| Capillary Electrophoresis-Elecreochemiluminescence(CE-ECL)has become an important and powerful detection technology due to its advantages of the high separation efficiency,short analysis time,low consumption of reagents for CE,and the low background,high sensitivity,wide linear range of ECL.CE-ECL has widely used in various fields such as biochemical,pharmaceutical and environment analysis.The application of CE-ECL in pharmaceutical analysis is a complement to the traditional analysis methods,which provides a new and sensitive analysis method to pharmaceutical analysis.The aim of this thesis was to widen the application of the technology of CE-ECL in pharmaceutical analysis.The novel methods for study of the drug-protein interaction,pharmacokinetics and the determination of alkaloids were developed by CE-ECL coupled with other techniques.The main contents and results were introduced as follows:1.A simple,sensitive and selective determination of trimetazidine dihydrochloride(TMZ)was developed using CE-ECL.Under the optimized experimental conditions,the linear range of TMZ was from 0.075 to 80 ?mol/L(R2 = 0.9974)and the detection limit was 26 nmol/L(3?).Designed a simple,convenient,reusable ultrasonic micro-dialysis device coupled with CE-ECL for studying the dialysis and combination equilibrium between human serum albumin(HSA)and TMZ.The time required for equilibrium by ultrasonic micro-dialysis was 45 min,which was far less than that by traditional dialysis(240 min).It took 80 min to achieve the required combination equilibrium by normal incubation and only 20 min by ultrasonic.Compared with traditional dialysis,ultrasonic micro-dialysis simplified experimental procedures,shortened experimental time.The interaction between HSA and TMZ was investigated and the number of binding sites and binding constant were 1.54 and 15.17 L/mol,respectively.2.A high sensitive method for determination of urapidil hydrochloride was developed using CE-ECL technique for the first time.Under the optimized experimental conditions,the ECL intensity was linear with the concentration of urapidil hydrochloride in the range from 0.050 to 50 ng/mL and the detection limit was 0.014 ng/mL(3?).The method was used for the determination of urapidil hydrochloride in rat plasma with the recoveries from 96.68?98.82%.It was used for studying pharmacokinetics of urapidil hydrochloride in rat plasma and the main pharmacokinetic parameters of the maximum concentration(Cmax),half life time(T1/2),peak concentration time(Tmax)were 240.45±21.15 ng/mL,0.58±0.16 h,1.08±0.13 h,respectively.3.Using n-propanol as an additive added into the running buffer,tlis work firstly developed a new method for simultaneous ECL determination of verapamil hydrochloride and its metabolite norverapamil hydrochloride coupled with CE.Under the optimized experimental conditions,the linearity of the method were 0.015?10.0 ?g/mL for verapamil hydrochloride and 0.060?10.0 ?g/mL for verapamil hydrochloride with the regression equation of y=581.2 x+19.94 and y=339.4x+29.16 respectively.The detection limits(3?)were 0.006 ?g/mL for verapamil hydrochloride and 0.024 ?g/mL for norverapamil hydrochloride.The proposed method was used for studying pharmacokinetics of verapamil hydrochloride and norverapamil hydrochloride in rat plasma and the main pharmacokinetic parameters of the maximum concentration(Cmax),half life time(T1/2),time to peak(Tmax)were 683.21±74.81 ng/mL,0.521±0.21 h and 2.49±0.32 h for verapamil hydrochloride and 698.42±71.45 ng/mL,1.14±0.26 h and 2.83±0.23 h for norverapamil hydrochloride after oral administration with 10 mg/kg verapamil hydrochloride for rats.4.Prepared a silica-sol/Nano-ZnO/PVP/Ru(bpy)32+ modified glassy carbon electrode as working electrode and n-propanol added into the running buffer as separation additive,a new method for simultaneous ECL determination of quinapril hydrochloride and its metabolite quinaprilat hydrochloride coupled with CE was developed.Compared with bare glassy carbon working electrode,ECL intensity of quinapril hydrochloride and quinaprilat hydrochloride increased dramatically when Silica-sol/Nano-ZnO/PVP/Ru(bpy)32+ modified glassy carbon electrode as working electrode.Under the optimized experimental conditions,the linearity concentration ranges of the method were 0.010?8.0 ?g/mL for quinapril hydrochloride and quinaprilat hydrochlorideion and the detection limits(3?)were 0.004 ?g/mL for the two drugs.The method was applied for the determination of quinapril hydrochloride and quinaprilat hydrochlorideion in human plasma with satisfactory result.5.Based on the separation by CE,using polyvinylpyrrolidone as an additive added into the running buffer to obtain good separating degree,this work firstly developed a simple and sensitive method for simultaneous ECL determination of galanthamine,homolycorine,lycorenine and tazettine in lycoris radiata with ultrasonic assisted extraction.Experimental conditions influencing the determination were studied,including additive,detection potential,separation voltage,injection voltage and injection time,the pH and concentration of running buffer.Under the optimized experimental conditions,the baseline separation of the four alkaloids was obtained within 16 min and the proposed method displayed the linear ranges for tazettine,lycorenine,galanthamine and homolycorine were 8.0-2500 ng/mL,5.0-1500 ng/mL,60-5000 ng/mL and 40-5000 ng/mL,respectively.The detection limits(3?)for tazettine,lycorenine,galanthamine and homolycorine were 3.1 ng/mL,1.8 ng/mL,14 ng/mL and 11 ng/mL,respectively.This method was successfully applied for determination of tazettine,lycorenine,galanthamine and homolycorine in lycoris radiata with the recoveries of 102.5%,98.20%,97.30%and 98.33%,respectively.6.The alkaloids in stephania epigaea grown in guangxi were extracted using ultrasonic in binary-water phase system.Based on the separation by CE and acetonitrile as an additive the work firstly developed a simple and sensitive method for simultaneous ECL determination of sinomenine,cepharanthine and tetrahydropalmatine in stephania epigaea.The extraction conditions in binary-water phase system assisted by ultrasonic and the detection conditions of CE-ECL were optimized.Compared with the extraction by ethanol and ethanol water,three kinds of alkaloids have highest extraction rate in binary-water phase system.Under the optimized experimental conditions,three kinds of alkaloids achieved better separation in 10 min and displayed the linear concentration ranges for sinomenine,cepharanthine and tetrahydropalmat were 0.08?4.0 ?g/mL,0.08-4.0 ?g/mL and 0.04?6.0 ?g/mL,respectively.The detection limits of sinomenine,cepharanthine and tetrahydropalmatine(3?)were 0.027 ?g/mL,0.024 ?g/mL and 0.013 ?g/mL respectively.This method was successfully applied for determination of sinomenine,cepharanthine and tetrahydropalmatine in stephania epigaea grown in guangxi with the recoveries of 98.4%?102.4%and 101.1%,respectively. |
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