Separation Of Central Nervous System Poisons And Drugs By Liquid Phase Microextraction

BackgroundThe samples for forensic toxicological analysis are complex biological or environmental samples,and spectrophotometer,chromatography,mass spectrometry and other instruments are commonly used for the analysis.Sample pretreatment is an important step before instrument analysis.However,due to the relatively cumbersome steps,low selectivity and weak purification capabilities,traditional liquid-liquid extraction and solid-phase extraction techniques are difficult to effectively eliminate the interference of the sample matrix and to achieve enrichment and separation of drugs/poisons in complex samples in one step.Especially when the sample amount is limited,analysts face several challenges when using traditional sample preparation techniques.Liquid phase microextraction(LPME)is a new type of sample pretreatment technique.Its basic principle is based on the passive diffusion of analyte concentration gradient and p H gradient.Membrane-based LPME technique has been developed,in which the physical isolation of the porous fibrous membrane and the special properties of the membrane solvent increase the selectivity of this sample pretreatment technique.Besides,LPME has been developed rapidly in the field of analytical chemistry in recent years with the characteristics of green,simple and fast.In addition,an electric field-assisted liquid-phase microextraction named electromembrane extraction(EME),is also playing an important role in the field of sample pretreatment.It is a new type of sample pretreatment technique originated from LPME.The principle of EME is to apply an electric field between the donor phase and the acceptor phase.The target analyte is in ionic form by adjusting the p H of the sample and the acceptor solution.Under the action of the electric field,the target analytes could be quickly extracted from the sample into an acceptor phase.EME is a green,fast and selective sample pretreatment technique.In the fields of separating and enriching central nervous system poisons and drugs from complex matrices,traditional sample pretreatment techniques are difficult to achieve rapid and highly selective separation and sufficient enrichment of target analytes.In response to the above problems,LPME was used to separate and enrich central nervous system poisons and drugs from wastewater,blood and urine,biological tissue.This research has broadly expanded the application of LPME in forensic toxicology analysis.The applicability of LPME in multiple forensic samples will lay the foundation for in vivo LPME.This study mainly includes the following three parts:Part Ⅰ:Separation and enrichment of drugs in wastewater by liquid-phase microextractionObjective:Based on the principle of LPME,this research intended to make a new LPME device named microextraction bag(MEB)for the separation and enrichment of drugs in environmental water and wastewater.Methods:In this study,polypropylene membrane was used to make disposable microextraction bags.The method of liquid phase microextraction based on microextraction bag(MEB-LPME)combined with high-performance liquid chromatography connected to ultraviolet detector(MEB-LPME-HPLC-UV)was used to optimize the parameters affecting LPME such as membrane solvent,p H value of sample phase and acceptor phase,volume of membrane solvent,salt,stirring rates and extraction time.Then,the method of liquid chromatography-tandem mass spectrometry(LC-MS/MS)were used to verify the applicability of MEB-LPME for the determination of the psychoactive substances of drugs in water samples.Finally,the application of the established method was applied to the determination of six drugs in wastewater and environmental water from lakes and rivers,which further proves the value of MEB-LPME-LC-MS/MS in analysis of drugs in environmental water samples and wastewater.Results:Under the optimum conditions,the recoveries of six drugs in spiked water were40-93%,the detection limits were 0.3-8.7 ng/L.The established analysis method has good repeatability,and the relative standard deviation(RSD)was in the range of 2.2-15%.The method has been successfully applied to the determination of the psychoactive substances of drugs in Zhongshan Park lake,Hanjiang River and hospital wastewater.And amphetamine,methamphetamine and codeine were quantitatively detected in hospital wastewater.The concentration of amphetamine,methamphetamine and codeine in wastewater were 30.8 ng/L,97.4 ng/L and 68.3 ng/L,respectively.In addition,the extraction device of this method could process 50 wastewater samples at the same time.The high throughput will greatly reduce the labor intensity.The established method could sensitively detect trace level drugs in wastewater and environmental water samples,which is of great significance for the government to carry out illicit drugs monitoring.PartⅡ:Separation of psychoactive substances of barbiturates from body fluids by electric field-assisted liquid phase microextractionObjective:Blood and urine are the most common biological samples in forensic toxicological analysis.Electric field-assisted liquid-phase microextraction(EME)is a new type of green liquid-phase microextraction technique,which has the advantages of high selectivity,short extraction time,and strong sample purification ability.Suicide or committing crimes by barbiturates abuse is often encountered in forensic toxicological analysis.Therefore,in this project,we used EME to separate the psychoactive substances of barbiturates from blood and urine samples,and finally combined EME with high-performance liquid chromatography-ultraviolet detector(HPLC-UV)and liquid chromatography-tandem mass spectrometry(LC-MS/MS)to establish methods for the determination of barbiturates in blood and urine.Methods:In this study,a flat membrane EME device was used.The method of EME-HPLC-UV was used to evaluate and optimize the parameters affecting EME efficiency such as membrane solvent,sample phase p H value,acceptor phase p H value,membrane solvent volume,salt,extraction voltage,stirring rates and extraction time.Then,the method of HPLC-UV and LC-MS/MS were used to verify the applicability of the EME method for the determination of barbiturates in blood and urine.Finally,the application of actual forensic cases further proves the value of EME-HPLC-UV and EME-LC-MS/MS in the field of forensic toxicological analysis.Results:Under the optimal conditions(membrane solvent:tributyl phosphate,solvent volume:4μL,extraction voltage:3 V,extraction time:20 min,stirring ratios:1000 rpm,p H of sample phase:12,p H of acceptor phase:2),the recoveries of barbital,phenobarbital and pentobarbital were 57%,60%and 67%for blood and 51%,50%and90%for urine,respectively.The relative standard deviation(RSD%)of all analytes were less than 15%.The detection limits of three drugs in blood and urine were 0.14-0.21μg/m L and 0.38-0.69μg/m L by EME-HPLC-UV,respectively.The detection limits of three drugs in blood and urine by EME-LC-MS/MS were 0.44-4.3 ng/m L and 0.87-2.92ng/m L,respectively.The established analysis method was successfully used for the determination of phenobarbital from blood and urine of suicide patient.It was the first time that tributyl phosphate was found to be a high-performance membrane solvent for the extraction of acidic analytes.The original results are as follows:Firstly,we evaluated accurately the contribution of LPME in the process of EME.What’s more,the established method could be used for the determination of three barbiturates in biofluids by high-performance liquid chromatography or high-performance liquid chromatography-mass spectrometry.The established method was successfully applied to actual cases and phenobarbital was quantified in the blood and urine.Part Ⅲ:Separation of antipsychotic and antidepressant medicines from tissues by electric field-assisted liquid phase microextractionObjective:The main objective of this study is to explore the feasibility of electric-field-assisted liquid-phase microextraction(EME)for the separation of antipsychotic and antidepressant drugs from tissues,which could be a start point for the study of poisons distribution using EME for the sample pretreatment.Methods:Based on the flat membrane EME device and the optimized EME condition for separation of theseantipsychotic and antidepressant drugs from standard buffer solution,the EME operation factors such as the type of acceptor phase,centrifugation speed and analytes concentration were further investigated and evaluated by HPLC-UV.Then,the established method of EME-LC-MS/MS was used to verify the applicability of the EME of antipsychotic drugs in fish tissues.Results:under the optimal conditions,the recovery of analytes in fish tissue was significantly lower than that in spiked solution.The recoveries of haloperidol,amitriptyline,fluoxetine and sertraline were 62%,60%,50%and 30%,respectively.The established method showed good linearity in the concentration range of 2-1000 ng/g(R~2>0.99)for amitriptyline,fluoxetine and sertraline,and 5-1000 ng/g(R~2>0.99)for haloperidol.The established analysis method has good repeatability(RSD<10%).The detection limits of the targets in fish were 0.3-0.8 ng/g.This developed method is of great significance for the study of the correlation between large-scale population health and antidepressant exposure in the future.What’s more,the attempt of this method is of great significance for the research of antipsychotic and antidepressant drugs in human tissues,postmortem metabolic distribution and toxicological research.This research will increase the possibility of the application of EME in the field of forensic toxicological analysis.