Method Development and validation for Determination of Nitrofuran Metabolites in Food by LC-MS/MS

Abstract

       Method for analysis of metabolites from nitrofuran drugs (furazolidone, furaltadone, nitrofurantoin and nitrofurazone) in food which are mainly employed for the treatment of bacterial diseases in livestock was studied and developed. Nitrofuran drugs are rapidly metabolized to form 3-amino-2-oxazolidinone (AOZ), 3-amino-5-morpholinomethyl-2-oxazolodinone (AMOZ), 1-aminohydantoin (AHD) and semicarbazide (SEM) and also strongly bound to proteins in target animals. Due to their carcinogenicity, nitrofuran drugs were prohibited to use as veterinary drugs in the EU. The development of an analytical method for the simultaneous determination of four nitrofuran metabolites was developed by LC-MS/MS. Sample was digested by diluted hydrochloric acid to release the metabolites from the protein in sample and then a derivatization step was performed. Next, the nitrofuran derivatives have been extracted with ethyl acetate and then defatted with hexane. Afterwards, LC-MS/MS was employed for qualitative and quantitative analysis of these compounds. Limits of detection (LOD) were 0.1 μg/kg for AOZ and AMOZ and 0.5 μg/kg for AHD and SEM. Limit of quantitation (LOQ) were 0.3 μg/kg for AOZ and AMOZ and 1.0 μg/kg for AHD and SEM. Method linear working range was 0.3-5.0 μg/kg with the correlation coefficient ranged from 0.9978-0.9999. The accuracy of method was presented in form of % mean recovery which equals to 65-120% while method precision (0.1-15.2%) was represented with % RSD value. From the validation results, this method was fit for intended use with for the regulations legislated by the Thai Food and Drug Administration and the EU commission. In 2011-2013, the proposed method was performed in meat, offal, aquatic animals, honey, milk powder, egg powder, and flour to inspect the nitrofuran metabolites residue. From 579 samples, the residues were found in 13 samples (2.2%). In 8 aquatic animal samples, AOZ was found in range of less than 1.0 to 2.4 and SEM was found at 2.2 μg/kg. Moreover, SEM was also found in range of less than 1.0 to 3.5 μg/kg in 5 samples of milk powder, egg powder, and flour.