| As an emerging technique, microanalysis served as a fast, efficient and economicalmean. Through microtubule (i.d.500μm～10μm) and accompanied by modern detectiontechnologies, analysis achieved in a restricted space using concentrated samples. Thus,high efficiency and sensitivity are guaranteed, minimizing the consumption of nano-level.From its dawn, microanalysis experienced all-round modifications and innovations. Fieldsof utility were also broadened to chemical engineering, bio-sample analysis, protomicsresearch, cytobiology and pharmacokinetic reseach, ect. Those advances contributedenormous research literatures, patents and new technologies.Natural products, including herb, food, and materials, are useful resource forhumanity that provide numerous help for our live and development. Since the very dawn ofeach civilization, researches of it keep emerging along with time. Complex as man itself,natural products contain various kinds of active components, expressing diverse usages andinteraction. Due to this diversity, analysis of natural products usually requires sophisticateddesigns in both instrumentation and methodology. Especially in determination oftherapeutic compounds in natural drug, which concealed in matrix with large interferenceand effects of some compounds of human body remain ambiguous.As for bio-samples, such as plasma, body fluid or interstitial fluid，extraction andpurification can strongly affected by proteins, ions and other interfering substances in theirmatrix. This would cause reduction in sensitivity and selectivity. Furthermore, adsorptionusually occurred when drugs are administrated, leaving fewer amount of unbound drugs inthe body fluid that easier to detect. Therefore, method development on separation,purification and concentration of in-vivo samples, and their sensitive and selectivedetermination would be a challenge to pharmaceutical researchers when dealing withcomplex samples.Efforts on providing efficient, sensitive and selective techniques always attractconsiderable attention in the fields concerned, resulting in emerging reports andinnovations. To date, the most mature and standard method for determination of complexmatrix are High performance liquid chromatography (HPLC), Mass spectrometry (MS)and Ultraviolet spectrometry (UV). Though a great number of research papers and modifications were presented for decades, drawbacks like difficulty in miniaturization,limited separation mode and constrained efficiency of the above mentioned technologiescan hardly be ignored yet.Capillary electrophoresis(CE), shared some similar features like othe microanalysistechnologies. Based on capillary and high voltage, it was considered mature in bothinstrumentation and methodology, and widely used it many fields. Principally, CE analysisis fulfilled by introducing electrical field, under which dissociated silanol group andelectrolyte in buffer forms double-layer. This layer is the source that produces a potentialnamed zeta potential, which forms electroosmotic flow (EOF), resulting in movement ofcharged compounds toward the other end of the capillary. Attributed to differences incharge-size ratio of the compounds, analytes was separated under certain conditions.The most obvious distinction of CE from conventional electrophoresis it that, thelatter operated in a fused-silica capillary (id25-100μm). Features such as miniaturevolume, large secar that makes high voltage stable, ability of using sol, flat flow profile,ect, enable CE outstand various types of techniques, having advantages as follow.Diversity in analytical modesFast separationGood performanceEconomical in both sample and reagent consumption.However，due to restriction of CE, such as limited separation mode, specific range ofsuitable sample and relatively poor reproducibility, revolution is needed. Various groupproposed their own solutions: addition of micellar or chiral selector as “moving solidphase”, introduction of pressure, or modification of inner structure by packing particle,coating or polymerizing solid phase. To some extent, efforts above do improveperformance of CE technologies.As pharmaceutical specialists, this research mainly focuses on determination of drugsin complex compounds. Based on experiences of previous researchers in our group, thecharacteristic of complex compounds, combing the advantages of CE, encumbers inanalysis of complex samples can be overcome. Thus, we dedicate to make contribution to various fields like quality control, fingerprint development and therapeutic drugmonitoring that analysis technique involved.Capillary eletrochromatography(CEC), as a hybrid of CE and HPLC, integrates theiradvantages. Therefore, column is the core of the technique. Among which, packed columnis one of the well-developed approaches, using particle as mobile phase. However,problem like strict packing and plunging experience requirement, introduction of bubblethat interrupt current in the plunging process and undeniable for such type of column.Monolithic column, on the other hand, are usually prepared by in-situ polymerizationreactions stimulated by heat or light. This column type frees researchers from laboriouspacking and plundering procedure, inner structure or functional groups can easily bechanged according to the very nature of the analytes. Monolithic column showed goodpotential in life science, protoemics, pharmaceutical analysis, developing rapidly in bothinstrumentation and methodology.Compared with HPLC, the nano flow of CEC guarantees its good selectivity andsensitivity, as well as considerably lower sample and solvent consumption. As to CE, CECover passed it for the reason that both charged and neutral compound can be separated,making interaction between analytes and solid phase possible.Conventional CEC solid phase was prepared by particles, which required laboriouspacking procedure, causing a lot of problem in both reproducibility and stability.Monolithic column, on the other hand, used organic monomer that prepared by in-situpolymerization, this reaction freed us from troubles in making particles and the followingpacking procedure. Besides, in monolithic column, mobile phase moves through pores thatdistributed throughout the monomer, which brings about mass transfer that seldomobserved in particle packed columns.One common thing in CEC is, the column plays critical role in separation and analysis,meaning that preparation of the capillary column is of great importance in CECexperiments. Judging from the formation of monomer in the capillary, columns can becategorized into3major types: open tubular capillary columns, packed capillary columnsand monolithic capillary columns.Monolithic capillary columns belongs to monolithic chromatographic column, itcomprises of monomer, cross-linker and porogen. Through certain polymerization reaction, mixture mentioned above could for continuous bed solid phase within the column.Monolithic column have some characteristics like easy preparation and modification, goodcolumn efficiency and permeability and the ability of fast separation, ect. Based ondifferent matrix of polymerization mixture, monolithic column can be further divided intoorganic and inorganic ones. The former has better reproducibility, larger range of suitedmonomer, more simplicity in preparation, faster separation and wider pH tolerance range;however, organic monomer cannot totally replace inorganic ones for its drawbacks inswelling, heat instability, poorer mechanical strength and difficulty in controlling pore sizeand pore distribution. In addition, monolithic column prepared by free-radical inducedreaction has poor reproducibility. In contrast, silica based monolithic column can easilytailor the porosity, also with better mechanical strength and solvent tolerance.In general, micro-analysis technologies based on monolithic capillaryelectrochromatography is fast, efficient, sensitive and selective. However, the utility ofthose methods seldom touched fields like analysis of traditional Chinese medicine orbiological samples, which prevents it from exploiting the full potential of suchtechnologies. So the aim of this thesis is to optimized those methods and transferred it topractical use of real pharmaceutical samples, especially the ones with complex matrix.The spefisic work in this thesis included:At first, a throughout and intentional research on development and innovation of thecapillary electrochromatography technologies should be carried out before real experimenton complex samples. For a deeper and wider scope of our insight into advantages anddrawbacks of this technique, and the wise selection of proper technology suiting the needfor different targets, major current separation modes in CEC should be comprehensivelytouched. Among developed columns, three types of them are dominating the knownexperiment concerning method development and optimization. They are, open-tubular,particle packed and monolithic columns. Investigation and imitation of methods reportedwas carried out first, then followed by certain modification in pursuits of determiningactive components in complex samples. Through our investigation, the principle ofimproving efficiency and introducing multiple separation mechanism can be evaluated andemphasized. All works followed should carried out around the principle, fulfilling the goalof broadening usage of CEC and exploiting its potentials. Then, according to our invetigation on previous reports and experiences, monolithiccapillary column was chosen as major type of column technology used in this thesis. Fordetermination of coumarins in A. Dahurica,4active compounds (imperatorin，isoimperatofin，phelloptorin and falcarindiol) were selected as targets when analyzing itscrude drugs. After successfully separation of the4compounds, the fact that several othercomponents with different but important activities still remain undetected cannot beignored. The reason for those compounds A. Dahurica also rich in lied in their similarity inchemical structure, making them harder to separate in either conventional methods or CEC.With the help of ethacrylate ester—based monolithic column, through certain optimizationprocedure, those analogs(namely byakangelicin, oxypeucedanin hydrate, xanthotoxol,5-hydroxy-8-methoxypsoralen and bergapten)can be baseline separated and determined. Inthe process of selecting optimum conditions, Surfactant sodium desoxycholate (SDC) wasintroduced into the mobile phase as the pseudostationary to dynamically increase theselectivity of analytes instead of increasing the hydrophobicity of stationary phase.However, the developed technology has its own confines that prevent the efficiency fromimproving: One of the problem was the range of target compounds suitable for separationin by this method was restricted attributed to the inner structure and electricity of themonomer. That is, this type of column performs much better when dealing with acidic orneutral target than the basic ones. Another problem lies in the fact that ethacrylateester—based monolithic column has a short carbon chain (C=4), thus, for better efficiency,monomers should be tightly incorporated， which was done at the cost of poorpermeability and much higher back pressure （>1500psi）. Structures with smaller porecaused frequent bubble formation, making current suddenly stop and conjestion of thecolumn. So improvement should be made to avoid such problems.In the field of ethacrylate ester—based monolithic column, though its effectivenesswas proven in coumarin analysis, few reports on its determination of natural drugs in-vivocan be seen in the field concerned. Such as Cnidium monnieri，a TCM rich in coumarins,still has much potential to be exploit. For its extraction, major methods were thoseconventional ones like ultrasonic, heat reflex or microwave. Accelerated SolventExtraction (ASE) provided a better option for its higher efficiency in extraction yields.Under ASE conditions, samples were more concentrated and purified and can beintroduced to the equipment directly. This saved a lot of time in herb analysis. Also, with apowerful extraction tool in hand, research based on monolithic CEC and be expanded to pharmacokinetic study of certain herb. ASE extracts was used for oral administration ofrats, and blood samples were consecutively collected for determination. After successfulmethod development and real sample determination, a fast, sensitive and efficient methodwas established for simultaneous determination of active compounds in Cnidium monnieriand their pharmacokinetic behavior.For exploitation of the perspective of CEC, a research on its utility in pharmacokineticresearch was carried out. On basis of our successful determination of Cnidium monnieri, itsactive components were determined in-vivo. After protein precipitation, samples wereanalyzed and concentrations of imperatorin, and osthole in rat plasma over time wereclarified so as the pharmacokinetic parameters. As results indicated, the monolithic columnwas proven effective, sensitive and suitable for complex and high throughput samples suchas body fluids.To solve the problem of poor permeability and bubble formation, suiting the need forfast and high-throughput analysis while precondition the column online, new type ofmonolithic column was introduced. This organic-inorganic hybrid column comprised ofsilica base and ADDA monomer, prepared by linking silicane and cationic monomer, thiscolumn has better mechanical strength and permeability while keep good performance likeethacrylate ester—based monolithic column. For its practical use, determination of cefdinirin rat with the help of microdialysis was carried out. As complementary methods for eachother, CEC and microdialysis successfully establish a system for real time and onlinemonitor of unbound drugs in the body fluid. As results indicated, this method was highlyautomatic, fast, efficient, sensitive and reproducible, obviously proven the utility of CEC incomplex compound determination.At last, based on a series research, some problems preventing CEC from analyzingcomplex samples were solvent while some others remains the same. Drawbacks likerelatively complicated preparation and preservation of monolithic column, difficulties inensuring reproducibility, longer precondition time and obstacles in miniaturization requiresfurther optimization and efforts. Therefore, based on our finished works, some speculationsand suggestions were proposed in the last chapter, all those thoughts dedicated inimprovement of stability, permeability, sensitivity and reproducibility of the technologies.For micro analysis, as long as innovations keep emerging, brighter future is a possibility tolook for. |
PDF Full Text Request