Study On Residue Detection Of Nitrofurantoin And Its Metabolites

Nitrofuran was synthetic broad-spectrum antibiotic,which had been previously used in the treatment of certain bacterial infections in animals and promoted the growth of animals as feed additives,for its excellent antibacterial effect and low price.They are rapidly metabolized in vivo and their major metabolites persist for considerable periods.Nitrofurans and their metabolites had been banned in animal husbandry worldwide due to their carcinogenic and mutagenic potency.However,they are still illegally used ofen by someone driven by economic benefits.In order to control the residues of veterinary drugs and to ensure the safety of animal food products,it is necessary to establish a rapid and effective method for the detection of residues.Nitrofurantoin(NFT)is often used as a feed additive to promote the growth of animals.It is metabolized rapidly in vivo and has a short half-life.In contrast,its metabolite could bind to tissue proteins closely and persist for considerable periods.Therefore,the AHD in animal tissues are usually tested to monitor the illegal use of NFT.In this paper,the NFT and its metabolite AHD were used as a research target.The residues of NFT in feed and the residues of AHD in animal tissues were detected by high performance liquid chromatography(HPLC)and indirect competitive enzyme-linked immunosorbent assay(ELISA).The main research results are as follows:(1)The HPLC method with the simple mobile phase acetonitrile-water was used to detect the residue of the original NFT on the basis of the traditional method.The chromatographic conditions were optimized and the method was validated.The limit of detection was 0.02 ?g/mL and the limit of quantification was 0.1 ?g/m L,respectively.In the feed,the limit of detection was 0.2 mg/kg and the limit of quantification was 0.7 mg/kg,respectively.The results are lower than the standards of issued by Ministry of Agriculture of the PRC(No.1486)that is the limit of detection of 0.3 mg/kg and the limit of quantification of 1.0 mg/kg.(2)The hapten,1-(4-carboxyphenylmethylene)-aminohydantoin(CPAHD),was synthesized by the reaction of metabolite AHD with p-carboxybenzaldehyde.And the preparation of hapten was successful by mass spectrometry,NMR and IR.The immunogens,CPAHD-BSA(Z)and CPAHD-BSA(L),were prepared by the activated ester method and the mixed anhydride method,and the immunogen were characterized by UV.The hapten-protein ratios were 12.5 and 4.4,respectively.In addition,polyclonal antibody was prepared by the immunized rabbit.And the antibody produced by immunogens CPAHD-BSA(Z)had good sensitivity and specificity.The result indicated that the synthesis of the immunogen was successful.The immunogen CPAHD-BSA(L)was not show a similar effect.The reason may be that the immunogen with a low hapten-protein ratio can not stimulate the body to produce antibodies against small molecules.(3)The growth performance and organ index for rabbits were investigated in the process of preparing antibodies.The results showed that the mean weight gain was increased significantly(p <0.05).The cardiac index and kidney index were increased significantly(p <0.05),and there was no significant effect on the liver(p> 0.05).In conclusion,the process of preparing antibodies improved the growth performance,cardiac index and kidney index of rabbits.And it was no significant effect on the liver.(4)The polyclonal antibodies were prepared by immunizing mice using immunogen CPAHD-BSA(Z)and CPAHD-BSA(L)with different doses of immunogens.The antibodies produced by immunogen CPAHD-BSA(Z)had high titer determined by ELISA when CPAHD-OVA(Z)and CPAHD-OVA(W)were used as coating antigens.Antiserum titers range from 1: 32000 to 1: 256000.The immunogen CPAHD-BSA(L)did not show a high titer compared to the immunogen CPAHD-BSA(Z).(5)The indirect competitive ELISA was used to detect the residual of AHD in animal tissues.The parameters of the method were optimized and the sensitivity of the antiserum produced by the different immunization dose of CPAHD-BSA(Z)was compared.The method has a good sensitivity with an IC50 value of 4.93 ng/m L and the limit of detection of 0.17 ?g/kg in pork tissue and 0.16 ?g/kg in chicken tissue,respectively.In addition,the antibody has good specificity for 1-(2-nitrobenzylidene)-aminohydantoin(NPAHD).And had no cross-reaction with other analogs in addition to CPAHD and NFT.The results of ELISA were confirmed by HPLC method,and showed good correlation(R=0.998).The molecular modeling was used to study the relationship between the affinity of the antibody and the structure of the small molecule.For the detection of AHD residues in the tissue,the existence of C=N double bond and phenyl ring could play an important role in the recognition of the antibody.