Studies On Determination Of Ractopamine Residues In Meat

Ractopamine belongs to ?-adrenegic agonist with phenyl ethanolamine,which can be used to treat asthma,muscular dystrophy and other diseases.But,in recent years,ractopamine has been used as a new type of clenbuterol in animal husbandry in illegal ways.It residues in animals that has a significant impact on consumers.Nowadays,ractopamine is listed in Catalogue of drugs prohibitedfrom use in feed and animal drinking water in China,the EU also explicitly prohibit its'use.However,27 countries including Japan,the United States,Canada allow its usage.Therefore,from the standpoint of food safety and the import and export trade,the detection of ractopamine residues has great value.In this paper,we established detection of ractopmine in meat by Ultraviolet Spectrophotometry combine with ?-cyclodextrin enrichment and direct competitive ELISA with biotin-streptavidin amplified system.We also prepared the immunomagnetic beads with biotin-streptavidin amplified system,the results are as follows.A new spectrophotometry for the determination of ractopamine was developed which was based on ?-cyclodextrin enrichment.It will be the optimum conditions of this detection method when inclusion time is 60 min,inclusion temperature is 30 ?,pH is 7.0.Under the best conditions,sensibility was 0.05mg/L,the linear range was 1.5-3.0mg/L(r=0.9998).The results indicated that the RSD of intra-day(n=6)and inter-day(n=3)were 0.87%-1.07%and 0.80%-1.28%.The average recoveries for ractopamine was in the ranges of 91.91%-102.30%that showed great precision,repeatability and stability.We prepared immunomagnetic beads for enrich ractopamine in meat by biotin-streptavidin labeling system.The results showed 81.6?g/mg immunomagnetic beads for ractopamine was prepared by 261 ?g biotinylated antibody added with 1 mg immunomagnetic beads coupled with 40 ?g streptavidin in pH7.2 PBS.Based on ractopamine solution(10ng/mL)as the research system,we added 0.3 mg immunomagnetic beads for lOmin when it got good enriching effect.The best eluent was methanol.The average adsorption efficiency was at 63.33%-80.7%with the RSD of 3.01%-13.19%.The direct competitive ELISA was established to detect ractopamine from the meat.The optimal reaction conditions were as follows:the 96 ELISA plate coated by RAC-BSA was 0.5?g per hole,the biotinylated antibody.was attenuated by 1:1000 for 1h,the blocking solution was 2.0%milk,the SA-HRP was also attenuated by 1:20000.1n this way,IC50=0.47ng/mL,the limits of detection was 0.02ng/mL.The intra-assay and inter-assay coefficients of variation were 3.62%-9.20%and 4.04%-12.39%which were all below 15%meeting high-precision requirements.The cross reaction rates of norepinephrine and epinephrine between ractopamine were 2.10%and 3.79%.We studied sample preparation methed by the direct competitive ELISA and immunomagnetic beads for enrich ractopamine.The average recoveries for ractopamine was in the ranges of 78.67%-86.75%and the RSD were 5.72%-9.01%which demonstrated that the direct competitive ELISA we developed could meet the requirements of realdetection.