Study On The Methods For Rapid Determination Of Nitrofurans In Animal Feeds

The genetic hapten of nitrofurans was synthesized with 5-nitrofurfural (NFF) as the template. Then two immunogens and two coating antigens were prepared by use of diazotization method and glutaraldehyde method, respectively. The coupling ratios of NFF to BSA in two immunogens were 13:1 and 20:l, respectively, and those of to OA in two coating antigens were 14:1 and 16:1, respectively. The two immunogens were used to immunize New Zealand white rabbits to produce the antibodies. The antisera were purified by acrylic acid-ammonium sulfate precipitation method and DEAE-cellulose anion-exchange chromatography to obtain the IgG fraction. The checkboard titration method was used to determine the antibody titer from diazotization method as 1: 320000 and the antibody titer from glutaraldehyde method was 1:640000. Both of the two antibodies showed broad specificity to seven nitrofurans (nitrofurazone, nifuroxazide, nifursol, furazolidone, furaltadone, and nitrofurantoin and nifurstyrenate sodium). After optimization of different coating antigen-antibody combinations, the coating antigen from diazotization method and the antibody from glutaraldehyde method were used to develop an indirect competitive enzyme linked immunosorbent assay (ELISA). The cross-reactivity to the seven nitrofurans were 95%,79%,76%,68%,61%,59%,42%, respectively, the IC50 values were 95,79,76,68,61,59,42ng/mL, respectively, and the limits of detection were 8.3,14.0,15.8,11.0,11.0,13.5,18.2ng/mL, respectively. The seven nitrofuran drugs were fortified into blank feeds at different levels and the recoveries were in a range of 78.0%-98.4% with coefficients of variation of 6.2%-13.8%. Furthermore, a HPLC method was developed to determine the 7 nitrofuran drugs. These drugs were in good linearity in the range of 0.01-1μg/mL. The limits of detection for the seven drugs in feeds were 21, 26, 28, 32, 17, 19, 20ng/g, respectively and the recoveries were in a range of 70.1%-99.6%.In this study, a rapid and sensitive ELISA method was developed to detect seven nitrofurans in animal feeds simultaneously and a HPLC method was established as the confirmatory method. The two methods could be used to determine the presence of nitrofuran drugs in animal feeds to monitor the illicit use of nitrofuran in animal husbandry and ensure the safety of animal feeds and animal-original food.