Determination Of 13 Metabolites Of Organophosphorus Pesticides By UPLC-Q Exactive Orbitrap HRMS

Organophosphorus pesticides（OPS）is a kind of synthetic phosphate compounds,which is one of the most commonly used insecticides in the world.It is also often used in suicide or homicide cases.The chemical stability of these substances is poor and they are easy to decompose,which brings some difficulties to the identification and detection of related cases.The metabolites of organophosphorus pesticides are one of the biomarkers of poisoning,and their chemical properties are relatively stable.The detection of them is the key to the identification of such cases and the diagnosis of poisoning.Therefore,based on the police practice,based on the precipitation protein and QuEChERS pretreatment method,combined with ultra-high performance liquid chromatography quadrupole/electrostatic field orbit trap high resolution mass spectrometry（UPLC-Q Exactive Orbitrap HRMS）technology,13 kinds of common organophosphorus pesticides metabolites,namely malathion dihydroxylic acid（MDA）,2-diethylamino-6-methyl-4-hydroxypyrimidine（deapy）,2-diethylamino-6-methyl-4-hydroxypyrimidine（deapy）,were analyzed 2-isopropyl-6-methyl-4-pyridyl alcohol（impy）,1,2,3-benzotriazin-4（3H）-one（BTA）,diethyl thiophosphate（DETP）,3,5,6-trichloro-2-pyridine（TCP）,3-chloro-4-methyl-7-hydroxycoumarin（CMHC）,trimethyl phosphate（TMP）,p-nitrophenol（PNP）,3-methyl-4-nitrophenol（MNP）,diethyl phosphate（DEP）,dimethyl phosphate（DMP）,and Dimethyldithiophosphate（dmdtp）is expected to open up a new idea for the public security organs to deal with organophosphorus pesticide poisoning cases,and provide a more comprehensive method for the judicial expertise of related cases.The main results are as follows:1.A UPLC-Q Ex Active Orbitrap HRMS method for simultaneous determination of 13organophosphorus pesticide metabolites was established.Acquity UPLC（?）HSS T3（2.1×100mm,1.8μm）column was used;Mobile phase A:0.1%formic acid solution,B:acetonitrile solution;Positive and negative ions are used to switch the acquisition mode;Acquisition mode:full MS/DD MS~2,Full scan resolution:70000;Scanning resolution of secondary mass spectrometry:17500;Normalized collision energy:50%,70%,90%.The correlation coefficients（R~2）were greater than 0.9950.The limits of detection（LOD）and limits of quantification（LOQ）were 0.1～2 ng/ml and 0.5～5 ng/ml,respectively;The RSD of intra day and inter day precision was less than 9.9%.2.For blood samples,two pretreatment methods,precipitation protein and QuEChERS,were investigated and evaluated,and two pretreatment methods,UPLC-Q Exactive Orbitrap HRMS,were established for the determination of thirteen organophosphorus pesticide metabolites in blood.There was a good linear relationship between the metabolites of 13organophosphorus pesticides in a certain concentration range（R~2≥0.9969）;The recoveries and matrix effects of protein precipitation method were 52.7%～128.8%and 67.9%～128.5%respectively at three concentration levels;The recoveries and matrix effects of QuEChERS were 60.6%～119.0%and 54.4%～122.1%,respectively.The limits of detection（LOD）and limits of quantification（LOQ）of 13 organophosphorus pesticide metabolites were in the range of 0.2～10 ng/ml and 0.5～10 ng/ml,respectively.The RSDS of intra day and inter day precision were less than 10.5%.3.For liver samples,two pretreatment methods,precipitation protein and QuEChERS,were investigated and evaluated,and two pretreatmeesticide metabolites had good linear relationship in a certain concentration range（R~2≥0.9952）;The recovery and matrix effect of protein precipitation method were 59.0%～123.6%and 67.3%～121.6%respectively at three concentration levels;The recoveries and matrix effects of QuEChERS were 68.2%～124.2%and 78.2%～122.1%,respectively.The limits of detection（LOD）and limits of quantitation（LOQ）of 13 organophosphorus pesticide metabolites were in the range of 0.2～10 ng/ml and0.5～10 ng/ml,respectively.The RSDS of intra day and inter day precision were less than10.6%.4.Through the actual case detection and animal experimental analysis,the feasibility of the established method was verified,and the metabolic law of toxic markers was explored.