Development and validation of a new method for determining nitrofuran metabolites in bovine urine using liquid chromatography - tandem mass spectrometry.

BACKGROUND: The use of nitrofurans as veterinary drugs in food-producing animals is banned throughout the European Union. Nevertheless, nitrofuran metabolites have been detected not only in animal products, but also in bovine urine. At present there are no methods yet published for the simultaneous detection of nitrofuran metabolites in bovine urine. OBJECTIVES: To develop and validate a method for determination of four key nitrofuran metabolites in bovine urine. MATERIAL AND METHODS: The four nitrofuran metabolites (nitrofurantoin, furazolidone, nitrofurazone and furaltadone), were determined in bovine urine using LC-ESI-MS/MS. The procedure required an acid-catalysed release of protein-bound metabolites, followed by their in situ conversion into 2-nitrobenzaldehyde (NBA) derivatives. The sample clean-up was performed using a polymer extraction cartridge before hydrolysis. Nitrofuran metabolites were then determined using electrospray ionization in the positive mode, that had previously been separated on a Phenomenex Luna C-18 column. RESULTS: The method was validated in accordance with the procedure outlined in the Commission Decision No. 2002/657/ EC. Urine samples were spiked with nitrofuran metabolite solutions at levels of 0.5, 1.0, 1.5 and 2.0 microg/kg. Recoveries ranged between 90 - 108% (inter standard-corrected), with a repeatability precision (RSD) of less than 19% for all four analytes. The decision limit (CC) and detection capability (DC) were obtained from a calibration curve and lay respectively within the following ranges: 0.11 - 0.34 microg/kg and 0.13 - 0.43 microg/kg. CONCLUSIONS: The developed and validated LC-ESI-MS/MS method allows four nitrofuran metabolites to be identified and quantitated in bovine urine. This analytical procedure meets the criteria defined in the Commission Decision No. 2002/657/EC.