Metabolomics reveals novel biomarkers of illegal 5-nitromimidazole treatment in pigs. Further evidence of drug toxicity uncovered.

The aim of the study was to investigate the potential of a metabolomics platform to distinguish between pigs treated with ronidazole, dimetridazole and metronidazole and non-medicated animals (controls), at two withdrawal periods (day 0 and 5). Livers from each animal were biochemically profiled using UHPLC-QTof-MS in ESI+ mode of acquisition. Several Orthogonal Partial Least Squares-Discriminant Analysis models were generated from the acquired mass spectrometry data. The models classified the two groups control and treated animals. A total of 42 ions of interest explained the variation in ESI+. It was possible to find the identity of 3 of the ions and to positively classify 4 of the ionic features, which can be used as potential biomarkers of illicit 5-nitroimidazole abuse. Further evidence of the toxic mechanisms of 5-nitroimidazole drugs has been revealed, which may be of substantial importance as metronidazole is widely used in human medicine.

Determination of imidocarb residues in bovine and ovine liver and milk by immunobiosensor.

Imidocarb (IMD) is a veterinary drug that has been used for more than 30 years to treat and prevent parasitic diseases. Pharmacokinetic studies have shown that substantial levels of IMD residues are retained in the edible tissues and milk of cattle and sheep for up to 6 months after administration. This has led to concern regarding the potential adverse effects posed through human consumption of edible tissue or milk from treated animals if the recommended withdrawal periods for the drug are not properly implemented. While MRLs have been established by the European Union, it is important that analytical methods are available to monitor food samples for potentially violative levels of IMD residues. A qualitative biosensor-based immunoassay was developed to allow the detection of IMD at less than the European Union MRLs of 50 µg kg(-1) for milk and 2 mg kg(-1) for bovine and ovine liver. Validation of the developed methods provided a detection capability of <25 µg kg(-1) in milk and <0.75 mg kg(-1) in liver. A comparison study was undertaken, with IMD incurred milk and ovine liver samples analysed by the newly developed procedures and results compared with those obtained by LC-MS/MS. The newly developed screening method was applied to both incurred milk and liver samples. This faster, cheaper and reliable screening method has potential use in sample analysis to ensure compliance with legislative requirements.

Biosensors for the analysis of microbiological and chemical contaminants in food.

Increases in food production and the ever-present threat of food contamination from microbiological and chemical sources have led the food industry and regulators to pursue rapid, inexpensive methods of analysis to safeguard the health and safety of the consumer. Although sophisticated techniques such as chromatography and spectrometry provide more accurate and conclusive results, screening tests allow a much higher throughput of samples at a lower cost and with less operator training, so larger numbers of samples can be analysed. Biosensors combine a biological recognition element (enzyme, antibody, receptor) with a transducer to produce a measurable signal proportional to the extent of interaction between the recognition element and the analyte. The different uses of the biosensing instrumentation available today are extremely varied, with food analysis as an emerging and growing application. The advantages offered by biosensors over other screening methods such as radioimmunoassay, enzyme-linked immunosorbent assay, fluorescence immunoassay and luminescence immunoassay, with respect to food analysis, include automation, improved reproducibility, speed of analysis and real-time analysis. This article will provide a brief footing in history before reviewing the latest developments in biosensor applications for analysis of food contaminants (January 2007 to December 2010), focusing on the detection of pathogens, toxins, pesticides and veterinary drug residues by biosensors, with emphasis on articles showing data in food matrices. The main areas of development common to these groups of contaminants include multiplexing, the ability to simultaneously analyse a sample for more than one contaminant and portability. Biosensors currently have an important role in food safety; further advances in the technology, reagents and sample handling will surely reinforce this position.

A competitive enzyme-linked immunosorbent assay for determination of chloramphenicol.

Chloramphenicol is a broad-spectrum antibiotic shown to have specific activity against a wide variety of organisms that are causative agents of several disease conditions in domestic animals. Chloramphenicol has been banned for use in food-producing animals for its serious adverse toxic effects in humans. Due to the harmful effects of chloramphenicol residues livestock products should be free of any traces of these residues. Several analytical methods are available for chloramphenicol analysis but sensitive methods are required in order to ensure that no traces of chloramphenicol residues are present in edible animal products. In order to prevent the illegal use of chloramphenicol, regulatory control of its residues in food of animal origin is essential. A competitive enzyme-linked immunosorbent assay for chloramphenicol has been locally developed and optimized for the detection of chloramphenicol in sheep serum. In the assay, chloramphenicol in the test samples and that in chloramphenicol-horseradish peroxidase conjugate compete for antibodies raised against the drug in camels and immobilized on a microtitre plate. Tetramethylbenzidine-hydrogen peroxide (TMB/H2O2) is used as chromogen-substrate system. The assay has a detection limit of 0.1 ng/mL of serum with a high specificity for chloramphenicol. Cross-reactivity with florfenicol, thiamphenicol, penicillin, tetracyclines and sulfamethazine was not observed. The assay was able to detect chloramphenicol concentrations in normal sheep serum for at least 1 week after intramuscular injection with the drug at a dose of 25 mg/kg body weight (b.w.). The assay can be used as a screening tool for chloramphenicol use in animals.

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.

Rapid screening for monensin residues in poultry plasma by a dry reagent dissociation enhanced lanthanide fluoroimmunoassay.

Monensin, a carboxylic acid ionophore, is commonly fed to poultry to control coccidiosis. A method for rapid analysis of unextracted poultry plasma samples has been developed based on a novel immunoassay format: one-step all-in-one dry reagent time resolved fluorimetry. All assay specific components were pre-dried onto microtitration plate wells. Only addition of the serum sample diluted in assay buffer was required to perform analysis. Results were available one hour after sample addition. The limit of detection (mean +/- 3s) of the assay calculated from the analysis of 23 known negative samples was 14.2 ng ml-1. Intra- and inter-assay RSD were determined as 15.2 and 7.4%, respectively, using a plasma sample fortified with 50 mg ml-1 monensin. Eight broiler chickens were fed monensin at a dose rate of 120 mg kg-1 feed for one week, blood sampled then slaughtered without drug withdrawal. Plasma monensin concentrations, as determined by the fluoroimmunoassay ranged from 101-297 ng ml-1. This compared with monensin liver concentrations, determined by LC-MS, which ranged from 13-41 ng g-1. The fluoroimmunoassay described is extremely user friendly, gives particularly rapid results and is suitable for the detection and quantification of plasma monensin residues. Data from medicated poultry suggest that analysis of plasma may be useful in predicting the extent of monensin liver residues.

Comparison of porcine urine and bile as matrices to screen for the residues of two sulfonamides using a semi-automated enzyme immunoassay.

Porcine urine enzyme immunoassays for sulfamethazine and sulfadiazine have previously been employed as screening tests to predict the concentrations of the drugs in the corresponding tissues (kidneys). If a urine was found positive (> 800 ng ml-1) the corresponding kidney was then analysed by an enzyme immunoassay and, if found positive, a confirmatory analysis by HPLC was performed. Urine was chosen as the screening matrix since sulfonamides are mainly eliminated through this body fluid. However, after obtaining a number of false positive predictions, an investigation was carried out to assess the possibility of using an alternative body fluid which would act as a superior indicator of the presence of sulfonamides in porcine kidney. An initial study indicated that serum, plasma- and bile could all be used as screening matrices. From these, bile was chosen as the preferred sample matrix and an extensive study followed to compare the efficiencies of sulfonamide positive bile and urine at predicting sulphonamide positive kidneys. Bile was found to be 17 times more efficient than urine at predicting a sulfamethazine positive kidney and 11 times more efficient at predicting a sulfadiazine positive kidney. With this enhanced performance of the initial screening test, the need for the costly and time consuming kidney enzyme immunoassay, prior to HPLC analysis, was eliminated.