| Traditional Chinese medicines(TCM)are one of the main products that people rely on for disease prevention and treatment,and the quality and safety of TCM is the basic guarantee to ensure the safety of TCMs consumers.At present,pesticide residues in TCM have caused widespread concern both at home and abroad.Pesticide residues not only affect the quality and safety of TCM,but also affect the economic trade of TCM in the world and pose a greater threat to the health of TCM intake population.Neonicotinoid insecticides are new types of insecticides.Since the launch of the first neonicotinoid insecticide imidacloprid product in 1991,many different types of neonicotinoid insecticides have been subsequently developed,registered and applied to the control of pests in the cultivation process of agricultural products.However,there are very limited reports on neonicotinoid insecticide residues in TCM,and there is a lack of analytical methods for comprehensive screening and rapid detection of neonicotinoid insecticide residues in TCM.In order to fill the data gap in this section,this paper has established a comprehensive screening and rapid detection analysis method applicable to the residues of neonicotinoid insecticides in different TCM samples,and the developed method was used to detect the residues of neonicotinoid insecticides in TCM samples and TCM planting base samples.The obtained residue results provide experimental data for understanding the neonicotinoid insecticide residues in TCM.In addition,this subject has applied nanozyme and biochar materials in the detection,sample pretreatment and removal of pesticide residues in TCM.which provided new research ideas for the analysis and removal of pesticide residues in TCM.1.Establishment of LC-MS/MS method for the detection of neonicotinoid insecticides in TCM and analysis of residue dataA green analytical method for the comprehensive screening of 20 neonicotinoid insecticides and their metabolites in TCM samples(Panacis quinquefolii radix,Arctii lappae radix,Carthami flos,Lycii fructus,Citri reticulatae pericarpium)and chrysanthemum plantations(chrysanthemum plants and planting soil)was established for the first time using QuEChERS sample pretreatment technique combined with LC-MS/MS analysis.The results showed that neonicotinoid insecticides and their metabolites were detected in lappae radix,Carthami flos,Lycii fructus,Citri reticulatae pericarpium samples,with a positive detection rate of 82.57%,and the residue of total neonicotinoid insecticides ranged from 0.26 to 139.28 μg kg-1.Acetamiprid(77.06%,≤85.95 μg kg-1),imidacloprid(67.89%,≤32.49 μgkg-1)and their metabolites(N-desmethyl-acetamiprid(44.04%,≤18.42 μg kg-1)and desnitro imidacloprid(48.62%,≤16.55 μg kg-1))were most frequently detected in herbs.There were significant positive correlations between the residual imidacloprid/acetamiprid and their metabolites in TCM samples.In addition,the test results in chrysanthemum plants and planting soil showed that the residual levels of imidacloprid and its metabolites(desnitro imidacloprid and imidacloprid urea)were high.The high concentration of imidacloprid residue in soil had a high ecological risk.There was also a significant positive correlation between the residual imidacloprid and its metabolites in the samples from the chrysanthemum planting base.Soil adsorption and degradation experiments have proved that imidacloprid,acetamiprid and their metabolites were difficult to adsorb in the soil,and were prone to the migration,with a degradation half-life of 20～36 days.2.Preparation of imidacloprid monoclonal antibodies and establishment of rapid detection methods for imidacloprid residuesA new imidacloprid hapten 6 was designed and synthesized,and it was used to prepare hapten 6-KLH immunogen,which could effectively immunize mice to produce imidacloprid antibodies.Using this immunogen,monoclonal antibodies(IMIB7C3)that could specifically recognize imidacloprid was obtained.The antibody subtype was IgG1,and the afflnity constant with antigen hapten 6-KLH was 5.7×109 L mol-1.In PBS buffer,the IC50 of the antibodies to imidacloprid was 1.3 ng mL-1,and it had 30.9%cross reaction with imidaclothiz,and 9.0%and 5.8%cross reaction with imidacloprid metabolites hydroxy imidacloprid and imidacloprid olefin,respectively.An ic-ELISA method for the determination of imidacloprid residues in TCMs(Notoginseng radix et rhizoma,Dioscoreae rhizoma,Lonicerae japonicae flos,Astragali radix,Jujubae fructus)was established with imidacloprid monoclonal antibodies.The results showed that the residual level of imidacloprid in Lonicerae japonicae flos(30.6～1205.3 μg kg-1)and the positive detection rate were high(88.57%).In order to validate the accuracy of the ic-ELISA detection results,LC-MS/MS method was established to determine the samples.The correlation coefficient R2 between the LC-MS/MS results and the ic-ELISA results was 0.9975,which proved that the established ic-ELISA could be used for the analysis of imidacloprid residues in different TCMs.In addition,the acute and chronic risks of imidacloprid ingested from Lonicerae japonicae flos were assessed,and the results showed that the acute and chronic risks were low.In addition,A colloidal gold immunechromatographic strip method for real-time,on-site and rapid detection of imidacloprid residues was developed based on imidacloprid antigens and antibodies.By optimizing the conditions of gold-labeled antibody conjunction,the concentration of C and T lines and the sample diluting solution.The prepared colloidal gold test strip was applied to the rapid screening of imidacloprid residues in 72 TCMs.The prepared imidacloprid test strip could be used to screen the imidacloprid residues in 60 different TCMs(including 26 kinds of root/stem medicines,20 kinds of seed/fruit/pericarp medicines,11 kinds of flower/leaf/whole herb medicines,3 kinds of peel/aboveground issues of herb medicines),and the limit of detection was 50 μg kg-1.In addition,the limit of detection and repeatability of the imidacloprid test strip in Coicis semen,Hordei fructus germinates,Nelumbinis semen,Zingiberis rhizoma and Lonicerae japonicae flos were also investigated,and the actual samples were determined.The results showed that the established colloidal gold immunechromatographic strip could be used for rapid screening and detection of imidacloprid residues in a variety of TCMs.3.Establishment of ELISA method for rapid detection of imidacloprid based on MIL100(Fe)@AuNCs nanozymeMIL-100(Fe)@AuNCs nanozyme with peroxidase activity was synthesized.The nanozyme had good stability and enzyme activity in a wide temperature range.The Vmax and Km of nanozyme to H2O2 substrate were 5.3 × 10-8 M s-1 and 0.20 mM,and the Vmax and Km of nanozyme TMB substrate were 11.3 × 10-8 M s-1 and 1.50 mM through steadystate enzyme kinetics study and Michaelis-Menten equation.The prepared nanozyme could be used to detect H2O2(LOD=1 μM),glutathione(LOD=5 μM),and cholesterol(LOD=3 μM).A rapid and on-site detection method for H2O2 was established by immobilizing nanozyme and TMB on paper and combining with RGB recognition technology.In addition,the prepared peroxidase-like nanozyme had the effect of antiEscherichia coli.Finally,an attempt was made to construct a direct competition ELISA for the detection of imidacloprid by coupling the nanozyme with imidacloprid antibodies.4.Application of biochar as a phytochrome purification agent in the pretreatment of traditional Chinese medicine for pesticide residue detectionBiochar derived from rice straw pyrolysis had high specific surface area and a variety of functional groups on the surface.The adsorption kinetics,adsorption isotherm and adsorption thermodynamics of biochar on malachite green,imidacloprid and chlorophyll were investigated,respectively.The results showed that the adsorption kinetics of biochar on the three adsorbates conformed to pseudo-second-order kinetics,belonging to chemical adsorption;The adsorption isotherm was consistent with Langmuir model,indicating that the surface of the adsorbent was uniform,the adsorption process was mainly monolayer adsorption;The adsorption thermodynamic results showed that the adsorption process of biochar on adsorbate was endothermic,irregular and spontaneous.The maximum adsorption capacity qm of biochar for malachite green,imidacloprid and chlorophyll were 2615.32,178.57 and 400.00 mg g-1,respectively,and the maximum adsorption capacity qm of GCB for chlorophyll was 69.25 mg g-1.It showed that the adsorption effect of biochar on chlorophyll was much higher than that of GCB.Biochar could be used to purify chlorophyll in 12 different aqueous and organic solutions.To investigate the practicability of biochar in sample pretreatment,biochar was applied to the established QuEChERS sample pretreatment process,and the results showed that biochar could not only effectively purify phytochrome,but also ensure the recoveries of 123 pesticides(including imidacloprid)in chrysanthemum samples and the recovery of imidacloprid in Lonicerae japonicae flos samples.On the other hand,the electrospinning film prepared from biochar was used as a sample pad for colloidal gold immunochromatographic strip.The results showed that the biochar electrospinning film could achieve online cleaning up of phytochrome to reduce background interference and improve the detection sensitivity of imidacloprid.5.Preparation of Fe/Zn-KOH-BBC material and its application in the removal of imidacloprid residues in Lonicerae japonicae flos decoctionThe modified biochar material Fe/Zn-KOH-BBC with higher adsorption capacity of imidacloprid was obtained by sugarcane bagasse pyrolysis and modification with KOH,ZnCl2 and FeCl3.Through adsorption kinetics,adsorption thermodynamics and adsorption isotherm experiments,it was proved that the adsorption of imidacloprid and chlorogenic acid by Fe/Zn-KOH-BBC belonged to pseudo-second order kinetics and chemical adsorption;The adsorption isotherm belongs to the Freundlich model,indicating that the adsorption process was multi-layer adsorption with uneven surface;The adsorption thermodynamic results showed that the adsorption process of biochar on adsorbate was endothermic,irregular and spontaneous.The Fe/Zn-KOH-BBC was used to construct a solid phase extraction device for removing imidacloprid residues from Lonicerae japonicae flos decoction.The results of LC-MS/MS showed that 71.01%of imidacloprid,65.70%of acetamiprid and 92.93%of desnitro imidacloprid in the Lonicerae japonicae flos decoction could be removed by the device,and 78.03%of chlorogenic acid could be retained;HPLC analysis showed that the similarity of the characteristic spectra before and after the removal of Lonicerae japonicae flos decoction was high(≥ 0.991),and the recoveries of the characteristic peaks was also high(the recovery of chlorogenic acid was 90.4%,the recovery of chiratin was 92.5%,the recovery of secoxyloganin was 100%,and the recovery of total phenolic acids(chlorogenic acid,isochlorogenic acid A and chlorogenic acid C)was 79.56%).Finally,the cytotoxicity experiment proved that low concentration of Fe/Zn-KOH-BBC material had little effect on cell activity. |
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