Toward a new European threshold to discriminate illegally administered from naturally occurring thiouracil in livestock.

Thiouracil is a thyrostat inhibiting the thyroid function, resulting in fraudulent weight gain if applied in the fattening of livestock. The latter abuse is strictly forbidden and monitored in the European Union. Recently, endogenous sources of thiouracil were identified after frequently monitoring low-level thiouracil positive urine samples and a "recommend concentration" (RC) of 10 µg/L was suggested by the EURL to facilitate decision-making. However, the systematic occurrence of urine samples exceeding the RC led to demands for international surveys defining an epidemiologic threshold. Therefore, six European member states (France, Poland, The Netherlands, United Kingdom, Norway, and Belgium) have shared their official thiouracil data (2010-2012) collected from bovines, porcines, and small livestock with 95 and 99% percentiles of 8.1 and 18.2 µg/L for bovines (n = 3894); 7.4 and 13.5 µg/L for porcines (n = 654); and 7.4 µg/L (95% only) for small livestock (n = 85), respectively. Bovine percentiles decreased with the animal age (nonadults had significantly higher levels for bovines), and higher levels were observed in male bovines compared to female bovines.

Approaches for the simultaneous detection of thiamphenicol, florfenicol and florfenicol amine using immunochemical techniques.

Thiamphenicol and florfenicol are antibacterial agents permitted for use as veterinary drugs in animals used for food production. However, as the EU has established maximum residue limits for both and the metabolite florfenicol amine, there is a requirement to monitor animal food products for their residues. In this study antisera were generated which can simultaneously detect thiamphenicol, florfenicol and florfenicol amine in an immunoassay. Details of the various coupling techniques employed to prepare immunogens and enzyme labels are provided and the antibodies produced have been assessed, in homologous and heterologous ELISA formats, with respect to sensitivity and specificity. It was found that while the antisera raised to thiamphenicol and florfenicol generally performed better in a heterologous set up, those raised to florfenicol amine were not only less affected by the assay format but also produced the most sensitive antibodies to all three target analytes. Antisera matched previous sensitivity (IC50 < 1 ng mL⁻¹) but had improved cross-reactivity (>100%) to thiamphenicol and florfenicol.

Maximum residue level validation of triclabendazole marker residues in bovine liver, muscle and milk matrices by ultra high pressure liquid chromatography tandem mass spectrometry.

Triclabendazole is the only anthelmintic drug, which is active against immature, mature and adult stages of fluke. The objective of this work was to develop an analytical method to quantify and confirm the presence of triclabendazole residues around the MRL. In this work, a new analytical method was developed, which extended dynamic range to 1-100 and 5-1000 µg kg(-1) for milk and tissue, respectively. This was achieved using a mobile phase containing trifluoroacetic acid (pK(a) of 0.3), which resulted in the formation of the protonated pseudomolecular ions, [M+H](+), of triclabendazole metabolites. Insufficient ionisation of common mobile phase additives due to low pK(a) values (<2) was identified as the cause of poor linearity. The new mobile phase conditions allowed the analysis of triclabendazole residues in liver, muscle and milk encompassing their EU maximum residue levels (MRL) (250, 225 and 10 µg kg(-1) respectively). Triclabendazole residues were extracted using a modified QuEChERS method and analysed by positive electrospray ionisation mass spectrometry with all analytes eluted by 2.23 min. The method was validated at the MRL according to Commission Decision (CD) 2002/657/EC criteria. The decision limit (CCα) of the method was in the range of 250.8-287.2, 2554.9-290.8 and 10.9-12.1 µg kg(-1) for liver, muscle and milk, respectively. The performance of the method was successfully verified for triclabendazole in muscle by participating in a proficiency study, the method was also applied to incurred liver, muscle and milk samples.

The occurrence of semicarbazide in the meat and shell of Bangladeshi fresh-water shrimp.

There is evidence that semicarbazide (SEM), a marker for the banned nitrofuran nitrofurazone, can arise from other, unrelated sources. Recently, Belgium rejected 54 consignments of Bangladeshi freshwater shrimp (Macrobrachium rosenbergii), following a laboratory decision to test meat and exoskeleton combined. To study the possible natural occurrence of SEM in wild shrimp, samples were collected and analysed from 29 sites across Bangladesh. SEM (<1.0 µg/kg) was detected in ∼65% of meat samples. However, SEM concentrations were approximately 100 times higher in the exoskeleton, and were unrelated to sampling location, strongly suggesting natural occurrence. In meat, most SEM was surface-associated. When the shrimp was shelled, some of the epidermal layer (which synthesises new exoskeleton) remained with the shell and some remained with the meat--leading to differing levels of natural SEM on the shrimp surface. This has implications for the use of SEM and the analytical strategy used to control nitrofuran use.

Screening method for the detection of a range of nitrofurans in avian eyes by optical biosensor.

An immunobiosensor assay was developed for the multi-residue screening of a range of nitrofuran compounds in avian eyes. A polyclonal antibody which binds at least 5 of the major parent nitrofurans was raised in a rabbit after inoculation with a nitrofuran mimic-protein conjugate. Sample homogenates were extracted into 0.1M hydrochloric acid and subjected to clean-up by solid phase extraction and micro-centrifugation prior to biosensor analysis. Validation data obtained from the analysis of 21 fortified samples has shown that the method has a detection capability (CCß) of less than 1 ng eye(-1) for nitrofurazone (NFZ). In addition, cross-reactivity data and the analysis of a smaller number of fortified samples have shown that the method will also detect a range of other major parent nitrofurans including furazolidone (FZD), furaltadone (FTD), nitrofurantoin (NFA) and nifursol (NFS). Intra-assay variation (n=10) was calculated at 12.9% and 10.1% at concentrations of 1 ng eye(-1) and 2 ng eye(-1) NFZ respectively. Inter-assay variation (n=3) was determined to be 10.8% and 4.7% at the same NFZ concentrations respectively. The cross-reactivity profile and validation data for the detection of these nitrofurans are presented together with the results obtained following the analysis of a small number of incurred samples using the developed method.

5α-Estrane-3ß,17ß-diol and 5ß-estrane-3α,17ß-diol: definitive screening biomarkers to sign nandrolone abuse in cattle?

17ß-Nandrolone (17ß-NT) is one of the most frequently misused anabolic steroids in meat producing animals. As a result of its extensive metabolism combined with the possibility of interferences with other endogenous compounds, detection of its illegal use often turns out to be a difficult issue. In recent years, proving the illegal administration of 17ß-NT became even more challenging since the presence of endogenous presence of 17ß-NT or some of its metabolite in different species was demonstrated. In bovines, 17α-NT can occur naturally in the urine of pregnant cows and recent findings reported that both forms can be detected in injured animals. Because efficient control must both take into account metabolic patterns and associated kinetics of elimination, the purpose of the present study was to investigate further some estranediols (5α-estrane-3ß,17ß-diol (abb), 5ß-estrane-3α,17ß-diol (bab), 5α-estrane-3ß,17α-diol (aba), 5α-estrane-3α,17ß-diol (aab) and 5ß-estrane-3α,17α-diol (baa)) as particular metabolites of 17ß-NT on a large number of injured (n=65) or pregnant (n=40) bovines. Whereas the metabolites abb, bab, aba and baa have previously been detected in urine up to several days after 17ß-NT administration, the present study showed that some of the isomers abb (5α-estrane-3ß,17ß-diol) and bab (5ß-estrane-3α,17ß-diol) could not be detected in injured or pregnant animals, even at very low levels. This result may open a new way for the screening of anabolic steroid administration considering these 2 estranediols as biomarkers to indicate nandrolone abuse in cattle.

Development of an optical biosensor based immunoassay to screen infant formula milk samples for adulteration with melamine.

The illegal adulteration of milk with melamine in 2008 in China led to adverse kidney and urinary tract effects in hundreds of thousands of children and the reported deaths of six. The milk had been deliberately adulterated to elevate the apparent protein content, and subsequently melamine was detected in many milk-related products which had been exported. This led to the banning of imports of milk and milk products from China intended for the nutritional use of children and to the implementation of analytical methods to test products containing milk products. An optical biosensor inhibition immunoassay has been developed as a rapid and robust method for the analysis of infant formula and infant liquid milk samples. A compound with a chemical structure similar to that of melamine was employed as a hapten to raise a polyclonal antibody and as the immobilized antigen on the surface of a biosensor chip. The sensitivity of the assay, given as an IC(50), was calculated to be 67.9 ng mL(-1) in buffer. The antibody did not cross-react with any of the byproducts of melamine manufacture; however, significant cross-reactivity was observed with the insecticide cyromazine of which melamine is a metabolite. When sample matrix was applied to the assay, a limit of detection of <0.5 µg mL(-1) was determined in both infant formula and infant liquid milk. The development of the immunoassay and validation data for the detection of melamine is presented together with the results obtained following the analysis of melamine-contaminated milk powder.

Enzyme immunoassay for semicarbazide--the nitrofuran metabolite and food contaminant.

Semicarbazide (SEM), the marker residue for the banned nitrofuran veterinary antibiotic nitrofurazone (NFZ), has been detected regularly in foods (47% of recent nitrofuran EU Rapid Alerts involve SEM). However, the validity of SEM as a definitive marker for NFZ has been undermined by SEM arising from other sources including azodicarbonamide, a plastics blowing agent and flour treatment additive. An inexpensive screening test for SEM in food matrices is needed--all SEM testing currently uses expensive LC-MS/MS instrumentation. We now report the first production of antibodies against derivatised SEM. A novel carboxyphenyl SEM derivative was used to raise a polyclonal antibody that has been incorporated into a semi-quantitative microtitre plate ELISA, validated according to the criteria set out in Commission Decision 2002/657/EC, for use with chicken muscle. The antibody is highly specific for derivatised SEM, cross-reactivity being 1.7% with NFZ and negligible with a wide range of other nitrofurans and poultry drugs. Samples are derivatised with o-nitrobenzaldehyde and simultaneously protease digested before extraction by cation exchange SPE. The ELISA has a SEM detection capability (CCbeta) of 0.25 microg kg(-1) when a threshold of 0.21 microg kg(-1) is applied to the selection of samples for confirmation (lowest observed 0.25 microg kg(-1) fortified sample, n=20), thus satisfying the EU nitrofurans' minimum required performance limit of 1 microg kg(-1). NFZ-incurred muscles (12) containing SEM at 0.5-5.0 microg kg(-1) by LC-MS/MS, all screened positive by this ELISA protocol which is also applicable to egg and chicken liver.

Development and validation of a lateral flow device for the detection of nicarbazin contamination in poultry feeds.

Concentrations of the coccidiostat nicarbazin as low as 2 mg/kg in feed can result in violative drug residues arising in poultry liver. A lateral flow device (LFD) was developed for the detection of contaminating concentrations of nicarbazin following solvent extraction of poultry feeds. Test results, as determined by both visual and instrumental measurement, are available within minutes. For 22 feed samples, nicarbazin-free and fortified at 2 mg/kg, the % relative inhibition ranged from 0 to 45% and from 53 to 85%, respectively. Nicarbazin contamination at the critical concentration (2 mg/kg) can be determined in all cases providing the sampling is representative. A wide range of feed samples taken at a mill that incorporated nicarbazin into poultry feed were analyzed. Data generated for these samples by both the LFDs and a mass spectrometric method were compared, and a significant correlation was achieved.

Nitrofurazone accumulates in avian eyes--a replacement for semicarbazide as a marker of abuse.

Intact nitrofurazone is present in whole eyes of chickens fed varying levels of this banned antibiotic and may therefore be used as an alternative to the controversial marker residue, semicarbazide, to monitor for abuse of this drug in primary production.

Nitrofuran antibiotic metabolites detected at parts per million concentrations in retina of pigs--a new matrix for enhanced monitoring of nitrofuran abuse.

Nitrofuran metabolite residues AOZ, AMOZ, AHD and SEM were detected at parts per million concentrations in retina of pigs fed therapeutic doses of nitrofuran antibiotics. Discovery of this residue depot may allow widespread technology transfer to laboratories lacking LC-MS/MS thus improving global monitoring of these drugs.

A specificity-enhanced time-resolved fluoroimmunoassay for zeranol employing the dry reagent all-in-one-well principle.

A simple dry chemistry time-resolved fluorescence immunoassay (TR-FIA) method was developed for the measurement of zeranol in bovine urine samples. The samples were purified by immunoaffinity chromatography and a specificity-enhanced zeranol antibody was employed in the immunoassay. This resulted in a highly selective method, which had only negligible reactivity with Fusarium spp. toxins. The all-in-one-well dry chemistry concept made the assay very simple to use because all the assay-specific reagents were already present in the reaction wells in dry form. Only the addition of diluted sample extract was required to perform the competitive one-step TR-FIA and the results were available in less than 1 h. The analytical limit of detection (mean + 3s) for the immunoassay was 0.16 ng ml(-1) (n = 12) and the functional limit of detection for the whole method, estimated by the analysis of zeranol-free samples, was 1.3 ng ml(-1) (n = 20). The recovery of zeranol at the level of 2 ng ml(-1) was 99% (n = 18) and the within-assay variation ranged between 4.5 and 9.0%.