| HPLC method, an fast separation and analysis technology, was developed on the basis of the classical column chromatography, which is widely used in the separation and analysis of the active ingredient in traditional Chinese medicine because of its features of wide scope,selectivity wide separation performance, analysis speed, high sensitivity, accurate results and operations automation features. Fluorescence analysis due to its advantages of high sensitivity,good selectivity and low detection limit and sampling less has gradually developed into a very important and effective means of spectroscopy for chemical analysis. The study using HPLC with fluorescence detector has made a qualitative and quantitative analysis about the active ingredients in Chinese medicine of Schisandra chinensis, Moslae herba and Artemisiae scopariae herba. The use of three-dimensional chromatogram retention time, excitation and emission wavelength peaks while makes the qualitative analysis, making the qualitative more realistic and accurate. The use of the characteristics of three-dimensional fluorescence chromatograms clearly observing the solvent peak and scattering peak makes the choice of mobile phase and detection wavelength effectively avoiding the impact on peak superimposed,making the quantitative analysis more accurate and reliable. While the lower detection limit of HPLC and the high-sensitivity of fluorescence detection were organically combined, so that the method detection level of fluorescence component basically reached the ng·m L-1,or even lower. The content of the relevant components of traditional Chinese medicine was verified through traditional HPLC with UV detection in this article, fully revealing the highly selective, the sensitivity and accuracy of HPLC with fluorescence detection, thus opening a new chapter about the determination of active ingredients in traditional Chinese medicine.This thesis falls into three parts:Part 1. An analytical method, based on RP-HPLC with ?uorescence detector, first has been developed and validated for the simultaneous determination of protocatechuic acid,schizandrin, anwuligan and γ-schisandrin in Schisandra chinensis.Using isocratic method, we chose methanol-acetonitrile-water(50:25:25) as the mobile phase, due to the limitation of the two mobile phase system of methanol-water or acetonitrile-water. The detection wavelengths of 244/356 nm was selected to overcome the shortcoming of only one component being determinated once in literature and separate of these four components within 20 minutes. We also carried out experiments on their qualitative and quantitative at the same time, which were simple and quick. It is turned out that this method has higher accuracy because the stability intraday and precision RSD results of four standards were between 0.25% and 0.81% and the recovery RSD results was from 95% to105%. It is turned out that this method has higher sensitivity and wider linear range because the limit of detection of three components reaches ng·m L-1. The method, which applied to the determination of the contents of the four standards in Schisandra commercial and control medicinal herbs, selected the best chromatographic conditions under the premise of stable nature and the strongest fluorescence intensity, whose results were accurate and reliable.Part 2. An analytical method, based on RP-HPLC with ?uorescence detector, has been developed and validated for the simultaneous determination of carvacrol and thymol in Moslae herba. Thymol and carvacrol was isomers with similar nature and little difference in chromatographic retention properties. Using isocratic method, we chose methanol-glacial acetic acid solution(45:55) as the mobile phase, 276/312 nm as wavelengths and 0.5 m L·min-1as flow rate and separate of the two components within 30 minutes overcoming the shortcoming of only determinating the total content in literature past. We also carried out experiments on their qualitative and quantitative at the same time, which were simple and quick. It is turned out that this method has higher accuracy because the stability intraday and precision RSD results of four standards was between 0.15% and 0.35% and the recovery RSD results was from 95% to 105%. It is turned out that this method has higher sensitivity and wider linear range because the limit of detection of three components reaches ng·m L-1. The method applied to the determination of the contents of the two standards in Moslae herba commercial and control medicinal herbs, whose results were accurate and reliable.Part 3. An analytical method, based on RP-HPLC with ?uorescence detector, first has been developed and validated for the simultaneous determination of scopoletin、scoparone and7-methoxy coumarin in Artemisiae scopariae. Using isocratic method, we chose methanol-glacial acetic acid solution(55:45) as the mobile phase, 356/428 nm as wavelengths and 0.8 m L·min-1 as flow rate and separate of the two components within 6 minutes overcoming the shortcoming of only determinating the total coumarin content in literature past. We also carried out experiments on their qualitative and quantitative at the same time,solving the the problem that scopoletin and scoparone can not be determined due to its low content in Artemisiae scopariae. It is turned out that this method has higher accuracy because the stability intraday and precision RSD results of four standards was between 0.5% and 2.0%and the recovery RSD results was from 95% to 105%. It is turned out that this method has higher sensitivity and wider linear range because the limit of detection of three components reaches ng·m L-1. The method applied to the determination of the contents of the three standards in Moslae herba commercial and control medicinal herbs, whose results were accurate and reliable. |
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