Bound residues and nitrofuran detection

Validation of analytical methods according to Commission Decision 2002/657/EC requires an assessment of the stability of the analyte(s) in solution and in samples. A study was undertaken to investigate these parameters, along with stability following cooking, for the acid-hydrolysable moieties derived from the tissue-bound metabolites of the nitrofuran drugs. This study showed that all of the nitrofuran metabolites are extremely stable. Solutions of the nitrofurans, either as stock solutions or as working standards, were all stable for at least 8 months and residues in frozen (-20°C) were similarly stable. Losses of the nitrofuran metabolites following a range of cooking treatments were low. Once this information is in the public domain, following publication of a refereed scientific publication (in preparation), this will facilitate residues analysts achieving validation of their analytical methods according to the provisions of this Decision. The result, concerning the stability of these residues following cooking, may facilitate risk assessment/management decisions when non-compliant residues are found in food of animal origin. The result is for information of scientists / regulators. It has no intrinsic commercial value and will not be the subject of any further commercial exploitation.

This result was intended to inform the consortium and the European Commission of the prevalence of nitrofuran residues in pork meat purchased at retail level throughout the EU. Consumers organisations and, in a limited number of cases, FoodBRAND partners were responsible for the collection of the samples. Following analysis details of the positive results (12) were reported to the European Commission and to the competent National Authorities in Portugal (10), Greece (1) and Italy (1) for further action as appropriate. This result complemented contemporaneous results, which showed massive misuse of nitrofurans at a global level. The results were disseminated by the consortium - In the form of a refereed scientific publication and - Through popular articles in consumer�s magazine published inside and outside the EU. The result facilitated direct communication of the work of the project to the European Consumer. The result is for the information of scientists / regulators / consumers. It has no intrinsic commercial value and will not be the subject of any further commercial exploitation.

Furazolidone, a nitrofuran antibiotic, is banned from use in food animal production within the European Union. Increasingly, compliance with this ban is monitored by use of analytical methods to detect a stable tissue-bound metabolite, 3-amino-2-oxazolidinone (AOZ). Widespread use of furazolidone in poultry and prawns imported into Europe highlighted the urgent need for development of nitrofuran immunoassay screening tests. Polyclonal antibodies were produced to detect AOZ, following derivatisation with o-nitrobenzaldehyde. A carboxyphenyl derivative of AOZ was prepared, purified and conjugated to immunogenic carrier protein. Six antisera were produced from the immunisation of seven rabbits using various immunogen doses and time-scales. IC50 values, as determined by competitive enzyme-linked immunosorbent assay (ELISA) suggested that reducing immunogen dose from 0.3 to 0.05 mg, while lengthening rest periods between booster immunisations from 2 to 8 weeks, increased the sensitivity of the antibodies to 3-{[(2-nitrophenyl)methylene]amino}-2-oxazolidinone (NPAOZ) from 3.8 to 0.3 µg.l−1. An IC50 of 0.065 µg.l−1 (AOZ in the form of NPAOZ) was achieved with antiserum R670 by altering ELISA conditions. This antibody was highly specific for NPAOZ and did not cross-react with various nitrofuran metabolites, their nitrophenyl derivatives or a range of veterinary drugs. Antibody R670 is suitable for incorporation into an immunoassay for AOZ with sufficient sensitivity to satisfy current criteria for monitoring of veterinary drug residues. A similar procedure was used to produce antisera against AMOZ the equivalent metabolite of furaltadone. Both assays were developed into commercial ELISA test kits, which were launched by the FoodBRAND team during 2003, in response to the growing international need for testing procedures for the nitrofuran drugs. This arose as a result of the finding of global misuse of these drugs in food animal production. The manufacturers validated the test kits in a range of matrices. The European Commission has established Minimum Required Performance Limit for the nitrofuran drugs in poultry meat and aquaculture products (1.0 µg/kg) by Commission Decision 2003/181/EC. This was subsequently extended to cover all matrices tested by Commission Decision 2005/34/EC. The tests kits developed by FoodBRAND meet fully and indeed exceed the Minimum Required Performance Limit requirements established by these decisions.

Depletion of the nitrofuran antibiotics furazolidone, furaltadone, nitrofurantoin and nitrofurazone and their tissue-bound metabolites AOZ, AMOZ, AHD and SEM from pig muscle, liver and kidney tissues is described. Groups of pigs were given feed medicated with one of the nitrofuran drugs at a therapeutic concentration (400mgkg_1) for ten days. Animals were slaughtered at intervals and tissue samples collected for analysis for six weeks following withdrawal of medicated feed. These samples were analyzed both for parent nitrofurans (using LC-MS/MS and HPLC-UV), and for tissue-bound metabolites (using LC-MS/MS). The parent drugs were detectable only sporadically and only in pigs subjected to no withdrawal period whatsoever. This confirms the instability of the four major nitrofuran antibiotics in edible tissues. In contrast, the metabolites accumulated to high concentrations in tissues (ppm levels) and had depletion half lives of between 5.5 and 15.5 days. The metabolites of all four drugs were still readily detectable in tissues six weeks after cessation of treatment. This emphasizes the benefits of monitoring for the stable metabolites of the nitrofurans.

Liquid chromatography, coupled to tandem mass spectrometry, (LC-MS/MS) is now firmly established as the method of choice for the detection, confirmation and quantification of a wide range of veterinary drug residues in food. A method to detect the stable, tissue-bound side chain metabolites of the nitrofuran drugs was one of the main objectives of the FoodBRAND project. The developed method was used, initially in two FoodBRAND partner laboratories to uncover global misuse of the nitrofuran drugs in poultry and aquaculture. The FoodBRAND method (in the form of two Standard Operating Procedures) was disseminated to the EU NRL/CRL network and 3rd countries during 2001 - 2003. In brief, the method consists of a simple derivatisation step (that may be preceded by a series of pre-washing steps if only tissue-bound residues are to be measured, followed by liquid:liquid extraction and detection using LC-MS/MS. Minced tissues (1.00 +/- 0.02 g) were homogenized in ice-cold methanol (8ml) and water (1ml). Following centrifugation (2000rpm at 4°C for 10min) the supernatant was discarded and the sample repeatedly washed by vortex mixing for 10 seconds in ice cold methanol (3 x 4ml), ethanol (2 x 4ml) and diethyl ether (2 x 4ml). Mixed internal standard (50ml of a 100ng.ml-1 solution) was added equally to all samples, controls and standards. Control tissues from nitrofuran-free pigs were fortified with mixed standard (100ml of a 10ng.ml-1 solution) to act as recovery control samples. Blank tissues were also included in every analytical run. To all tubes were added de-ionized water (4ml), 1M hydrochloric acid (0.5ml) and 2-nitrobenzaldehyde (150ml, 50mM in DMSO). After vortex mixing, tubes were incubated overnight (approximately 16 h) in a water bath held at 37°C. All tubes were then adjusted to pH 7.4 +/- 0.2 with 0.1M di-potassium hydrogen orthophosphate (5ml) and 1M sodium hydroxide (approximately 0.4ml). Liquid-liquid extraction was carried out using ethyl acetate (2x5ml, mixing for 1min and centrifugation at 2000rpm for 15min at 4°C) and the organic phase evaporated to dryness at 50°C under nitrogen. Residues were re-dissolved in methanol: water (50:50 v/v; 200 ml) and transferred to HPLC microvials (200ml). LC-MS/MS analysis was carried out using electrospray ionisation. The analytical methods were validated according to Commission Decision 2002/657/EC. In all cases, the determined values of CCa and CCb were well below the Minimum Required Performance Limit of 1.0 µg/kg established for nitrofuran metabolites in poultrymeat and aquaculture products by Commission Decision 2003/181/EC and amended by Commission Decision 2005/34/EC to cover all matrices. This method, with minor variations, is now in use throughout the world for the control of the nitrofuran antibiotics in food animal production.

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