Studies On The Analysis Methods And Metabolic Mechanism Of Several Kinds Of Systemic Insecticides In Tea

Systemtic insecticides can be permeated through the plant epidermis,and transmitted to other parts of plant by apoplast and symplast the two pathways,which having the great control effect on pests and diseases.At the same time,systemtic insecticides in the plant will be metabolized to different structure and toxic metabolites,and some metabolites are also used in the prevention and control of pests and diseases.Due to the systemtic peculiarity,the residual analysis of systemtic insecticides in the plant is faced with the difficulty of extraction and purification,thereby suffer from the matrix effects.And the highly toxic metabolites of some systemtic insecticides bring the unavoidable security issues to the human and environment.Thus,it is of great practical significance to establish the residual analysis method of systemtic insecticides and to study its mechanism of absorption and metabolism in tea plant.As tea contains the rich polyphenols,alkaloids,amino acids and other ingredients,there is a great difficulty in the analysis of trace parent and residual metabolites.In this paper,the residual analysis methods of organophosphorus and the neonicotinoid pesticides the two kinds of insecticides which are commonly used in tea garden were developed in tea and tea cell suspension.In order to study the absorption,transportation and metabolism of the systemtic insecticides more conveniently and quickly in the tea plant,the tea cell suspension platform which was cultured by callus of tea sterile seedling and excised leaf were established to study the metabolism of organophosphorus and neonicotinoid pesticides in tea cell suspension and the absorption and transportation of organophosphorus pesticides in tea plant.The main research contents are as follows:1.Development of Eight Neonicotinoid Pesticide Residue Analysis Methods in Six Types of Tea.Liquid chromatography—electrospray ionization tandem mass spectrometry(LC-ESI-MS/MS)is a primary tool for analysis of low volatility compounds in complex matrices.However,complex matrices,such as different types of tea contained different ingredients,even for the residual analysis of the same pesticide,there will be different matrix enhancement or weakening effect.The matrix effect seriously affects the accuracy of the analytical method.Therefore,optimizing the pretreatment method to eliminate matrix interference is the key to the establishment of pesticide residue analysis method.In this study,tea samples were soaked with water and extracted with acetonitrile,cleaned up with a combination of PVPP(160 mg)and GCB(20 mg),and dried.Dried extracts were diluted with 20 mL acetonitrile/water(15:85,v/v)before analysis by UPLC-MS/MS.The average recoveries of eight neonicotinoid insecticides(dinotefuran,nitenpyram,thiamethoxam,imidacloprid,clothianidin,imidaclothiz,acetamiprid,and thiacloprid)ranged from 66.3 to 108.0%from tea samples spiked at 0.01-0.5 mg/kg.Relative standard deviations were below 16%for all recovery tests.The limit of quantification ranged from 0.01 to 0.05 mg/kg.The optimal combination of dilution method and modified QuEChERS were used to remove the substrate effect of tea.A method for the determination of eight neonicotinoid insecticides in six types of tea by ultra performance liquid chromatography tandem mass spectrometry was established,and the accuracy,the precision and detection limits of the method were consistent with the requirements for pesticide residue analysis.2.Development of Organophosphorus Pesticides Residue Analysis Methods in Tea Leaves and Tea Cell Suspension.A method for the determination of acephate and methamidophos racemic compounds and their chiral enantiomers in tea leaves and tea cell suspension were established by ultra performance liquid chromatography and gas chromatography.Tea samples were extracted with 20mL acetonitrile for 2min,then adding 5g sodium chloride,centrifuge the supernatant and dried.The residues were dissolved with 10 mL Methanol/water(5:95,v/v)and 10mL acetone,taking 1mL to filter the 0.22μm PTFE hydrophilic membrane before analysis by UPLC-MS/MS and GC.The average recoveries ranged from 75.9 to 93.0%and 68.4-94.0%from tea samples spiked at 0.01-0.5 mg/kg.Relative standard deviations were below 15%for all recovery tests.The matrix effect of racemic acephate and methamidophos were assessed at 0.05mg/kg spiked level by UPLC-MS/MS,the values were only 7%and 8%respectively.Pesticide toxicity and environmental attribution with chiral enantiomeric structure are different,which is the frontier and hotspot of analytical chemistry.The residue analysis methods of chiral enantiomers acephate and methamidophos in tea cell suspension was established,the chiral enantiomers of acephate and methamidophos were well separated by using chiral capillary column CYCLOSIL-B with gas chromatography.The target compounds in cell suspension were extracted with acetone/ethyl acetate(3:7,v/v),and the average recoveries ranged from 86.0 to 96.0%from tea samples spiked at 0.01-0.5 mg/kg.Relative standard deviations were 0.3-3.2%.The three residue analysis methods established in this experiment are not only simple,efficient and low-cost,but also provide a technical basis for the later metabolic experiments of acephate and methamidophos in tea leaves and tea cell suspension.3.Study on Absorption and Metabolism of Acephate in Tea Plant by Excised Leaves.In order to study the digestion dynamics and mechanism of acephate and its chiral enantiomers in tea,after spraying the acephate in the tea garden,the degradation rates of acephate were 67.98%and 77.42%on day 4 and day 6 respectively,at the same time the production rate of metabolites methamidophos were 2.64%and 4.62%.In order to further prove that methamidophos is the metabolite of acephate metabolized in tea plant inside,the mechanism of absorption and metabolism of acephate and its chiral enantiomers was studied by using sterile tissue culture plantlet and field excised leaf.The results show that no matter in the light or dark condition,acephate and its chiral enantiomers can be absorbed through the tea stem and transferred to the terminal buds,at the same time in stem and leaf parts of the tea excised leaf can be detected metabolite methamidophos.There is no significant enantioselectivity of the chiral enantiomers of acephate during the process of absorption,while the metabolite methamidophos has the enantioselectivity in the process of metabolism and degradation,and(+)-methamidophos produced more than(-)-methamidophos.This experiment provides a reference for the safety evaluation of acephate in tea.4.The Establishment of Tea Cell Suspension Metabolism Platform of Organophosphorus and Neonicotinoid Pesticide.The raw material come from tea leaf callus induced by sterile tissue culture seedlings of Longjing No.33,which was cultivated by the State Key Laboratory of Tea Plant Biology and Utilization,Anhui Agricultural University.Selected the several subculture,growing well,loose,and fragile callus,then cut them into small particles and inoculated into a liquid medium for shaking culture under dark condition.After 5-7 days,stabilized the large pieces of callus and the supernatant suspension culture was obtained as a seed suspension cell to carry out subculture.In this study,the effects of the selection of basic culture medium,the proportion of suspended cells,the ratio of plant growth hormone and the amount of liquid on the growth of cell suspension were studied.The growth conditions of the cell suspension were obtained finally:Inoculation amount 4g/mL,subculture ratio 1:(2～3)(cell suspension mother liquor:fresh medium),the amount of liquid 40mL/150mL Erlenmeyer flask,B5+2,4-D(1mg/L)+KT(0.1mg/L),25 ℃,120rpm constant temperature oscillation dark culture.The TTC method was used to test the cell viability,and the color was more red,which indicated that the growth was more vigorous and the activity was higher.Furthermore,the cell suspension with good dispersion were observed under the microscope imaging system,which has established a good platform for the study of cell location and metabolic mechanism of pesticides.5 μg/mLof 3 neonicotinoid(thiamethoxam,imidacloprid,acetamiprid and imidaclothiz)and 4 organophosphorus pesticides(acephate,methamidophos,dimethoate and omethoate)were added to tea cell suspension for metabolic studies respectively.The results show that in addition to thiamethoxam,the other three neonicotinoid pesticides in the cell suspension had no degradable phenomenon,thiamethoxam entered into the degradation period from the 10th day,the degradation rate reached to 55.3%in the 75 days.While organophosphorus pesticide have a good metabolism in the cell suspension,omethoate had the fastest degradation,degraded all in 30 days.The degradation of chiral acephate and methamidophos in suspension cells was consistent with the first order kinetic model.The half-lives of(+)-acephate and(-)-acephate in suspension cells were 22.8 days and 22.6 days,the half-lives of(+)-methamidophos and(-)-methamidophos in suspension cells were 17.0 days and 16.9 days respectively,and metabolites of acepahte methamidophos had a certain enantioselectivity in cell suspension during the metabolism vigorous period,and the production of(-)-methamidophos is greater than that of(+)-methamidophos.The metabolites of thiamethoxam,acephate and dimethoate,which were degraded in cell suspension,were screened by high resolution mass spectrometry(QTOF).The metabolites were identified by secondary mass spectrometry.There are six kinds of metabolites detected in the cell suspended for thiamethoxam,there are two metabolites for acephate and dimethoate.