Study On The Novel System Of Pharmic Analysis By FI-CE

The combination of the analysis instruments and the hybrid of analysis techniques are one of the trends of the development of analytical chemistry.The combination of flow injection with capillary electrophoresis(FI-CE)is representative of the trend.The combined technique that integrates the essential favorable merits of FI and CE can significantly expand the application scope of CE by exploring the various on-line sample pretreatments and preconcentration,and improves the sampling frequency of CE.However,hitherto,the ability of on-line sample pretreatments of FI is not widely applied,and many theories and applications in pharmic analysis need to deeply study by FI-CE.Additional,the poor concentration sensitivity is one of the reasons of confined development of FI-CE in trace analysis. To exert the advantages of FI-CE,solve the shortcoming of poor concentration sensitivity in CE and extend the applied fields of FI-CE,the following major innovative work are carried out in this dissertation on the basis of the previous literatures:1.A novel method on separation and determination of four active compounds in Leimengxin by FI-capillary zone electrophoresis(FI-CZE)was developed for the first time,and the quantitative analysis respectively in the same system for two compounds which contents have large difference was performed.2.A head-column field amplified sample stacking(HC-FASS)combined with large volume sample injection in FI-CZE was developed using electroosmotic flow to introduce a short water plug.The four active compounds in Leimengxin were separated and enriched.The two compounds which contents have large difference in Leimengxin were quantified simultaneously for the first time.3.The three water-soluble compounds in Salvia miltiorrhiza Bunge were firstly separated and determined by FI-capillary zone electrophoresis(FI-CZE).The optimum condition for extracting was put forward.4.Flow injection(FI)-capillary electrophoresis(CE)coupled with frontal analysis(FA)was applied to the study of stereoselectivity binding of amlodipine(AL) to human serum albumin(HSA).The binding parameters of AL enantiomers to human serum albumin were determined for the first time.5.A fast and simple method for the study interactions of a series of drugs used in the treatment of hypertension to human serum albumin(HSA)at near physiological conditions was developed by flow injection-capillary electrophoresis(FI-CE)based on the principle of frontal analysis(FA).The results were compared with those obtained by equilibrium dialysis-UV-visible spectrophotometer and fluorescence spectra.The molecular simulation method was used to investigate the relationship between the binding constants of drug-protein and structural descriptors.The effects of all kinds of metal ions existing in human plasma on protein binding were investigated.A binding synergism effect between drugs was also investigated completely.This dissertation consists of six chapters.In chapter 1,the combination of FI and CE was discussed from the necessity of the combined technique,the principle of this technique,split-flow interfaces and the basic FI-CE system setup,the detection means and sample injection models of this technique and the methods of stacking.More importantly,the techniques for studying the interaction of drug and prorein were reviewed in detail.In chapter 2,an unmodified effective length of 4.5 cm fused-silica capillary was used as separation capillary.A novel method for the separation and determination of paracetamol(Par),pseudoephedrine hydrochloride(Pse),dextromethorphan hydrobromide(Dex)and chlorphenamine hydrogen maleate(Chl)was developed by FI-CZE for the first time.The running buffer was composed of 75 mmol/L sodium borate-15%(v/v)ACN.Under the optimized conditions,separation of the compounds could be achieved within 5 min.The sample throughput rate could reach up to 19 h^-1. The repeatability(defined as relative standard deviation)was 0.6%,1.0%,2.1%,1.9% with peak height evaluation and 0.7%,1.8%,0.7%,1.1%with peak area evaluation for Par,Pse,Dex and Chl,respectively.The limits of detection(S/N=3)were 0.22μg/mL,0.29μg/mL,0.42μg/mL and 0.70μg/mL for Par,Pse,Dex and Chl, respectively. In chapter 3,a simple,effective,and sensitive online concentration method for the detection of dextromethorphan hydrobromide(Dex),chlorphenamine hydrogen maleate(Chl),pseudoephedrine hydrochloride(Pse)and paracetamol(Par)based on flow injection-capillary electrophoresis(FI-CE)analysis with head-column field-amplified sample stacking combined with large-volume sample stacking was developed.The orthogonal design was used to optimize the separation conditions.The optimum background electrolyte(BGE)was a solution composed of 55 mM borate-15%(v/v)aeetonitrile(ACN)(pH 9.3).Under the optimum conditions, 27.3-99.0-fold improvement in the concentration sensitivity for four analytes relative to normal CE methods was achieved,giving low limits of detection(LOD)of 1.94×10^-5,0.64×10^-5,1.16×10^-5and 2.84×10^-5mg/mL for Dex,Chl,Pse and Par, respectively.The repeatability(defined as RSD)was 1.01,1.91,0.89 and 0.92%with the peak-area evaluation and 1.94,3.98,2.66 and 3.27%with the peak-height evaluation for Dex,Chl,Pse and Par,respectively.In chapter 4,a sensitive and reproducible method was developed for the separation and determination of three water-soluble components-protocatechuic aldehyde(PAH), 13-(3,4-dihydroxyphenyl)lactic acid(DSS)and protocatechuic acid(PA)in medicine plant Salvia miltiorrhiza Bunge and two related traditional medicinal preparations using flow injection-capillary electrophoresis system.This analysis was carried out by using an unmodified fused-silica capillary(28.4 cm×75μm i.d.×375μm o.d.,25 cm effective separation length)and direct ultraviolet detection at 214 nm,7.0 kV applied voltage.With boric acid(200 mM)adjusted to pH 7.8 as a background electrolyte.The separation was achieved in 9 min.The sample throughput rate could reach up to 15 h^-1.Calibration curves showed good linearity with correlation coefficients(r)more than 0.9986.The repeatability(defined as RSD)were 0.20%, 0.46%,0.47%with migration time evaluation and 0.62%,3.66%,1.50%with peak area evaluation for PAH,DSS and PA,respectively.The limits of detection(S/N = 3) were 0.36μg/mL,0.84μg/mL,and 0.73μg/mL for PAH,DSS,and PA,respectively. The mean recoveries of PAH,DSS and PA were 103.2%,98.1%and 100.5%, respectively.The most suitable extraction method was that the analytes were extracted by 25 mL 70%methanol for 50 min.This method has been applied successfully to monitor these three components in Salvia miltiorrhiza Bunge and its two traditional medicinal preparations.In chapter 5,flow injection(FI)-capillary electrophoresis(CE)coupled with frontal analysis(FA)was applied to the study of stereoselectivity binding of amlodipine(AL)to human serum albumin(HSA).Hydroxypropyl-β-cyclodextrin (HP-β-CD)(10 mM)was used as a chiral selector in pH 3.7,60 mM phosphate buffer. The binding constants of two enantiomers,K_R-ALand K_S-AL,were 9910-11200 M^-1and 90200-104000 M^-1,respectively.L-tryptophan(L-try)and ketoprofen(Ket)were used as displacement reagents to investigate the binding sites of AL to HSA.A binding synergism effect between hydroehlorothiazide(QL)and AL was observed and the results suggested that QL can destroy binding equilibrium of R-AL and S-AL towards HSA and they can occupy the same binding site of HSA(siteⅠ).The reproducibility was confirmed by RSD(RSD＜1.5%)of the plateau height determined by flow injection-capillary electrophoresis frontal analysis(FI-CE-FA).In chapter 6,a fast and simple method for the study interactions of a series of drugs used in the treatment of hypertension to human serum albumin(HSA)at near physiological conditions was developed by flow injection-capillary electrophoresis (FI-CE)based on the principle of frontal analysis(FA).The binding parameters were determined and the results were compared with those obtained by equilibrium dialysis-UV-visible spectrophotometer and fluorescence spectra.The results suggested that the binding paratemers obtained by different methods were a little different.The molecular simulation method was used to investigate the relationship between the binding constants of drug-protein and structural descriptors.The results suggested that the important descriptors were quantum chemical descriptor and constitutional descriptor.The MLR model proposed could identify and provide some insight into what structural features were related to the drug-protein interaction in this experiment and afford some instruction for further investigation other drugs and protein interaction.The effects of all kinds of metal ions existing in human plasma on protein binding were investigated.A binding synergism effect between drugs was also investigated completely.