A Novel Analytical Strategy to Detect Specific Markers of Nitrofurazone from Protein-Bound Metabolites in Food Animals.

Accurate detection of nitrofuran residues, as an indicator of banned drug use, in animal-derived food products is vital for ensuring food safety. The accepted regulatory approach for nitrofurans produces a residue marker from their protein-bound metabolites, which is nonspecific for nitrofurazone (i.e., semicarbazide (SEM)). Since SEM may originate from non-nitrofurazone sources, it is therefore not conclusive for nitrofurazone. Our main objective was to apply novel deconjugation/substitution reactions, which have been used to reverse protein binding, to identify a specific nitrofurazone marker. Bound nitrofurazone metabolites, produced using S9 from bovine liver or homogenized bovine liver tissue, were subjected to basic media and thiol reagents. Analyses carried out using high-resolution mass spectrometry showed that substitution of protein with thiol reagents produced thiol-metabolite conjugates whose structures were supported with stable isotopically labeled experiments. These conjugates can serve as new, specific markers for conclusive detection of nitrofurazone abuse in food animals.