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 (CC alpha) and detection capability (CC beta). The calibration curves were linear, with typical (R-2) 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 (CC alpha) and detection capability (CC beta) were 0. 12-0.29 mu g/kg and 0. 15-0.37 mu g/kg, thus below the minimum required performance limit (MRPL) set at 1 mu g/kg by the UE. This validated method was successfully applied for the determination of nitrofuran metabolites in a large number of milk samples. (C) 2008 Elsevier B.V. All rights reserved.