The nitrofuran drugs furaltadone, nitrofurantoin and nitrofurazone were banned from use in food animal production in the EU in 1993, and the use of furazolidone was similarly prohibited in 1995, because of concerns about their carcinogenicity and mutagenicity. There is evidence of illegal abuse of these compounds within the EU, since a nitrofuran preparation, intended for illegal use in animal production, was seized in a Member State and bound residues of AOZ have been detected in a minor food species (rabbit) in the same country. In a recent survey, EU National Reference Laboratories (NRLs) expressed the view that testing for bound residues of the nitrofurans was superior to testing for parent drugs (92%); stated that they had inadequate information on the best matrix to test (83%); expressed a need for internal standards (85%); expressed a need for analytical calibrants (92%); and stated that the organisation of a workshop was a high priority (92%). The analytical capability of NRLs to monitor compliance with the EU ban on nitrofurans is poor. While a significant proportion of EU NRLs have screening and confirmatory tests for the parent drugs (57 and 29%, respectively) only 7% of NRLs can screen for bound residues of the nitrofurans. No broad-spectrum screening tests (immunochemical or chemical) or Reference Methods exist.
The nitrofurans are banned from use in food animal production in the EU. Monitoring compliance with the ban by measuring residues of the parent drug is of limited value because of their short biological half-life and marked in vitro instability. The project is using novel and innovative approaches to result in effective control by developing well-validated tests in accredited laboratories to measure tissue-bound residues that persist in tissues for up to 6 weeks after cessation of illegal treatment. The consortium includes 4 NRLs with GLP/EN45000 accreditation, one SME, one research laboratory and consumer representation. Some EU NRLs prefer to screen using in-house chemical methods and others prefer to use commercially available immunochemical screening methods. The project will enable NRL end users to choose a screening method according to their preferences, and will also develop validated Reference Methods for the confirmation of these drugs.
Delays in the recruitment of staff in some Partner laboratories have meant that progress during the first year has been more limited than anticipated. However, FoodBRAND has been able to deliver a supply of reference standards for the moieties released from tissue-bound residues of the nitrofurans. These compounds are not commercially available. Problems were encountered in the identification of a suitable derivatising reagent that was needed for the experimental approach to antibody production adopted by Partner 1. Reagents, originally scheduled to be used as derivatising agents, proved to have very low reactivity with the target analytes. However, these difficulties were overcome when 5-hydroxy-o-nitrobenzaldehyde was examined. This compound proved to be reactive with the nitrofuran moieties and possessed a reactive hydroxyl group to permit the subsequent conjugation of the HNBA-nitrofuran derivative to carrier proteins for immunisation into animals. Partners 1 and 2-2 have adopted different strategies to raise the desired antisera. A large number of animals (108) are currently undergoing immunisation in an attempt to produce the desired antibodies. Early results suggest that Partner 2-2 has produced the required antibodies, but these do not appear to show any displacement. Further work is necessary. No data are currently available on the animals immunised by Partner 1. Partner 2-3 has concentrated initially on possible clean-up methods for the nitrofuran moieties. Although some difficulties were encountered, there are many alternative approaches that must be explored. Partner 2-1, working on the development of an HPLC-UV screening test for the nitrofuran drugs has been able to detect peaks corresponding to 3 out of the 4 target compounds. To date they have been unable to detect NPAMOZ (derived from furaltadone). Investigations continue, in collaboration with Partner 1, to ensure detection of all compounds. FoodBRAND has delivered deuterated internal standards, necessary for accurate quantification in the Refe rence Method, based on LC-MS-MS, being developed by Partner 2. Working on 3 out of the 4 target compounds, Partner 2 has made good progress in obtaining chromatographic resolution of the target analytes. Partner 2 has shown that an adequate number of fragments can be produced for NPAMOZ and NPSEM using electrospray LC-MS-MS to permit unambiguous identification of these compounds. Initial experiments have shown that recovery of NPAOZ and NPAMOZ from spiked liver samples should be satisfactory. Dissemination of the work of the project has occurred at several conferences, through the establishment of the project web site and at a NRL-and a Consumer workshop.