A high performance liquid chromatography with fluorescence detection (HPLC-FLD) method for the simultaneous determination of total nitrofuran metabolite residues (furazolidone, furaltadone, nitrofurantoin, and nitrofurazone) in shrimp was developed. The method involves the acid hydrolysis of protein-bound metabolites, followed by the derivatization of the freed metabolites with the new fluorescent derivatization reagent 2-hydroxy-1-naphthaldehyde (HN) and subsequent liquid-liquid extraction (LLE). Separation is achieved on a YMC-Pack Polymer C18 column under alkaline conditions, and the high fluorescence intensity of the derivatives at an emission wavelength Em=463nm (Ex=395nm) enables, for the first time, their simultaneous determination in shrimp at concentrations as low as 1μg/kg by HPLC-FLD. The method was validated using blank shrimp fortified with all four metabolites at 0.5, 1.0 and 2.0μg/kg. Recoveries were >87% with relative standard deviations of <8.1% for all four metabolites. Furthermore, the results obtained by HPLC-FLD were in very good agreement with those obtained by LC-MS/MS analysis.
Keywords: HPLC–FLD; LC–MS/MS; Nitrofuran metabolites; Shrimp.
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