Rapid Detection Method Of Illegal Additives And Heavy Metals In Health Food

In recent years,the health food industry has been developing rapidly,and there are many products.However,due to imperfect regulations and limitations of testing methods,health food safety problems are increasing,and the safety problems mainly come from two aspects:artificial addition and contamination of raw materials.With the rapid expansion of the industry,the scope of safety supervision has also increased,and the traditional instrument detection methods cannot meet the rapid detection of large-scale samples on site.Therefore,there is an urgent need to develop a rapid,accurate and simple detection method for the detection and analysis of harmful substances in health food.In this study,the artificially added illegal drugs（CBD,THC,ZOP）and heavy metal contamination（Hg,Cr）in raw materials were used as research objects to prepare highly sensitive m Abs by synthesizing antigens,and then to establish a rapid colloidal gold immunoassay method,which was applied to protein powder of plant origin and functional drinks.1、Design and modification of semi-antigen and synthesis of artificial antigenCannabidiol（CBD）does not have its own structure-COOH or-NH₂ and other groups,choose to start from its parent nucleus structure,using the phenolic hydroxyl group on the benzene ring and 4-bromobutyric acid ethyl ester substitution reaction to obtain the linkage arm with ester group,in high temperature alkaline conditions,the ester group hydrolysis to generate-COOH,CBD hapten;through CBD and MMC（methyl Methoxy magnesium carbonate）to obtain cannabidiolic acid（CBDA）,as the second hapten.Δ⁹-Tetrahydrocannabinol（Δ⁹-THC）,zopiclone（ZOP）have no-COOH or-NH₂ in their structure and no reactive groups that can be modified and modified（e.g.-OH,-Cl,-CN）.For such compounds,the synthesis of tetrahydrocannabinol and zopiclone hapten was chosen from their intermediates.Starting with theΔ⁹-THC intermediate,the tetrahydrocannabinol hapten（THC hapten）with-COOH and linker arm was obtained in a 6-step reaction;6-（5-chloro-2-pyridyl）-6,7-dihydro-7-hydroxy-5H-pyrrolo[3,4-b]pyrazin-5-one was used as the starting material,and the-OH on the pyridine ring was activated using the succinic anhydride method to introduce the-COOH bearing carbon chain.The above derivatized product was identified by LC-MS,and the molecular weight of the reaction product remained consistent with that of the target compound,indicating successful derivatization of the hapten.The above hapten with-COOH was activated by carbodiimide method（EDC method）and then coupled with-NH₂ of carrier proteins（KLH,BSA,OVA）to synthesize the corresponding immunogen and coating antigen.The heavy metals mercury and chromium have small molecular weights and are unable to elicit immune responses,requiring the use of bifunctional chelating agents to link carrier proteins and heavy metal ions.The chelator ITCBE was used to synthesize the mercury immunogen Hg-ITCBE-KLH and the chelator MNA to synthesize the coating antigen Hg-MNA-BSA;the synthesis of chromium immunogen and coating antigen was carried out under weak acidic conditions,and since Cr⁶⁺could not chelate with ITCBE,the Cr³⁺standard was chosen to synthesize the immunogen Cr-ITCBE-KLH and the coating antigen Cr-ITCBE-BSA.The synthesized complete antigen was characterized by UV spectrophotometry,and a broadening or red-shift blue-shift of the UV absorption peak of the antigen compared with that of the carrier protein indicated the successful synthesis of the immunogen or the encapsulant.2、Monoclonal antibody preparationA single hybridoma cell line capable of secreting specific antibodies was obtained by ic-ELISA serum screening and cell fusion techniques,followed by multiple subcloning:1H6（CBD）,1C10（Δ⁹-THC）,4E4（ZOP）,4D7（Hg）,4H4（Cr）,with m Abs IC50s of 2.03,4.15,0.099,0.606 ng/m L,and the affinity constants of the five monoclonal antibodies,KD,were between 107 and 1012 L/mo L,which were high affinity antibodies.The isotypes of antibodies1H6,1C10,4E4,4D7 and 4H4 were Ig G2a,Ig G2a,Ig G1,Ig G2b and Ig G1,respectively.in the antibody specificity identification,antibody number 1H6 showed slight cross-reactivity to CBDA and CBN,1C10 showed 69.3%cross-reactivity toΔ⁸-tetrahydrocannabinol,and the remaining three antibodies were all high specificity monoclonal antibodies.3、Establishment of colloidal gold immunochromatographic method in health foodThe more popular health foods-plant-derived protein powder and functional drinks were selected as substrates and identified as negative by the instrument.In the establishment of the test strip detection method,the concentration of antigen antibody,surfactant and other parameters were optimized;for heavy metals mercury and chromium in solid and liquid samples,a special extraction method was designed and the parameter conditions（p H,EDTA）in the process of heavy metal detection were optimized.A rapid method was developed for the colloidal gold immunochromatographic detection of three illegal additives,cannabidiol,tetrahydrocannabinol,zopiclone and two heavy metals,mercury and chromium,in health food products.The visual limits of detection（v LOD）of cannabidiol,tetrahydrocannabinol,zopiclone,mercury and chromium in protein powder of plant origin were 250,100,25,20 and1000 ng/g;v LOD in functional drinks were for 100,50,25,10,20 ng/m L,the developed colloidal gold test strips can meet the limit requirements in the national standards.In order to verify the accuracy and stability of colloidal gold test strips in real samples,spiked recovery experiments of five hazards were conducted in negative plant-derived protein powder and functional beverage matrix,and the results showed that the accuracy and stability of the test strips were consistent with those of the instrumental methods and could achieve rapid detection of large-scale samples in the field.The results showed that the accuracy and stability of the test strips were consistent with those of the instrumental method,and could achieve rapid detection of large-scale samples in the field.