| Chemiluminescence is a light emission from chemical reactions.Chemiluminescence analysis has some advantages such as high sensitivity,wide linear range and simple instrument. It has become an attractive analytical tool in biological and chemical analysis.However,the CL analysis has also some disadvantage;the main disadvantage is very poor selectivity which limited its application widely.How to solute this problem?One of the approaches is combining the CL analysis with high performance separation method such as the high performance liquid cheromatograpy or capillary elecrophorecence.The another approach is combining with specific molecular recognation such as enzyme reaction including pure enzyme,plant and animal tissue or cell,antibody,antigen or receptor,nucleic acid,aptamer,DNA or RNA and molecular imprinting polymer.As a powerful modern separation technique,High Performance Liquid Chromatography (HPLC) has been widely used for the analysis of many analytes in diverse fields.Detectors are one of the most important components of HPLC system since they produce a wealth of information about the separated components which can be stored in computers and manipulated as desired to assist solute identification.In recent years,chemiluminescence(CL) as a detection technique of HPLC is very attractive due to higher sensitivity,wider linear dynamic ranges and simpler instrumentation.The advance of CL detection has greatly catalyzed the growth and popularity of HPLC-CL application,and made trace analysis possible owing to its capability of measuring pictogram or femtogram quantities of compounds in the column eluate.The investigations and applications of HPLC-CL technology are currently also an important subject, and a hot point in analytical science.The combination of HPLC separation,which is versatile and robust,and CL-based reactions,which are extremely sensitive,is promising for numerous applications in recent years.However,the number of the practical CL methods so far reported are not so many owing to some limitation as follows:in HPLC-CL methods,the reagent such as oxidizing reagent is delivered for the post-column CL reaction using additional pump.Thus,the dilution of the analytes by mixing with reagent sometimes results in the lower sensitivity.So far the CL reagents used in HPLC are still limited,the complicated chemistry involved makes application of these reagents in HPLC even more difficult.Although many types of reactors(interfaces) have been developed;however,most of them are still in the optimization phase and mainly suited to the fast kinetic reaction.In addition,analysis of real samples in HPLC-CL is unsatisfactory probably due to matrix interference.To solve these problems,we designed a simple reactor so extraordinary that it can obtain the most luminescent intensity as soon as the CL reaction was carried out.Moreover,there wasn't any dead volume and dilution effect.The new assemble technique based on HPLC coupled with CL detection has been successfully applied in determination of some medicinements or neurotransmitters.The effects of several parameters on the HPLC resolution and CL emission were studied systematically.Several novel methods based on HPLC with chemiluminescence detectetor have been developed for the determination of in pharmaceutical and biological samples.Coupled with microdialysis,one of the HPLC-CL systems has been applied to study the pharmacokinetics of some drugs for in-vivo,on-line and real time determination.Microdialysis sampling has become a powerful technique for studying biochemical events in the extraceilular fluid of actually any tissue,organ or biological fluid in recent years.It is a standard technique in the neurosciences and has been extended to use in many other fields including pharmacokinetic,bioprocess monitoring etc.Microdialysis is a dynamic sampling method based on analyte diffusion across a semipermeable membrane driven by a concentration gradient.The advantage of using microdialysis sampling is that relatively low numbers of experimental animals can be used and the physiological and anatomical features of their tissue remain intact during sampling with the microdialysis probe.In addition, microdialysis sampling coupled with appropriate analytical chemical technology can be applied to continuously monitor chemical changes within the ECF with respect to time at one implantation site.Microdialysis has also the advantage that the technique is easy to automate and can be on-line coupled with many analytical techniques.On-line HPLC with microdialysis perfusion provides simplified sample preparation and automated analyses.In this research work,we have developed a new HPLC-analysis-CL method and successfully applied it to monitor levodopa in rabbit blood in vivo and on-line.This dissertation consists of four chapters.Chapters 1 is a review and Chapters 2 to 4 are research reports including 8 research works.In Chapter 1,the development and tendency of the HPLC with CL detection are reviewed. It covers the principles of the HPLC with CL detector,the design of CL detectors,CL detection systems(including luminol,peroxyoxalates,Ru(bipy) 32+,KMnO4),the applications of analytical methods for many kinds of inorganic,organic and biologic samples,and the trends of HPLC-CL in environmental,life science,pharmacy and clinical medical science.The research works includes:(1) High Performance Liquid Chromatography-Chemiluminescence Detection for the in vivo on-line determination and study of the pharmacokinetics of Levodopa in blood with microdialysis samplingA simple,reliable and reproducible method for in vivo on-line separation and determination of ievodopa based on high performance liquid chromatography with chemiluminescence detection and microdialysis is described.The perfusate is perfused at a flow rate of 5μL/min. The concentration of levodopa in the dialysate is determined on line with a chemiluminescence system.The dialysate sample volume is about 20μL.The system is linearly related to the concentration of levodopa in the range 1×10-8-1×10-6g/mL(r2 =0.9995) with a detection l imit(3σ) of 3×10-9 g/mL and the sample throughput of 12h-1,RSD is 2.8%(n=11).The method has been successfully applied to study the pharmacokinetics of levodopa in vivo,and the Cmax,AUC0-t and Tmax value of the pharmacokinetics parameter are 16.60ng/mL,20.92ng/mL,90min respectively.(2) Simultaneous determination of Epinephrine,Noradrenaline and Dopamine in human serum samples by high performance liquid chromatography with chemiluminescence detectionA simple,rapid and accurate high performance liquid chromatographic(HPLC) technique coupled with chemiluminescence(CL) detection was developed for the simultaneous determination of epinephrine(E),noradrenaline(NA) and dopamine(DA).It was based on the analyte enhancement effect on the CL reaction between luminol and potassium ferricyanide.The effects of various parameters,such as potassium ferricyanide concentration,luminol concentration,pH value and component of the mobile phase on chromatographic behaviors of the analytes(E,NA and DA) were investigated.The separation was carried out on C18 column using the mobile phase of 0.01 mol/L potassium hydrogen phthalate solution and methanol(92:8,ν/ν).Under the optimum conditions,E,NA and DA showed good linear relationships in the range of 1×10-8-5×10-6,5.0×10-9-1.0×10-6 and 5.0×10-9-1.0×10-6g/mL respectively.The detection limits for E,NA and DA were 4.0×10-9,1.0×10-9 and 8.0×10-10g/mL.The proposed method has been applied successfully to the analysis of E,NA and DA in human serum samples.(3) Determination of Inosine in human serum using high-performance liquid chromatography with chemiluminescence detectionBased on the sensitizing effect of formaldehyde on the chemiluminescence(CL) reaction of Inosine with acidic potassium permanganate and the combination technique of high-performance liquid chromatography(HPLC),a sensitive,selective and simple post-column CL detection method for determining Inosine is described.The optimal conditions for the CL detection and HPLC separation were carried out.The linear range is 0.4-12μg mL-1 for Inosine.The detection limit of is 0.1μg mL-1 and the relative standard deviation with a concentration of 0.15×10-6 g/mL was 2.9%respectively(n=9).The method has been satisfactorily applied to the determination of lnosine in pharmaceutical preparations and human serum samples(4) High Performance Liquid Chromatography-Chemiluminescence Detection for Vitamin B6 in human serumThis paper described a sensitive,selective and simple post-column CL detection method for determining VB6.It was based on the sensitizing effect of formaldehyde on the chemiluminescence(CL) reaction of VB6 with acidic potassium permanganate and the combination technique of high-performance liquid chromatography(HPLC).The optimal conditions for the CL detection and HPLC separation were carried out.The linear range is 0.3-10μg mL-1 for VB6,the detection limit of is 0.1μgmL-1 and the relative standard deviation with a concentrati on of 1.0×10-6 g/mL was 3.7%for VB6(n=7).The method has been satisfactorily applied to the determination of VB6 in pharmaceutical preparations and human serum samples(5) Determination of Promethazine Hydrochioride in human urine using high-performance liquid chromatography with chemiluminescence detectionA sensitive,selective and simple post-column CL detection method for determining Promethazine Hydrochloride is described based on the sensitizing effect of formaldehyde on the chemiluminescence(CL) reaction of Promethazine Hydrochloride with acidic potassium permanganate and the combination technique of high-performance liquid chromatography (HPLC),The optimal conditions for the CL detection and HPLC separation were carried out.The linear range is 1--100μg mL-1 for Promethazine Hydrochloride,the detection limit of is 0.3μgmL-1 and the relative standard deviation with a concentration of 1.0×10-6 g/mL was 2.7% for Promethazine Hydrochloride(n=11).The method has been satisfactorily applied to the determination ofPromethazine Hydroehloride in human urine samples(6) Chemiluminescent detection of fruoside in serum and urine samples after HPLC separationA novel method was developed for the determination of fruoside by high-performance liquid chromatography(HPLC) coupled with chemiluminescence(CL) detection.The separation was carried out with an isocratic elution using the mobile phase of 50:49:1(ν/ν/ν) water-methanol -phosphate buffer(containing 7.0×10-4M Potassium dihydrogen phosphate,pH 3.0) at a flow rate of 1.1 mL/min.The CL intensity was linear within the range of 1～50×10-7g/mL to the fruoside's concentration,and the detection limits at a signal-to-noise of 3 was 3.0×10-8g/mL.The method was successfully applied to the determination of fruoside in serum and urine samples.The possible mechanism of the CL reaction was also discussed briefly.(7)Development of a HPLC-chemiluminescence assay for diethylstilbestrol(DES) in tablets and serum samplesA new,highly sensitive chemiluminescence method for measurement of diethylstilbestrol (DES) in various substances such as human serum and tablets has been developed.The method is based on reverse-phase high performance liquid chromatographic separation and subsequent tris(2,2'-bipyridine)ruthenium(Ⅱ)-Ce(Ⅳ) chemiluminescence detection.It was confirmed that DES show strong chemiluminescence upon mixing with tris(2,2′-bipyridine) ruthenium(Ⅱ)-Ce(Ⅳ). A calibration graph,based on a standard DES solution,was linear over the range 8×10-8～1×10-5g/mL and the detection limit was 3.0×10-8g/mL(signal-to-noise ratio=3).This highly sensitive and selective determination method can be applied to selected samples without purification or pre-concentration procedures.The proposed method is easier,more sensitive,and time-saving.(8)Analysis of phentolamine by HPLC with Ru(bipy)3Cl2-Ce(Ⅳ) chemiluminescence detectionA simple,selective and sensitive method for the determination of phentolamine has been developed using high-performance liquid chromatography with chemiluminescence detection. The phentolamine were separated on a C18 reversed-phase column with an isocratic elution using the mobile phase of 50:49:1(ν/ν/ν) water-methanol-phosphate buffer(containing 7.0×10-4M Potassium dihydrophosphate,pH 3.0) at a flow rate of 0.8mL/min.The eluate was mixed with tris(2,2′-bipyridyl)ruthenium(Ⅱ)-Ce(Ⅳ),and the generated chemiluminescence was detected. Calibration graphs,based on standard solutions,were linear over the range 1～20×10-7g/mL.The detection limits at a signal-to-noise ratio of 3 ranged 3.0×10-8g/mL.The relative standard deviations was 3.1%.This HPLC system was successfully applied to the determination of phentolamine in serum and urine samples.
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