Ultrasonic microdialysis coupled with capillary electrophoresis electrochemiluminescence study the interaction between trimetazidine dihydrochloride and human serum albuminShuangjiao Sun,Chanjuan Long,Chunyao Tao,Sa Meng,Biyang Deng *Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources (Ministry of Education of China),School of Chemistry and Pharmaceutical Sciences,Guangxi Normal University,Guilin 541004,ChinaH I G H L I G H T SG R A P H I C A L A B S T R A C TA homemade ultrasonic (US)micro-dialysis (MD)device was described. The equilibrium speed was improved about 6times using USMD.The binding speed was improved 4times using US technique.New method for the determination of binding sites number and binding constant wasdeveloped.A R T I C L E I N F OArticle history:Received 12June 2014Received in revised form 1August 2014Accepted 6August 2014Available online 13August 2014Keywords:Ultrasonic microdialysis Capillary electrophoresis Electrochemiluminescence Trimetazidine dihydrochloride Human serum albuminA B S T R A C TThe paper describes a homemade ultrasonic microdialysis device coupled with capillary electrophoresis electrochemiluminescence (CE-ECL)for studying the interaction between human serum albumin (HSA)and trimetazidine dihydrochloride (TMZ).The time required for equilibrium by ultrasonic microdialysis was 45min,which was far less than that by traditional dialysis (240min).It took 80min to achieve the required combination equilibrium by normal incubation and only 20min by pared with traditional dialysis,the use of ultrasonic microdialysis simpli fied experimental procedures,shortened experimental time and saved consumption of sample.A simple,sensitive and selective determination of TMZ was developed using CE-ECL and the parameters that affected ECL intensity were optimized.Under the optimized conditions,the linear range of TMZ was from 0.075to 80m mol/L (r 2=0.9974).The detection limit was 26nmol/L with RSD of 2.8%.The number of binding sites and binding constant were 1.54and 15.17L/mol,respectively.ã2014Elsevier B.V.All rights reserved.1.IntroductionTrimetazidine dihydrochloride (TMZ,Fig.S1in Supporting Information)is an effective and well-tolerated anti-anginal meta-bolic agent used for optimizing myocardial energy metabolism,protecting the adenosine triphosphate generation process,reducing the intracellular acidosis,preventing the accumulation of sodiumand calcium and maintaining stable environment in the cell.Meanwhile,it is an antiadrenergic drug that can reduce vascular resistance,increase blood flow of coronary artery and improve heart function.It also has a curative effect in the treatment of angina pectoris,acute myocardial infarction,ischemic heart disease,idiopathic dilated heart,heart failure,arrhythmia and other diseases [1–8].The increasing interests in metabolic modulation in recent years need to develop quick analytical methods for the determina-tion of TMZ.Supplementry material related to this article found,in the online version,at /10.1016/j.aca.2014.08.012.*Corresponding author.Tel.:+867735845726;fax:+867732120958.E-mail address:dengby16@ (B.Deng)./10.1016/j.aca.2014.08.0120003-2670/ã2014Elsevier B.V.All rights reserved.Analytica Chimica Acta 851(2014)37–42Contents lists available at ScienceDirectAnalytica Chimica Actaj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /a caSeveral methods have been reported for the determination of TMZ such as HPLC[9],RPLC[10],voltammetry[11],mass spectrometry[12,13],spectrophotometry[14–16],chemilumines-cence[17].The method for the determination of TMZ using CE-ECL has not been reported.There are two types of molecular structures in blood.One is in molecular state(free drugs),and the other one is reversibly associated with plasma proteins.Only the free drug molecules are able to cross membrane barriers and be distributed to tissues and organs and exert pharmacological effect[18].When free drugs are delivered into tissues and organs or are cleared away,the combined drugs could separate from combining site to make up for the free drugs.Thus,the reversible combination of drugs with protein is critically involved in drug pharmacokinetics and pharmacodynamics.Therefore,it is important to study the combination of drugs and HSA[19].Equilibrium dialysis[20], ultrafiltration method[21],ultracentrifugation,gelfiltration and microdialysis[22]are main methods to study the interaction between drugs and protein.Among these techniques,the equilibrium dialysis is regarded as the reference method.The traditional dialysis device contains three units(dialysis bag,vessel loaded dialysate,and mechanical stirrer).It has the disadvantages of low efficiency and time consuming due to low speed of mechanical stirrer.The normal incubation was carried out in shaking table.Ultrasound is helpful when small molecules pass through the dialysis membrane and reach the equilibrium quickly. Therefore,dialysis assisted with ultrasound can improve the analysis speed,effectively shorten the equilibrium time[23]. CE-ECL is simple,fast,highly sensitive and has better precision and wide linear range.In recent years,many papers have been reported regarding the application of CE-ECL on drug determination [24–31],but only some of them concerned about the interaction between drugs and protein[32,33].Besides,it takes several hours for some drugs to reach the combination equilibrium with HAS by traditional method,which is time-consuming[32,33].This work designed an ultrasonic microdialysis device for studying the combination balance and dialysis equilibrium between HSA and TMZ.In37 C,the time for combination balance and dialysis equilibrium of HSA with TMZ were80min and240min, respectively.However,they were only20min and45min when assisted with ultrasonic.The paper chose the condition of ultrasonic to study the combination balance and equilibrium dialysis between HSA and TMZ at the same time,which simplified the experimental steps,shortened experimental time and obtained the binding-sites number and binding constant.2.Experimental2.1.Reagents and apparatusThe CE-ECL system was produced by Xi’an Remex Electronic Science-Tech Co.,Ltd.(Xi’an,China)and consists of four main parts: a numerical control capillary electrophoresis high-voltage power supply,a multifunction chemiluminescence detector,a multichan-nel data collection analyzer and a numerical controlflow injection sample injector.The end-column ECL cell is composed of a three-electrode system:a Pt disk as work electrode,a Pt wire as auxiliary electrode and Ag/AgCl(saturated KCl)as the reference electrode. An uncoated fused-silica capillary(75m m I.D)with the length of 50cm was obtained from Yongnian Optical Conductive Fiber Plant (Hebei,China).The dialysis membrane(15kDa)was obtained from Union Carbide Corporation(Chicago,IL,USA);Model HSJ-4A pH meter was produced by Shanghai Precision and Scientific Instrument Corporation(Shanghai,China);Model SK3200H ultrasonic cleaner was produced by Shanghai Kudos Ultrasonic Instrument Co.,Ltd.(Shanghai,China).Cary60UV–vis spectro-photometer was produced by Agilent Technologies,Australia Pty Ltd.(Australia).TMZ was obtained from the National Institute for the Control of Pharmaceutical and Biological Products(Beijing,China)and HAS (>96%,MW65.3kDa)was obtained from Sigma–Aldrich(St.Louis, MO,USA).Tris(2,20-bipyridyl)ruthenium(II)chloride hexahydrate was purchased from Alfa Aesar(Johnson Matthey,Ward Hill,MA, USA).Na2HPO4,Na3PO4and NaH2PO4were purchased fromXilongFig.1.Exploded view of microdialysis device(a);microdialysis device(b);ultrasonic microdialysis device(c).1forfixing cover with a sample-introducing hole;2for dialysis bag;3forfixing sleeve;4for centrifuge tube;5for thermometer;6for water outlet;7for water inlet;8for sample;9for dialysis liquid;10for ultrasonic cleaning instrument; 11forfloating frame.38S.Sun et al./Analytica Chimica Acta851(2014)37–42Chemical Co.,Ltd.(Guangdong,China).All reagents used were of analytical grade.Double-distilled water was used throughout. 2.2.ProceduresNew capillary was activated by0.1mol/L NaOH for12h.For daily use,the activated capillary was subsequentlyflushed with 0.1mol/L NaOH,filtered water and separation buffer for10min each.Between runs,the capillary wasflushed by separation buffer. The surface of the working electrode was polished with0.3m m alumina power and cleaned with water in an ultrasonic cleaner before use.The Ru(bpy)32+–phosphate solution was replaced every 2h during the experiments to maintain good reproducibility of the ECL measurements.In all experiments,samples were introduced into the capillary by electrokinetic injection at10kV for10s, separated in the capillary at9kV.Detection potential wasfixed at 1.25V.The separation buffer(pH7.5)contained15mmol/L phosphate.The potential of the photomultiplier tube(PMT)was operated at800V.2.3.Ultrasonic microdialysis deviceThe microdialysis device consists of a centrifuge tube,dialysis bag,fixing sleeve andfixing cover(see Fig.1a).In order to reduce the dead volume in dialysis bag and enhance dialysis efficiency, thefixing button was designed to be hard and thefixing part has the same height as thefixing sleeve.In the center of thefixing button,a hole was made for sample introduction.For the device, the volume of the centrifuge tube can be adjusted according to the experiment requirement.The volume of the centrifuge tube with the dialysis solution of500m L is750m L.The microdialysis equipment was shown in Fig.1b.In this equipment,the volume of dialysis bag is50m L.Normally,the temperature of human body is about37 C and too high temperature may cause the protein denaturation.In order to maintain a temperature with37 C for ultrasonic dialysis,the flowing water supplied by a pump with two pipes was used as ultrasonic media.In order to prevent the dialysis equipment from move,a foam was used forfixing the equipment.The whole ultrasonic dialysis device was shown in Fig.1c.3.Results and discussion3.1.Cyclic voltammetric scan and detection potentialWhen there was only Ru(bpy)32+in ECL detection,ECL intensity was weak,as shown in Fig.2a.An obvious enhancement to the ECL intensity was detected after adding TMZ into the ECL detection cell, as shown in Fig.2b.Therefore,based on this examination,a new sensitive method for determination of TMZ has been established.3.2.Optimization of detection potentialThe effect of detection potential on the ECL intensity of TMZ was investigated.As shown in Fig.S2(Fig.S2in Supporting Informa-tion),when detection potential changed from1.10V to1.40V,the ECL intensity reached a maximum at1.25V,so1.25V was selected as the ECL optimum detection potential.Supplementry material related to this article found,in the online version,at /10.1016/j.aca.2014.08.012.3.3.Optimization of pH in the ECL cellFor CE-ECL detection system,the reaction between Ru(bpy)32+ and analyte depends on the pH in the ECL cell,so the pH could directly affect the ECL intensity.Previous report indicated that the optimum concentration of Ru(bpy)32+and buffer were5mmol/L and50mmol/L in the ECL cell,respectively[34],so5mmol/L Ru (bpy)32+and50mmol/L buffer were chosen as experimental concentration.The effects of buffer pH on the ECL intensity was investigated with a range of6.0–9.0.The results indicated that the ECL intensities reached the maximum at pH7.5and then decreased with the increase in the pH(Fig.3).Therefore,pH7.5was selected.3.4.Optimization of the concentration and pH of running bufferThe effects of concentration and pH of running buffer on the ECL intensity were studied.The results were shown in Fig.S3(Fig.S3in Supporting Information)and Fig.4.When the buffer concentration was set at10mmol/L,the pH was investigated from6.0to8.5and the maximum intensity was observed at7.5.When pH value of the running buffer was kept at pH7.5,the buffer concentration was augmented gradually from5to25mmol/L.The results indicated that the highest ECL signals appeared when the buffer concentrationwasFig.2.The profile of electrochemiluminescence.Detection conditions:scan rate:100mV/s;50mmol/L pH7.5phosphate buffer containing5mmol/L Ru(bpy)32+(a),50mmol/L pH7.5phosphate buffer containing5mmol/L Ru(bpy)32+and20m mol/LTMZ(b).Fig.3.Effect of pH in the ECL cell on ECL intensity.Detection conditions:detectionpotential of1.25V;other conditions are the same as conditions in Fig.S2(Fig.S2inSupporting Information).S.Sun et al./Analytica Chimica Acta851(2014)37–423915mmol/L.Therefore,15mmol/L phosphate buffers at pH 7.5was chosen for following experiments.Supplementry material related to this article found,in the online version,at /10.1016/j.aca.2014.08.012.3.5.Optimization of separation voltageThe separation voltage directly in fluences the migration time and the ECL intensity of analyte.The increase of separation voltage would shorten analysis time.However,too high voltage led to Joule ’s heat.The effects of separation voltage on the ECL intensity was investigated varying from 7kV to15kV.As shown in Fig.S4(Fig.S4in Supporting Information),the ECL intensity reached its maximum values,when the separation voltage was 9kV.In this paper,9kV separation voltage was selected.Supplementry material related to this article found,in the online version,at /10.1016/j.aca.2014.08.012.3.6.Optimization of injection voltage and injection timeInjection voltage and injection time are other two important factors in fluencing ECL intensity of the tested analyte.The effect of injection time and injection voltage were investigated by altering injection voltage from 4kV to 14kV while keeping injection time constant and changing injection time from 4s to 14s when injection voltage was invariable.As shown in Fig.S5(Fig.S5in Supporting Information),the system had higher ECL signal when injection voltage was 10kV.Therefore,10kV was selected as the optimized injection voltage.ECL intensity became stronger with the increasing of the injection time,however,when too much sample was injected,electrophoresis peak was broadened.Considering the peak width and the peak shape,10s was selected as the optimized injection time.Supplementry material related to this article found,in the online version,at /10.1016/j.aca.2014.08.012.3.7.Linearity,detection limit and reproducibility of TMZUnder the optimized experimental conditions,as shown in Fig.5,with the increase of TMZ concentration,the ECL intensity enhanced.The linear concentration range and the detection limit were studied.The ECL intensity was linear with the concentration of TMZ in the range from 0.075to 80m mol/L,with a regression equation of y =56.14x (m mol/L)+14.83(R 2=0.9974).The detection limit was 26nmol/L (3s ).When six consecutive injections of20m mol/L TMZ were performed,the relative standard deviations (RSD)of the peak height and the migration time were 2.8%and 2.6%,respectively.4.Interaction between TMZ and HSA4.1.Study on dialysis timeThe combination of HSA with TMZ was studied by dialysis method.Dialysis bag was soaked in 15mmol/L phosphate buffer solution at pH 7.5before use and was fixed by the fixing device.A 50m L phosphate buffers at pH 7.5containing 20m mol/L TMZ and 10m mol/L HSA was injected into dialysis bag through the sample-introducing hole.The dialysis solutionwas 450m L phosphate buffers (15mmol/L)at pH 7.5.Both traditional dialysis and ultrasonic microdialysis were investigated using CE-ECL method by determin-ing the ECL intensity of dialysate every 15min or 30min.As shown in Fig.6,the ECLintensity increased withthedialysis timeandreached a stable value at 45min for ultrasonic microdialysis and at 240min for traditional dialysis.The stable value indicated the arrival of dialysis equilibrium and TMZ concentration would no longer change.Therefore,the time required for equilibrium by ultrasonic micro-dialysis was 45min,which was far less than that by traditional dialysis (240min).UV –vis is the most common method for studying the combination of protein with drugs,the combinationofFig.4.Effect of concentration of the running buffer on ECL intensity.Detection conditions:pH of separation phosphate buffer for pH 7.5;other conditions are the same as conditions in Fig.S3(Fig.S3in SupportingInformation).Fig. 5.CE-ECL electropherograms of TMZ.Detection conditions:50mmol/L phosphate buffer (pH 7.5)containing 5mmol/L Ru(bpy)32+in ECL cell;detection potential of 1.25V;electrokinetic injection of 10kV Â10s;separation voltage of 9kV;separation buffer of 15mmol/L phosphate (pH 7.5);5m mol/L TMZ (a);10m mol/L TMZ (b);20m mol/L TMZ(c).Fig.6.Effect of dialysis time on ECL intensity.Normal dialysis (a);ultrasonic dialysis (b).40S.Sun et al./Analytica Chimica Acta 851(2014)37–420.10mmol/L HSA with 0.10mmol/L TMZ also investigated by UV –vis under the condition of normal incubation and ultrasonic incubation,respectively.As shown in Fig.7,it took 80min to achieve the required equilibrium by normal incubation.However,it was only 20min by ultrasonic incubation.As a result,the condition of ultrasonic can greatly improve the binding rate of HSA with TMZ and shorten the experimental period.4.2.The ultraviolet spectrum of HSA with TMZThe binding of HSA with TMZ was characterized by ultraviolet spectrum.As shown in Fig.S6a and Fig.S6b (Fig.S6in Supporting Information),both HSA and TMZ had UV absorption.The UV absorption enhanced as TMZ was added into the HSA solution (Fig.S6c),however,the absorption became weak after ultrasound for 20min (Fig.S6d).The decrease of absorption indicated the combination of HSA with TMZ.Supplementry material related to this article found,in the online version,at /10.1016/j.aca.2014.08.012.4.3.Study on the number of binding sites and binding constantThe dialysis membrane (<15kDa)allows the free TMZ passage and prevents the passage of HSA as well as the combined TMZ with HSA.The concentrations of free TMZ would be equivalent inside and outside of the dialysis bag after dialysis equilibrium.The quantity of the combined TMZ with HSA is equal to the total amount of TMZ minus the quantity of free TMZ.In order to study the number of binding sites and binding constant,a series of different concentrations of TMZ were mixed with 0.01mmol/L HSA in the microdialysis bag and different concentrations of free TMZ were determined after the equilibrium of ultrasonic microdialysis.In most cases,it is supposed that the drug (D )is bound to m types of independent binding sites on the protein (P ).The fraction (r )of bound drug molecules per protein molecule can be represented as [35]:r ¼½D bound ½P total ¼X mi ¼1n iK i ½D free1þK i ½D free (1)In the expression,[D bound ,P total ]and [D free ]represent the concen-trations of bound drug,total protein and free drug,respectively.The n i is the number of sites of class i involved in binding and K i stand for the binding constant.Considering that the nature of protein binding sites often remain unclear.Eq.(1)can be simpli fiedinto Eq.(2)by accounting for one type of binding sites on the protein:r ¼nK ½D free I þK ½D free (2)In the expression,n and K are the number of binding sites and the binding constant,respectively [36].If different [D free ]and the corresponding r were given,the binding curve could be established to estimate the number of sites and binding constant by non-linear fitting using origin 8.0.In this paper,the number of binding sites and the binding constant of HSA with TMZ was obtained by non-linear fitting with origin 8.0.The binding curve was shown in Fig.8and the fitting non-linear equation as follows:r ¼1:53Â15:15½D free 1þ15:15½D freeIt can be seen from the equation,the number of binding sites and the binding constant of HSA with TMZ were 1.53and 15.15L/mol,respectively.5.ConclusionsIn this work,a new CE-ECL method for the determination of TMZ was developed.A simple device for ultrasonic microdialysis was designed.The number of binding sites and binding constant between HSA and TMZ were investigated using the device coupled with CE-ECL.The number of binding sites and binding constant were 1.53and 15.15L/mol,pared with the other reported methods,the ultrasonic microdialysis in the paper simpli fied the experimental procedure,shortened experimental time and improved analysis ef 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