliforms than medium without DOX. Transconjugants of EC 682 were isolated more frequently in the presence of 1 and 4 mg l-1 DOX compared to medium without DOX. CONCLUSIONS: Subtherapeutic intestinal DOX concentrations have the potential to select for DOX-resistant E. coli, and promote the selection of transconjugants in a porcine caecum model. SIGNIFICANCE AND IMPACT OF THE STUDY: Cross-contamination of feed with antimicrobials such as DOX likely promotes the spread of antimicrobial resistance. Therefore, it is important to develop or fine-tune guidelines for the safe use of antimicrobials in animal feed and its storage.

Can the use of coccidiostats in poultry breeding lead to residues in vegetables? An experimental study.

The aim of this study was to provide information on the dietary exposure of the European public to coccidiostats via vegetable consumption. Five groups of poultry followed a three-phase feeding schedule with feed containing the maximum allowed level of a coccidiostat: monensin, lasalocid A, salinomycin, diclazuril, and nicarbazin/narasin, plus one control group. Vegetables were cultivated on soil amended with manure (10 g of fresh weight/kg of soil) from the treated poultry. To mimic a worst-case scenario, vegetables were also grown on soil spiked with coccidiostats. For each vegetable/treatment combination, samples were harvested, freeze-dried, and analyzed using a validated liquid chromatography-tandem mass spectrometry method. Analysis of the vegetables demonstrated that these plants are capable of taking up these coccidiostats from the soil. However, the results indicate that these low incorporation levels, coupled with food consumption data and acceptable daily intakes, are unlikely to pose a direct threat to public health.

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.

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.

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.

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).

Development and validation of an UPLC-MS/MS method for the determination of ionophoric and synthetic coccidiostats in vegetables.

In poultry farming, anticoccidial drugs are widely used as feed additives for the prevention and treatment of coccidiosis. Because coccidiostats and veterinary medicines, in general, are often poorly absorbed, manure from treated animals may contain high concentrations of these compounds. Experimental studies have shown that the uptake of veterinary medicines by plants from soil containing contaminated manure may occur. This leads to several questions regarding the impact on the environment, resistance problems, and public health and allergy issues. This work describes the development of a quantification method for coccidiostats in vegetables. Vegetables were spiked at 100 µg kg(-1) (dry weight) with coccidiostats (monensin, narasin, lasalocid A, salinomycin, diclazuril, and nicarbazin) in order to optimize the extraction and clean-up. Possible critical factors (e.g., extraction solvent) were statistically examined by linear regression with the use of Plackett-Burman and full factorial designs. Final extracts were analyzed with ultra-performance liquid chromatography tandem mass spectrometry operating in multiple-reaction monitoring mode. Both the synthetic and ionophoric coccidiostats could be determined in a single run with an analysis time of 5 min. The developed method was validated taking into account the requirements of the Commission Decision 2002/657/EC as a guideline. The method is regarded as applicable for its intended purposes with quantification limits between 0.30 and 2.98 µg kg(-1). This method could be used to establish possible maximum residue limits for coccidiostats in vegetables, as already exist for eggs, meat, and milk.

Validation of the betaeta-s.t.a.r. 1 + 1 for rapid screening of residues of beta-lactam antibiotics in milk.

The 2-min protocol (1 + 1) for the betaeta-s.t.a.r. (manufactured by Neogen Corporation, Lansing, MI, USA) was validated at the Technology and Food Science Unit of the Institute for Agricultural and Fisheries Research according to Commission Decision 2002/657/EC. The test was very selective for the group of beta-lactam compounds: the only interference found was by clavulanic acid at 2500 microg kg(-1) and above. The modified protocol (betaeta-s.t.a.r. 1 + 1) detected all beta-lactams with a maximum residue limit (MRL) in milk, but not all these compounds were detected at their respective MRL. The detection of cefalexin (detection capability = 6000 microg kg(-1); MRL = 100 microg kg(-1)) and penethamate (detection capability = 80 microg kg(-1); MRL = 4 microg kg(-1)) was especially poor, and also ceftiofur was only detected from 500 microg kg(-1) (MRL = 100 microg kg(-1)). The repeatability of the reader and of the test was very good. The test was very robust: test results were not significantly influenced by small changes in the test protocol, by the milk composition or by the type of milk. The test was also suitable to test the milk of animal species other than cow. Favourable results were obtained in testing monitoring samples, in two national ring trials, and in an international proficiency test. The betaeta-s.t.a.r. 1 + 1 is a very fast, simple, and reliable test that could be used at the farm level to prevent tanker milk contamination by beta-lactams.

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.

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.

Distribution and depletion of flubendazole and its metabolites in edible tissues of guinea fowl.

1. We measured the distribution and depletion of residues of flubendazole and its major metabolites in breast muscle, thigh muscle and liver of guinea fowls during and after oral administration of the veterinary medicine Flubenol 5% at two doses. 2. The guinea fowls were treated orally with normal feed, medicated at doses of 56 and 86 mg per kg feed for 7 successive days. Afterwards, depletion was observed for 8 d. Just before slaughter, body weights were measured. Thigh muscle, breast muscle and liver of three female and three male birds were sampled. The concentrations of the flubendazole-derived residues were determined by a liquid chromatographic-mass spectrometric method. 3. The highest residue concentrations were obtained for the reduced metabolite. With the therapeutic dose, the maximum mean residue concentrations obtained for this compound in thigh muscle, breast muscle and liver were 312, 288 and 1043 microg/kg, respectively. The values for flubendazole, the parent molecule, were 114, 108 and 108 microg/kg, respectively. The residues of the hydrolysed metabolite were negligible in the sampled muscle tissues. After 24 h of depletion, the sum of the residues of parent and metabolites in muscle tissue still exceeded 50 microg/kg. After 8 d of depletion, flubendazole-derived residues at low concentrations could still be measured in both muscle tissues and liver. Generally, the disposition of residues in breast and thigh muscle was comparable. 4. The European Union has not established a maximum residue limit (MRL) for flubendazole in edible tissues of guinea fowl. In contrast, the existing MRLs for other bird species are expressed as the sum of parent flubendazole and its hydrolysed metabolites. An estimated withdrawal period of three days will assure residue safety in the edible tissues of guinea fowl treated with flubendazole at therapeutic dose. After this withdrawal period following treatment of the guinea fowl, the residues were approximately constant, very low and far below the established safe MRL level for other bird species.

Determination of halofuginone in eggs by liquid chromatography/tandem mass spectrometry after cleanup with immunoaffinity chromatography.

A sensitive and very selective high-performance liquid chromatography/tandem mass spectrometric (LC/MS/MS) method for the detection of halofuginone in whole egg has been developed. After deproteinisation with acetonitrile and evaporation of the organic solvent, halofuginone was further isolated by applying immunoaffinity chromatography. The concentrated eluent was injected into the LC/MS/MS system on a C18 column. The precursor ion ([M+H]+) produced by positive electrospray ionisation was selected for fragmentation with argon. Validation parameters such as recovery, linearity and repeatability, decision limit (CCalpha) and detection capability (CCbeta) were determined.

Determination of flubendazole and its metabolites in eggs and poultry muscle with liquid chromatography-tandem mass spectrometry.

The optimization of a quantitative and sensitive LC-MS/MS method to determine flubendazole and its hydrolyzed and reduced metabolites in eggs and poultry muscle is described. The benzimidazole components were extracted from the two matrices with ethyl acetate after the sample mixtures had been made alkaline. The HPLC separation was performed on an RP C-18 column with gradient elution, using ammonium acetate and acetonitrile as mobile phase. The analytes were detected after atmospheric pressure electrospray ionization on a tandem quadrupole mass spectrometer in MS/MS mode. The components were measured by the MS/MS transition of the molecular ion to the most abundant daughter ion. The overall extraction recovery values for flubendazole, the hydrolyzed metabolite, and the reduced metabolite in eggs (fortification levels of 200, 400, and 800 microg kg(-1)) and muscle (fortification levels of 25, 50, and 100 microg kg(-1)) were, respectively, 77, 78, and 80% and 92, 95, and 90%. The trueness (fortification levels of 400 and 50 microg kg(-1), respectively, for eggs and muscle), expressed as a percentage of the added values for these analytes, was, respectively, 89, 100, and 86 and 110, 110, and 98%. The proposed MS detection method operating in the MS/MS mode is very selective and very sensitive. The limits of detection for flubendazole and its hydrolyzed and reduced metabolites in egg and muscle were, respectively, 0.19, 0.29, and 1.14 microg kg(-1) and 0.14, 0.75, and 0.31 microg kg(-1). The limits of quantification were, respectively, 1, 1, and 2 microg kg(-1) and 1, 1, and 1 microg kg(-1). The discussed method was applied to a pharmacokinetic study with turkeys. Residue concentrations in breast and thigh muscle of turkeys orally treated with flubendazole were quantified. Medicated feed containing 19.9 and 29.6 mg kg(-1) flubendazole was provided to the turkeys for seven consecutive days. For the trial with the recommended dose of 19.9 mg kg(-1), one day after the end of the treatment, the mean sum of the flubendazole plus hydrolyzed metabolite residue values in thigh and breast muscle declined to below the maximum residue limit (50 microg kg(-1)) and were, respectively, 36.6 and 54.1 microg kg(-1). The corresponding values with the higher dose of 29.6 mg kg(-1) were, respectively, 101.7 and 119.7 microg kg(-1).

Development and validation of a liquid chromatography-electrospray tandem mass spectrometry method for mebendazole and its metabolites hydroxymebendazole and aminomebendazole in sheep liver.

A quantitative liquid chromatography-electrospray tandem mass spectrometry method for the determination of mebendazole and its hydrolysed and reduced metabolites in sheep liver has been developed and validated. The benzimidazole substances were extracted with ethyl acetate after the sample mixture had been made alkaline. The HPLC separation was performed on a reversed-phase C18 column with gradient elution using a mobile phase consisting of water containing 0.1% formic acid and acetonitrile. The analytes were detected after atmospheric pressure electrospray ionization on a tandem quadrupole mass spectrometer in MS-MS mode. The components were measured by the MS-MS transitions of the molecular ion to the two most abundant daughter ions. The detection limits are lower than 1 microg kg(-1). For this application, the validation limit was set at 50 microg kg(-1). The examined validation parameters were in accordance with the permitted tolerances ranges stipulated in the proposed new European validation criteria for residue surveillance. For the three analytes, the overall recovery was higher than 90%. The RSD for the repeatability ranged from 5 to 11%. The range for the within-laboratory reproducibility was between 2 and 17%. The decision limits for mebendazole, the hydrolysed and the reduced metabolite were 56.6, 61.8 and 64.2 microg kg(-1), respectively. The detection capabilities for these substances were 60.0, 86.1 and 90.9 microg kg(-1), respectively.

Confirmatory assay for the simultaneous detection of penicillins and cephalosporins in milk using liquid chromatography/tandem mass spectrometry.

A high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the detection of residues of penicillins and cephalosporins in milk has been developed. After a simple extraction with acetonitrile, the extract was directly injected into the LC/MS/MS system on a C(18) column. A gradient consisting of acetonitrile and water, each containing 0.1% formic acid, was applied. The abundant parent ions [M + H](+) produced by positive electrospray ionisation were selected for fragmentation with argon. For each compound at least one fragment was recorded with multiple reaction monitoring. The limits of detection ranged from 1.5 to 25 microg/kg and the limits of quantification ranged from 4 to 50 microg/kg. Recoveries were examined at three levels (MRL, 0.5 x MRL, 2 x MRL) and ranged from 57 to 88%. The coefficients of variation obtained for the repeatability experiments were in agreement with those specified by the Horwitz equation. Linearity was checked by injecting extracts of samples spiked with increasing amounts of the different standards ranging from 0 to 150 microg/kg. The advantage of this method over existing methods is the very simple sample pre-treatment which makes the method very suitable for routine analysis.

Determination of the hop-derived phytoestrogen, 8-prenylnaringenin, in beer by gas chromatography/mass spectrometry.

A method was developed to determine 8-prenylnaringenin, a novel hop-derived phytoestrogen, in beer. Matrix purification involved solid-phase extraction on octadecyl silica followed by liquid/liquid extraction on a ChemElut 1010 column connected to a Florisil adsorption/desorption cartridge. 8-Prenylnaringenin was eluted from the tandem columns using a 1:1 mixture of diethyl ether and ethyl acetate and subsequently determined as tris(trimethylsilyl) ether by GC/MS-SIM. The recovery of 8-prenylnaringenin in beer samples was between 61.1 +/- 6.6 and 82.2 +/- 8.8% for levels of 37 and 92.5 microg L(-1), respectively, and the detection limit was approximately 5 microg L(-1). Although most beers do not contain 8-prenylnaringenin in detectable quantities, the highest concentration found was 19.8 microg L(-1). The concentration of 8-prenylnaringenin in beers and, possibly, its absence depend on the selection of particular hop varieties, the hopping rate, or the type of hop product used in brewing. The efficiency of transfer of 8-prenylnaringenin from hops to beer is between 10 and 20%.

Development of a simple method for the determination of genistein, daidzein, biochanin A, and formononetin (biochanin B) in human urine.

A simple method was developed for the determination of free and/or total isoflavones daidzein, genistein, and their respective 4'-methoxy derivatives biochanin A and formononetin (biochanin B) at low levels in human urine. A solid-phase extraction on octadecyl silica (C(18)) columns was used for the isolation of the phytoestrogens from the matrix. An extraction on a ChemElut 1010 column connected on-line to a Florisil cartridge by a Teflon stopcock was used for effective eluate purification. A mixture of dichloromethane and ethyl acetate was used for elution of the isoflavones from the columns in tandem. The isoflavones were determined as trimethylsilyl (TMS) ethers using GC/MS-SIM after separation on an HP-5MS fused silica column. TMS ethers were obtained by using BSTFA containing 1% of TMCS. For the determination of free isoflavones 6-hydroxyflavone was used as internal standard, whereas robigenin was used in the case of total isoflavone determination. Recoveries for free isoflavones under study varied from 63.5 to 89.6% at the 25 ng mL(-)(1) level and from 63.5 to 89. 2% at the 5 ng mL(-)(1) level in urine. Analytical curves were linear between 5 and 25 ng mL(-)(1). Detection limits varied from 1 ng mL(-)(1) for formononetin to 2.3 ng mL(-)(1) for daidzein. Recoveries for total isoflavone determination after enzymatic hydrolysis with glucuronidase from Helix pomatia ranged from 56.5 to 77.1% at the 25 ng mL(-1) level.

Validation of multi-residue methods for the detection of anabolic steroids by GC-MS in muscle tissues and urine samples from cattle.

The use of anabolic steroids is prohibited in the European Community by Directive 96/22/EC and the control of compliance is regulated by Directive 96/23/EC. Multi-residue methods are necessary for screening for the use of forbidden substances. Because accreditation is gaining more and more importance, validation of the methods used and of the results obtained has become indispensable. The developed GC-MS methods for the detection of anabolic steroids in urine and muscle tissue were validated with regard to the following parameters: specificity, recovery at the 2 micrograms kg-1 level and limit of detection. For urine the recoveries ranged from 17 to 81% and for muscle tissue from 26 to 65%. The limit of detection ranged from 0.1 to 2.6 micrograms kg-1 for urine and from 0.3 to 4.6 micrograms kg-1 for muscle tissue. Specificity was guaranteed in both matrices by the selection of four specific ions. Blank samples were evaluated for interferences and it could be concluded that in no case did the four selected ions appear simultaneously at the correct retention time. The practicability of the criteria for low resolution mass spectrometry set in Decision 93/256 in the low micrograms kg-1 range is also briefly discussed.