Simultaneous and fast determination of antibiotics as nitrofuran metabolites in fish and shrimp muscle using Enzyme-Linked Immunosorbent Assay (ELISA)

Abstract

A simultaneous and fast analytical method was developed for the identification of four
nitrofuran metabolites from the fish and shrimp samples. Homogenized samples were hydrolyzed
and derivatized with 4-nitrobenzaldehyde. Subsequently, extracted with ethyl acetate, evaporated
to dryness and the residue was re-dissolved in Hexane. Commercial enzyme-linked
immunosorbent assay (ELISA) method was applied for the analysis of nitrofuran metabolites. The
method was validated in shrimp and fish matrix according to the criteria defined in Commission
Decision 2002/657/EC for qualitative screening method following the guidelines set by the
community reference laboratories residues (CRLs) 2010. Characteristic’s parameters as detection
capability (CC?), specificity/selectivity, stability, recovery and precision were determined.
Detection capability (CC?) for nitrofuran metabolites (AMOZ, AOZ, AHD and SEM) in Fish
and shrimp matrix was in the range of 0.5- 0.75?g/kg, which were less than the Minimum
Required Performance Limit (MRPL) of 1?g/kg set by European Union. The proposed method is
suitable for semi-quantitative screening analysis of antibiotics in the fish and shrimp muscle in
conformity with the current EU performance requirements before exporting to EU and other
countries. Results from analysis of unknown samples by the developed ELISA method were
comparable to those obtained by a liquid chromatography-tandem mass spectrometry (LCMS/MS)

method. Accuracy and precision of the method had also been checked through participation of International Proficiency Testing (PT) having a very satisfactory performance score.