Immunochromatographic Assays For Quantitative Determination Of Nitrofuran Metabolite Residues In Animal-derived Food

Food safety has always been a focus of concern and need to be addressed,which is closely related to people's health.Animal-derived food safety is an important part of it.In livestock and aquaculture,nitrofuran antibacterials were widely used for prophylactic and therapeutic purposes,or promote animal growth.Due to the carcinogenic and teratogenic side effects of these drugs and their metabolites,nitrofurans have been completely banned from animal husbandry and prohibited from being detected in animal-derived food by many countries around the world.Commonly used drugs,including nitrofurazone?NFZ?,nitrofurantoin?NFT?,furaltadone?FTD?and furazolidone?FZD?,are respectively metabolized to semicarbazide?SEM?,1-aminohydantoin?AHD?,3-amino-5-morpholino-methyl-2-oxazolidone?AMOZ?and 3-amino-2-oxazolidone?AOZ?soon after being absorbed into animal body and existed as stable protein-bound metabolites.Therefore,the detection of metabolites is usually used to reflect the residual state of nitrofurans.The European Union and the United States have established the minimum required performance limit?MRPL?at 1?g/kg for nitrofuran metabolites in animal-derived food.The rapid,sensitive and efficient immunochromatographic assays for detection of nitrofuran metabolites in animal-derived food were developed.The hapten 4-??3-benzaldehyde-semicarbazone-nitro?phenol?butyric acid?SEM-NP-BBA?was prepared by the derivatization of nitrofurazone metabolite?semicarbazide,SEM?with the substitution and hydrolysis reaction product of 2-nitro-5-hydroxybenzaldehyde and ethyl 4-bromobutyrate,the coated hapten 4-carboxybenzaldehyde semicarbazone?4-CPSEM?was prepared based on aldehyde amine condensation reaction between 4-carboxybenzalde-hyde and SEM.The immunogen and coating antigen of SEM were synthesized by coupling with bovine serum albumin?BSA?and ovalbumin?OVA?through active ester method,and the immunogen was identified by MALDI-TOF with a hapten coupling ratio of about 8.5.Using this immunogen to immunize BALB/c mice,a monoclonal antibody?mAb?SEM-9A6-F1 was prepared based on hybridoma technology.The mAb subtype was IgG2a,the light chain was?type.The concentration of mAb was 4.25 mg/mL after purified by Protein A affinity column.The results of competitive indirect enzyme-linked immunosorbent assay?ciELISA?showed that the antibody titer was 1:27000,the IC₅₀ value was 0.013?g/L,there is no cross-reaction with other structurally similar drugs and residual drugs commonly found in livestock and aquatic products.A background fluorescence colloidal gold immunochromatographic assay?bFQICA?combined with a portable fluorescent immunoassay analyzer and a two-dimensional code with a built-in standard curve for the rapid and on-site quantitative determination of four major nitrofuran metabolites in animal-derived foods?egg,chicken,fish and shrimp?was developed.The limits of detection?LODs?for bFQICA in egg,chicken,fish and shrimp were0.09,0.10,0.12,0.15?g/kg for SEM,0.12,0.15,0.20,0.20?g/kg for AHD,0.15,0.15,0.20,0.20?g/kg for AMOZ,0.07,0.10,0.10,015?g/kg for AOZ,respectively.Blank animal samples were fortified at concentration of LOD,2LOD,4LOD and 1?g/kg,recoveries of intra-assay ranged from 73.7%to 107.4%with coefficient of variation?CV?ranged from 3.2%to 12.2%,recoveries of inter-assay ranged from 75.7%to 108.0%with CV ranged from 3.0%to 12.8%.Analysis of field animal-derived food samples by bFQICA was in accordance with that of LC-MS/MS,proving that this bFQICA method was a promising method for the rapid detection of nitrofuran metabolites in animal-derived foods.A rapid and sensitive high-throughput latex bead immunochromatographic assay?LB-ICA?combined with a multi-channel immunochromatographic quantitative reader for simultaneously quantitative detection of four major nitrofuran metabolites in animal-derived food?egg,chicken,fish and shrimp?was developed.The LODs for LB-ICA in egg,chicken,fish and shrimp were 0.02?0.02?0.04?0.05?g/kg for SEM,0.04?0.05?0.08?0.07?g/kg for AHD,0.10?0.10?0.15?0.12?g/kg for AMOZ,0.02?0.02?0.02?0.03?g/kg for AOZ,respectively.Blank animal samples were fortified at concentration of LOD,2LOD and 4LOD and 1?g/kg,recoveries of intra-assay ranged from 73.5%to 109.2%with CV ranged from3.5%to 12.4%,recoveries of inter-assay ranged from 75.8%to 105.5%with CV ranged from2.3%to 12.9%.Analysis of field animal-derived food samples by LB-ICA was in accordance with that of LC-MS/MS.Therefore,this proposed multiplex LB-ICA was a promising method for on-site screening of multiple analytes in animal-derived food.