Three-dimensional Chromatogram Assists The Research On High Performance Liquid Chromatographic-fluorescence Detection Of Traditional Chinese Medicine

High Performance liquid chromatography (HPLC) as an important modern measurementof separation and analysis is developed on the basis of classical Liquid Chromatography. Dueto it has been widely used in the areas of biology engineering,pharmacy, foodstuff,environment and petroleum so far. This thesis developed a new method for the qualitative andquantitative analysis of active ingredients in Chinese herbal medicine by reverse-phase highperformance liquid chromatography with fluorescence detection (RP-HPLC-FLD) of three-dimensional chromatogram. The detection sensitivity is improved because that the overlapsof fluorescence peaks with solvents or Rayleigh scattering peaks is avoided according tothree-dimensional chromatogram select detection wavelength. The three-dimensionalchromatogram have an advantage over the two-dimensional fluorescence chromatographicabout the choice of chromatographic conditions. The method is reliable, accurate, specific andreproducible. This work provided a new approach for the determination of Chinese herbalmedicine.This thesis falls into three parts:Part1. An analytical method, based on HPLC with fluorescence detector, has beendeveloped and validated for the simultaneous determination of catechin and epicatechin inAcacia catechu and teas. The three-dimensional chromatogram and two-dimensionalfluorescence chromatogram allows the comparison of the results obtained with the twoqualitative and quantitative means and this grants a higher level of confidence to the resultsfor the selection of chromatographic conditions of three-dimensional chromatogram. Throughthe three-dimensional chromatogram found the disadvantages of methanol-acetic acid mobilephase, so this experiment chose the methanol-0.1%phosphoric acid (40:60) as mobile phase.With respect to previously published methods, the proposed one has the advantage of havingbeen validated also for the selection of detection wavelength. Selecting235nm as theexcitation wavelength is able to avoid the interference of Rayleigh scattering which grantsvery good selectivity to the assay. The RSD of stability and precision value for the peak-areasand fluorescence intensity were between0.57%and1.71%, the overall mean recovery ranged from100.0%to10.1%, showing the developed method is reproducible with good accuracy.Finally, it does not use gradient elution or multiple solvent extractions and separate of the twostandards within5minutes, with obvious advantages in terms of time, costs, feasibility andenvironmental compatibility. The method is used for the qualitative and quantitative analysisof Acacia catechu and teas.Part2. An analytical method, based on HPLC with fluorescence detector, has beendeveloped and validated for the simultaneous determination of five anthraquinonesderivatives(rhein, aloe-emodin, emodin, chrysophanol, physcion). The three-dimensionalchromatogram and two-dimensional fluorescence chromatogram allows the comparison of theresults obtained with the two qualitative and quantitative means and this grants a higher levelof confidence to the results for the selection of chromatographic conditions ofthree-dimensional chromatogram. This experiment choose the methanol-water(85:15) asmobile phase without acid. The RSD of stability and precision value for the peak-areas andfluorescence intensity were between0.536%and3.10%, the overall mean recovery rangedfrom95.8%to100.1%(expert emodin), showing the developed method is reproducible withgood accuracy. Finally, it does not use gradient elution or multiple solvent extractions andseparate of the two standards within8minutes, with obvious advantages in terms of time,costs, feasibility and environmental compatibility. The method is used for the qualitative andquantitative analysis of three kinds of contrast medicinal materials and three kinds ofcommercial medicinal materials.Part3. An analytical method, based on HPLC with fluorescence detector, first has beendeveloped and validated for the simultaneous determination of aesculin, aesculetin andfraxetin in Cortex Fraxini. The three-dimensional chromatogram and two-dimensionalfluorescence chromatogram allows the comparison of the results obtained with the twoqualitative and quantitative means and this grants a higher level of confidence to the resultsfor the selection of chromatographic conditions of three-dimensional chromatogram. TheRSD of stability and precision value for the peak-areas and fluorescence intensity werebetween0.670%and2.40%, the overall mean recovery ranged from96.0%to102.0%,showing the developed method is reproducible with good accuracy. Finally, it does not use gradient elution or multiple solvent extractions and separate of the two standards within3minutes, with obvious advantages in terms of time, costs, feasibility and environmentalcompatibility. The method is used for the qualitative and quantitative analysis of three kindsof contrast medicinal materials and three kinds of commercial medicinal materials.