Method validation and a Survey for Total Nitrofuran Metabolites in Animal Tissues by LC-MS/MS

Abstract

       Nitrofurans were used effectively as medication of animals in the past. Due to the evidences of their properties as carcinogenicity and mutagenicity, Thailand and many countries revised
the legislation and regulations to prohibit the drugs used in food producing animals. The European Union established the Minimum Required Performance Limits (MRPLs) of analytical method for nitrofuran metabolites namely 3-amino-2-oxazolidinone (AOZ), 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), 1-aminohydantoin (AHD) and semicarbazide (SEM) in animal products at 1.0 μg/kg. The determination method has been developed and validated. The four isotope-labelled analogues were added in the sample as internal standard. Acid hydrolysis to release the side chains of metabolites from protein-bound residues was required and metabolites were derivatised. After pH adjustment to 7.0 ± 0.5 and liquid-liquid extraction, the residues were confirmatively determined by LC-MS/MS. The method validation showed the linearity of working range between 0.1-2.0 μg/kg with correlation coefficient valued 0.9860 -1.0000. The accuracy (%recovery) ranged within 88.9-112.0% and the precision was evaluated by HORRAT method and given the values lower than 2. The limit of detection (LOD) and limit of quantitation (LOQ) were 0.05-0.3 μg/kg and 0.1-1.0 μg/kg, respectively.
       The survey research on nitrofuran metabolites abuse from 402 samples of edible animal tissues, sold in the local markets and supermarkets in Bangkok was conducted during 2008-2011. The result showed that AOZ was found in pork and visceras 12.2 % and SEM in shrimp 4.2%. The amount range of AOZ and SEM were <0.1-44.5 μ g/kg and <0.3-2.3 μ g/kg, respectively. The detections of AOZ and SEM were not significantly different (p>0.05) at the survey period, indicating both of drugs were still used in animal farms. Therefore, monitoring of drug residues and strictly law enforcement are further needed.