Screening And Validation Of Markers For Pesticide Exposure In Plants By Metabolomics

With the improvement of standards in living of people,who more and more realized the importance in the food safety and increased demands of organic food day by day.The organic food refers to that in their growth without any pesticides,fertilizers,growth regulators,antibiotics,genetically modified technologies.However,after vegetables and fruit which have been sprayed pesticides,in particular,in a safe period of spraying pesticides behind,the pesticides degrading or metabolized by plant.moreover,the current detectable methods and Indexes are targeted to testing the residence of pesticides in food,and there are no methods to detect whether pesticides had been used to food.Therefore,to provide the new detection methods and judge indexes for pesticides identify and certification of organic food,in this paper,we utilized metabolomics to screened the differential Chemical compounds as crucial markers that plants exposure to pesticides in the drug withdrawal period,In this experiment,methyl tert-butyl ether(MTBE):methanol:water system was adapted.And optimized ratios of extractant by response surface methods were the material-liquid ratio of methyl tert-butyl ether.(V:W)2.4;ultra-pure water-to-liquid ratio(V:W)5.8;methanol-to-liquid ratio(V:W)3.2.It provided a feasible sample preparation scheme for related research through plant metabolomics experiments.Multivariate data analysis was performed on the data of 320 crop samples exposed to pesticides and 80 unexposed pesticides.According to distribution characteristics of each metabolite in the metabolite's volcano map,combined with VIP values of metabolic PLS-DA,four pesticides were selected respectively to expose to different crops and different drug withdrawal periods VIP>1,FC>2 metabolites,which were used as differential metabolites,and then 11 differential metabolites co-owned of four pesticide exposures were screened by Venn analysis as potential markers of pesticide exposure with different drug withdrawal periods to satisfy multiple crops and various pesticides,According to the information from mass spectrum,after searching and comparing with the mass spectrometry library,and it was identified that the devise metabolite that the exact mass is m/z 287.1553(the characteristic fragment ions are m/z213.0565,m/z153.1029,m/z 121.1286)is kaempferol,and then it was verified in terms to its standards.Since the kaempferol in the four crops showed significant reduction in different degrees after exposure to pesticides,in order to determine whether the threshold of pesticide exposure was passed by the content of kaempferol in different crops.The MRM quantitative detection method of kaempferol was established by triple quadrupole mass spectrometry.Detection linear range 10~2000?g/kg,R~2=0.996;low of detection(LOD)is 0.1?g/kg(S/N=3.3),low of quantification(LOQ)1?g/kg(S/N=10),The recovery rate in different substrates of the standard addition is greater than 90%,the daytime and intraday CV both are less than 10%,and the ion ratio of the quantitative ion pair and the qualitative ion pair in standards and spiked samples are greater than 50%.The above data shows that the method is stable and reliable and meets the quantitative requirement.After quantitative analysis of the content of kaempferol in the sample,the determination threshold determined by ROC analysis that was 30.78?g/kg in cherry tomatoes,5.05?g/kg in amaranth;227.23?g/kg in okra,respectively;and in pea seedlings,200.42?g/kg.above data are regarded as the judgment threshold,judgmental accuracies in the verification samples of four crops are greater than 90%,which indicating that the method is accurate and reliable.Sum up,this study screened and identified the common pesticide exposure marker is kaempferol of three types of plants,which exposed to different types of pesticides after different drug withdrawal periods,and determined the threshold for the determination of 4plants.It provides a new testing basis for pesticides exposure to organic food,and lays a certain foundation for monitoring to use pesticides in the production process.