Determination of nitrofuran metabolites in milk by liquid chromatography–electrospray ionization tandem mass spectrometry

An LC–ESI–MS–MS method for the analysis of metabolites of four nitrofurans (furazolidone, furaltadone, nitrofurazone and nitrofurantoin) in raw milk has been developed. The samples were achieved by hydrolysis of the protein-bound drug metabolites, derivatization with 2-nitrobenzaldehyd (2-NBA) and clean-up extraction liquid–liquid with ethyl acetate. LC separation was achieved by using a Phenomenex Luna C-18 column. The mass spectrometer operated in multiple reaction monitoring mode (MRM) with positive electro-spray interface (ESI). The method validation was done according to the criteria laid down in Commission Decision No. 2002/657 EC. The validation includes the determination of linearity, repeatability, within-laboratory reproducibility, accuracy, decision limit (CCα) and detection capability (CCβ). The calibration curves were linear, with typical (R2) values higher than 0.991. The coefficient of variation (CV, %) was lower than 9.3% and the accuracy (RE, %) ranged from −9.0% to 7.0%. CV within-laboratory reproducibility was lower than 13%. The limits of decision (CCα) and detection capability (CCβ) were 0.12–0.29 μg/kg and 0.15–0.37 μg/kg, thus below the minimum required performance limit (MRPL) set at 1 μg/kg by the UE. This validated method was successfully applied for the determination of nitrofuran metabolites in a large number of milk samples.