Investigation Of The Combination Of Molecular Imprinting And Electromembrane Extraction

Background:Antidepressants are currently the main treatment of depressive disorder.However,antidepressant exposure has become the fourth most common poisoning-related event,especially due to deliberate or accidental overdose of antidepressants.The diagnosis rate of depressive disorder in China is gradually increasing,and thus the subsequent cases of poisoning caused by overdose of antidepressant drugs intentionally or accidentally will become a practical problem that must be faced and urgently solved by the forensic medicine in China.In order to overcome the shortcomings of traditional analytical methods,such as cumbersome operation,low efficiency,and dependence on large-scale detection instruments,as well as to improve the sensitivity of detection methods and meet the analysis needs of increasing target analyte species,it is urgent to develop new forensic toxicological analysis methods.Solid-phase extraction technology and sensor detection methods based on molecularly imprinted polymer（MIP）have become the current research hotspots.However,the current preparation methods of MIP materials for antidepressants are time-consuming and have limited selective adsorption performance.In addition,there was a problem that the complex sample matrix interfered with the specific adsorption capacity in the practical application of MIP materials,which limited the application of traditional MIP materials in complex samples.Objective:（1）Based on the reflux precipitation polymerization method,a kind of MIP microspheres would be rapidly prepared to achieve selective adsorption of five antidepressant drugs,including venlafaxine（VEN）,citalopram（CIT）,amitriptyline（AMI）,fluoxetine（FLU）,sertraline（SER）.And then the adsorption properties of the MIP microspheres for various antidepressants would be study for the subsequent combination of MIP technology and EME technology.（2）Electromembrane extraction（EME）technology would be developed for extraction of common antidepressant drugs（including VEN,CIT,AMI,FLU,SER,paroxetine（PAR）,trazodone（TRA）,and bupropion（BUP））,and would be combined with liquid chromatography-tandem mass spectrometry（LC-MS/MS）to achieve the determination of antidepressant drugs from blood and urine samples.The above experiments would provide a basis for the combination of MIP technology and EME technology.（3）Combined with the influence characteristics of extraction conditions on the separation performance of EME and the adsorption environment of MIP microspheres,the combination of MIP technology and EME technology would be studied.The experiment would help for the development of MIP sensors with strong resistance to matrix interference in the future.Methods:（1）Five antidepressants（VEN,CIT,AMI,FLU,and SER）and a substance with similar structure（4-bromo-N,N-dimethylaniline,BNND）were used as templates to synthesize single-template MIP microspheres by reflux precipitation polymerization,and two substances were used as templates to synthesize multi-templated MIP microspheres.And then,the physical property of MIP microspheres was studied;the adsorption thermodynamic process of MIP microspheres to five antidepressant drugs was studied;the adsorption kinetic process of MIP microspheres to five antidepressant drugs was studied;the selective adsorption capacity of MIP microspheres and the stability of MIP microspheres to adsorpt trace amount of the antidepressants were studied.（2）A flat-membrane EME method was developed for detection of eight common antidepressant drugs（VEN,CIT,AMI,FLU,SER,PAR,TRA,and BUP）.After optimization of the parameters that affected EME（including support liquid membrane solvent,extraction voltage,acceptor phase composition,sample phase composition,and extraction time）,EME was coupled with LC-MS/MS to detect the antidepressants in complex biological samples（blood and urine）which were commonly found in forensic toxicology analysis.（3）The influence of parameters in the adsorption solution,including organic solvent ratio and pH value,on the adsorption of five antidepressants by MIP microspheres was studied.The optimal adsorption solution of MIP microspheres was used as the acceptor phase of EME,and the parameters of EME（including sample phase composition,pH value of acceptor phase,salt concentration of acceptor phase solution,volume ratio of acetonitrile in acceptor phase solution,extraction current and extraction time）was optimized.The combination of MIP and EME technology was studied.Results:（1）Multi-templated MIP microspheres were synthesized with SER and VEN（mole ratio 1:2）as templates by reflux precipitation polymerization method,which had great uniformity of morphology and particle size,and the diameter was 1.4±0.2μm.The maximal effects In optimal adsorption solution(pH 6 citrate buffer(25 mmol L^-1)mixed with acetonitrile（50:50,v:v）),the maximum adsorption capacities of MIP for five antidepressants（VEN,CIT,AMI,FLU,and SER）were 6.31,14.31,27.32,21.41,and 36.90 mg g^-1,respectively.And the maximum adsorption capacities of MIP were 1.4-2.1 times than that of NIP.The initial adsorption rates of MIP were 0.57,0.89,1.88,1.02,and 2.13 mg g^-1 min^-1,respectively.And the initial adsorption rates of MIP were 1.2-2.1 times than that of NIP.At the same time,the MIP microspheres showed good selective adsorption ability and stable adsorption ability for the trace amout of targets.（2）Under optimal conditions(membrane solvent:NPOE;membrane solvent volume:5μL;extraction voltage:80 V;extraction time:50 min for blood samples and 40 min for urine samples;rotation speed:1000 rpm;sample phase:400μL blood/urine mixed with 400μL10 mmol L^-1 HCl solution;acceptor phase:200μL 10 mmol L^-1 trifluoroacetic acid solution),the extraction recoveries of the targets（TRA,BUP,VEN,CIT,PAR,AMI,FLU and SER）from blood samples were 94.1%,101.3%,100.1%,95.5%,78.0%,90.9%,85.1%and 68.7%,respectively.And the extraction recoveries of the targets from urine samples were 95.2%,105.2%,100.9%,96.1%,99.9%,94.9%,97.7%and 95.0%,respectively.The established method has good linearity（R²>0.99）in a certain concentration range,and the relative standard deviation（RSD%）was less than 13%.The LODs of the established EME-LC-MS/MS method for the determination of eight antidepressant drugs from blood samples were 0.02-0.96μg L^-1;and the LODs of the established EME-LC-MS/MS method for the determination of eight antidepressants from urine samples was 0.07-1.08μg L^-1.Generally,the established method was successfully used for the qualitative and quantitative analysis of eight antidepressants in spiked blood and urine samples.（3）Under optimal conditions（membrane solvent:NPOE;membrane solvent volume:5μL;extraction current:80μA for blood samples and 50μA for urine samples）;extraction time:25 min;rotational speed:1000 rpm;sample phase:200μL blood/urine mixed with200μL 10 mmol L^-1 HCl solution and 400μL acetonitrile;acceptor phase:100μL pH 6citrate buffer(75 mmol L^-1)mixed with 100μL acetonitrile),the extraction recoveries of five target compounds（VEN,CIT,AMI,FLU and SER）from blood samples were 60.7%,70.6%,75.2%,73.6%and 65.8%,respectively;and the extraction recoveries of five antidepressants from urine sampels were 81.2%,89.3%,76.7%,82.7%and 77.7%,respectively.The MIP microspheres prepared previously were combined with the established acetonitrile-EME system to extract five antidepressants from urine samples.The extraction recoveries of MIP microspheres in the combined system were 10.7%,20.5%,36.7%,20.7%and 56.5%,respectively.The above experiments confirmed the feasibility of the combination of MIP technology and EME technology.Conclusions:（1）The multi-templated MIP microspheres synthesized with two templates have a wider range of selective adsorption properties,and the reflux precipitation polymerization method could complete the rapid preparation of MIP microspheres in 2 hours.（2）NPOE was an effective support liquid membrane solvent for a variety of antidepressants,laying a foundation for the combination of MIP technology and EME.（3）Using the constant current EME method,the combination of MIP technology and EME technology was preliminarily realized.The research provided a new idea for the application of sensors based on MIP materials in complex biological samples.