High-performance liquid chromatography with fluorescence detection for the determination of nitrofuran metabolites in pork muscle

A simple and sensitive HPLC method with fluorescence detection (HPLC-FLD) is reported for the simultaneous determination of metabolites of four nitrofuran drugs (furazolidone, furaltadone, nitrofurantoin and nitrofurazone) in pork muscle. The method involves acid hydrolysis of the protein-bound drug metabolites and the conjugation of the released side-chains with a novel fluorescence agent 2-hydroxy-1-naphthaldehyde. After liquid-liquid extraction and effective separation of the derivatives on a YMC-Pack Polymer C18 column at 40 degrees C under alkaline conditions, the high fluorescence intensity of these derivatives at emission wavelength (em)=463nm enables their simultaneous determination in pork muscle at concentrations as low as 1 mu gkg(-1). The method was validated using blank pork muscle fortified with all four metabolites at 0.5, 1.0 and 2.0 mu gkg(-1). Recoveries were >92.3% with RSDs <8.5% for all four metabolites. The results obtained with HPLC-FLD and LC-MS/MS methods showed very good agreement for pork muscle samples.