Determination of the metabolites of nitrofuran antibiotics in animal tissue and primary animal products by liquid chromatography-tandem mass spectrometry (LC-MS/MS)

The nitrofurans (furazolidone, furaltadone, nitrofurazone and nitrofurantoin) have been widely used antibiotics for the treatment of infections caused by Salmonella end Escherichia coli in cattle, swine, poultry and fish. Very often they have been used as feed additives as well as growth promoters. Following data which classified furaltadone, nitrofurazone and nitrofurantoin as a mutagenic and cancerogenic drugs, since 1993 (Council Regulation of EU EEC No 2901/93) their use has been forbidden in animal breading. Since 1995 furazolidone has been banned, too (Commission Regulation of EU EC No 1442/95). However, regardless of the ban of nitrofurans use because of high toxicity and possibility of illegall animal treatment, EU requires strict control of nitrofurans and their matabolites (Council Directive of EU 96/23/EC). Due to a very fast metabolism, the in vivo half-life of the nitrofuran type antibiotics is of only few hours. For this reason, the metabolites of nitrofuran antibiotics, with longer half-life (between 4 and 9 days), are determined as a markers for the illegal use of nitrofurans. In accordance with Commission Decision 2003/181/EC EU has set Minimum Required Performance Limit (MRPL) for nitrofurans and their matabolites of 1μg/kg. Clasifi cation of the nitrofuran antibiotics in the group of banned veterinary drugs, requires sofisticated analitical technique for their determination and confirmation. According to Commission Decision of EU 2002/657/EC, LC-MS/MS was set as the confirmative technique for unambiguos determination of the nitrofurans and their matabolites. Mass detector with two hexapoles fulfilles the most severe demands, by detection at least two daughter ions per one parent ion. In accordance with the mentioned, we accepted the method: Determination of the metabolites of nitrofuran antibiotics in animal tissue by high-performance liquid chromatograpy-tandem mass spectrometry, Journal of Chromatography A, 2001. After validation based on requirements of Commission Decision of EU 2002/657/EC we extended application of the mentioned method to tissues (muscle and kidney), urine, as well as to primary animal products (eggs and milk). After a fast and simple sample preparation procedure the analysis has been performed on Waters 2695 Separation Module with Micromass Quattro Micro mass detector working in positive electrospray ionization mode (ESI+). Chromatography was performed on X- Terra MS C18 (2.1x100 mm, 3.5 μm) column.