Nitrofuran antibiotics, employed for the treatment of bacterial diseases in livestock production, were banned from use in the European Union (EU) in 1995 due to concerns about the carcinogenicity of their residues in edible tissue. This review provides an overview of nitrofuran toxicity, metabolism, and also specific aspects of legislation surrounding their prohibition. Special attention is devoted to semicarbazide -a nitrofuran metabolite and food contaminant. Analytical procedures for nitrofuran analysis in various matrices and validation requirements for screening and confirmation methods with respect to EU regulations are also reviewed.Keywords: nitrofurans; tissue bound metabolites; semicarbazide; bioavailability; mutagenicity; legislation; sample preparation; validation; detection methods List of abbreviations AHD = 1-aminohydantoin; AOZ = 3-amino-2-oxazolidinone; AMOZ = 3-amino-5-morpholino-methyl-1,3-oxazolidinone; CC α = decision limit; CC β = detection capability; EC = European Commission; EFSA = European Food Safety Authority; ELISA = enzyme linked immuno-adsorbent assay; ESI = electro-spray ionisation; EU = European Union; FTD = furaltadone; FZD = furazolidone; HPLC = high performance liquid chromatography; IC = inhibition concentration; LC = liquid chromatography; LOD = limit of detection; MS = mass spectrometry; NFT = nitrofurantoin; NFZ = nitrofurazone; NP = nitrophenyl; NPAHD = 3-(2-nitrobenzylidenamino)-2,4-imidazolidinedione); NPAMOZ = 5-(morpholinomethyl)-3-(2-nitrobenzylidenamino)-2-oxazolidinone); NPAOZ = [3-(2-nitrobenzylidenamino)-2-oxazolidinone]; NPSEM = 3[(2-nitrophenyl)methylene]-hydrazinecarboxamide; o-NBA = ortho-nitrobenzaldehyde; RASFF = Rapid Alert System for Food and Feed; SE = solvent extraction; SEM = semicarbazide; SPE = solid phase extraction; UV = ultraviolet

Development of antibodies with broad specificity recognition for sulfonamide drugs was found to be surprisingly difficult when conventional immunochemical strategies were applied to hapten design. To improve the cross-reactivity pattern of antibodies for the family of sulfonamide drugs, a novel strategy based on the single-ring (fragment-derived) hapten moieties with different spacer substituent lengths was employed for the preparation of immunogens, coating conjugates, and enzyme competitors. The rabbit antibodies raised against a common (one-ring) p-aminobenzenesulfonamide hapten moiety (attached to a carrier protein through the N-1 position) in combination with a homologous hapten-peroxidase tracer allowed the detection of 15 sulfonamide species at the maximum residue limit level using direct ELISA. The two-ring 6-(4-aminobenzensulfonylamino)hexanoic hapten mimics, previously reported in the literature as a weak generic antigen, generated surprisingly superior immune responses in rabbits. The antibodies raised against this two-ring hapten were capable of detecting at least 19 and 17 sulfonamides in a direct ELISA system at the regulatory level with sensitivities corresponding to 20 and 50% binding inhibition, respectively. A negligible cross-reaction with N4 metabolites makes it possible to measure responses of parent sulfonamides in the presence of their metabolized forms. In skimmed milk, the highest limit of detection (LOD) for sulfacetamide defined as 20% inhibition was 65.2 microg x L(-1) (IC20 value), whereas the additional 18 sulfonamides tested exhibited LODs in the range of 0.2-36.8 microg x L(-1). This sensitivity allows simple multisulfonamide tests to be established for use in the laboratory or on site.

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ABSTRACT:A monoclonal-based ELISA, coupled with an assay buffer, solvent and solid phase extraction procedures, was validated for use in the monitoring of egg samples for 3-amino-2-oxazolidinone (AOZ). The procedures allow the detection of protein bound AOZ in the form of 2-nitrophenyl derivative (NPAOZ) in sample supernatant or extract after acid hydrolysis and derivatisation with o-nitrobenzaldehyde. The assays were validated according to criteria set down by Commission Decision (2003) for the performance and validation of analytical methods for chemical residues. The detection capability of ELISA's for AOZ in eggs (set on the basis of acceptance of no false negatives) was 0.6, 0.3 and 0.3 µg/kg for buffer, solvent and solid phase extraction, respectively. These values are well below the maximum required performance limit (MRLP) of 1 µg/kg for tissue bound residues of nitrofuran antibiotics. An excellent correlation of results (r = 0.99, n = 14) obtained by the ELISA and LC-MS/MS techniques within the concentration range of 0-5 µg/kg was found in the incurred egg samples. The eggs collected from layer chickens fed 30 and 400 mg/kg of furazolidone for 10 days were monitored by ELISA until AOZ concentrations approached the LoD.