Detection of 3-amino-2-oxazolidinone (AOZ), a tissue-bound metabolite of the nitrofuran furazolidone, in prawn tissue by enzyme immunoassay

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. The first enzyme-linked immunoabsorbant assay for detection of AOZ residues in prawns (shrimps) is now described. Prawn samples were derivatized with o-nitrobenzaldehyde, extracted into ethyl acetate, washed with hexane and applied to a competitive enzyme immunoassay based on a rabbit polyclonal antiserum. Assay limit of detection (LOD) (mean + 3 s) calculated from the analysis of 20 known negative cold and warm water prawn samples was 0.1 µg kg^-1. Intra- and interassay relative standard deviations were determined as 18.8 and 38.2%, respectively, using a negative prawn fortified at 0.7 µg kg^-1. The detection capability (CC_β), defined as the concentration of AOZ at which 20 different fortified samples yielded results above the LOD, was achieved at fortification between 0.4 and 0.7 µg kg^-1. Incurred prawn samples (n = 8) confirmed by liquid chromatography coupled with tandem mass spectrometry detection to contain AOZ concentrations between 0.4 and 12.7 µg kg^-1 were all screened positive by this enzyme-linked immunoabsorbant assay. Further data are presented and discussed with regard to calculating assay LOD based on accepting a 5% false-positive rate with representative negative prawn samples. Such an acceptance improves the sensitivity of an ELISA and in this case permitted an LOD of 0.05 µg kg^-1 and a CC_β of below 0.4 µg kg^-1.