| Pyraoxystrobin is a novel strobilurin fungicide that was independently developed by the Shenyang Research Institute of Chemical Industry,which shows highly efficient,broad spectrum and low toxicity activities.It shows highly efficient broad spectrum fungicidal activities against Pseudoperonospora cubensis,Blumeria graminis,Erysiphe cichoracearum,Plasmopara viticola and Phyricularia grisea.In this study,the high performance liquid chromatography(HPLC)method was developed for determination of pyraoxystrobin formulation;the liquid chromatography tandem mass spectrometry(LC-MS/MS)method was established and validated for assay of pyraoxystrobin in rat pasma,tissues,feces and urine,and the toxicokinetics of pyraoxystrobin in rat was revealed;the metabolites of pyraoxystrobin in rat feces and urine were tracked by 13C labeled and the structure of metabolites was deduced.The primary metabolism pathway of pyraoxystrobin was acquired.The enzyme kinetics of pyraoxystrobin in rat liver microsomal was investiged,and the enzyme kinetics characteristic of pyraoxystrobin was clarified.The research contents are as follows:1.The analysis of pyraoxystrobin formulationThe HPLC method was developed for determination of pyraoxystrobin formulation.Chromatographic separation was achieved on a Waters Symmetry C18 column,with 70%acetonitrile-water as mobile phase,and pyraoxystrobin was detected by ultraviolet detector.The results indicated the accuracy of pyraoxystrobin suspension liquid in 0.5%sodium carboxymethyl cellulose(5 mg/mL,10 mg/mL,50 mg/mL and 100 mg/mL)used for administraton to rats in toxicokinetics study was between 87.2%-96.8%and the suspension liquid was with good homogeneity,which could reach the acceptable criteria.2.The study of pyraoxystrobin toxicokintics and tissue discribtion in rat2.1 A sensitive,specific and accurate LC-MS/MS method had been developed for determination of pyraoxystrobin in rat plasma.The quantification method of pyraoxystrobin in rat plasma has been validated in the concentration range from 0.5 to 200 ng/mL,and the standard curve displays good linearity.The intra-day and inter-day accuracy of QC samples was between 90.7%-108%and 99.9%-104%,respectively.The intra-day and inter-day precision of QC samples was between 0.7%-8.4%and 4.2%-8.1%,respectively.The matrix effect was between 86.6%-97.4%and extract recovery was between 101.4%-108.2%,with precision≤6.1%.The stability of plasma sample could meet the requirement of bio-sample analysis.Finaly,the developed method was applied to determine the concentration of pyraoxystrobin in rat plasma.2.2 A simple and high efficiency LC-MS/MS method had been developed for determination of pyraoxystrobin in rat tissues.The LC-MS/MS method had been validated in the concentration range from 1 to 200 ng/mL,and the standard curve displays good linearity.The intra-day and inter-day accuracy ranged from 88.7%to 110.7%and 93.2%to 108.7%,respectively.The intra-day and inter-day precision was between 0.9%-9.9%and 0.3%-8.9%,respectively.The matrix effect and extract recovery was between 84.5%-103.5%and 49.1%-59.4%respectively with the RSD≤10.6%.The stability of tissue sample could meet the requirement of bio-sample analysis.Finaly,the developed method was applied to determine the concentration of pyraoxystrobin in rat tissue.3.The study of pyraoxystrobin metabolism and excretion in rat3.1 A LC-MS/MS method was established for determination of pyraoxystrobin in rat feces and urine.The LC-MS/MS method had been validated in the concentration range from 0.4 to 20 ng/mL and the standard curve displays good linearity.The intra-day and inter-day accuracy for QC in feces was between 89.5%-106.6%and 94.3%-104.6%,respectively;the intra-day and inter-day precision was between 2.6%-10.7%and 2.2%-7.6%,respectively.The intra-day and inter-day accuracy for QC in urine was between 87.5%-107.8%and 93.8%-102.4%,respectively;the intra-day and inter-day precision was between 0.6%-10.7%and 2.9%-8.4%,respectively.The stability of feces and urine samples could meet the requirement of bio-sample analysis.Finaly,the developed method was applied to determine the concentration of pyraoxystrobin in rat feces and urine.3.2 The metabolites structure of pyraoxystrobin was deduced according to parent ion peak and product ion peak information by EPI and EMS mode of LC-20A/API4000 Q Trap.According to parent ion peak and product ion peak information of pyraoxystrobin and 13C labeled pyraoxystrobin,the metabolites structure of pyraoxystrobin was deduced.4.The enzyme kinetics study of pyraoxystrobin in rat liver microsomal The rat liver microsomal was prepared by calcium precipitate method and the total protein of rat liver microsomal was determined.The quantity of CYP450 was determined by CO reduction differential method.A LC-MS/MS method was established for determination of pyraoxystrobin in rat liver microsomal.The LC-MS/MS method had been validated in the concentration range from 50 to 25000 ng/mL,and the standard curve displays good linearity.The intra-day and inter-day accuracy for QC was between 90.1%-109%and 91.8%-105%,respectively;the intra-day and inter-day precision was between 1.9%-5.0%and 2.1%-3.3%,respectively.The stability of rat liver microsomal sample could meet the requirement of this study.Finaly,the developed method was applied to determine the concentration of pyraoxystrobin in rat liver microsomal. |
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