Micellar Electrokinetic Chromatography Analysis Of Several Types Of Active Ingredients Of Traditional Chinese Medicine

By employing of the surfactant micelles as pseudo-stationary phases, micellar electrokinetic capillary chromatography (MECC), which is an important branch of capillary electrophoretic (CE) techniques, is amenable for the separation of ionic analytes and also electrically neutral analytes or analytes with similar charge-to-size ratios. In addition, MECC possesses several advantages such as high efficiency, selectivity, sensitivity, easy set-up procedures and great economy of operation, and has generated much interest in the areas of importance, including pharmaceutical analysis, biochemistry and life science. In this dissertation, an overview on the basic principles and influencing factors of MECC, as well as current studies about the application of MECC to Chinese traditional medicine analysis was presented. After this, Four MECC methods have been developed for the separation and determination of active components in Chinese traditional medicine, the results showed as follows:1. Mixed micelles of Tween-20and sodium dodecyl sulfate (SDS) have been used as mobile-phase modifiers to develop a MECC method for the simultaneous separation and determination of loureirin A, loureirin B and cochinchinenin C in resina draconis. The effects of the solvent of samples, pH of buffer, the concentration of electrolyte as well as Tween-20and SDS on migration behavior were studied. Optimum separation condition was achieved using a20mmol/L borax buffer (pH9.98) contained22.5mmol/L SDS and0.35%(v/v) Tween-20as electrolytic solution. The sample solution was diluted with electrolytic solution and injected for5.0s with0.5psi. The applied voltage was20kV and the capillary temperature was kept constant at25℃. Under the conditions described above, the complete separation of these analytes was obtained within18min with UV detection at206nm. Method validation revealed that the developed assay is repeatable and sensitive. The linear range for loureirin A, loureirin B and cochinchinenin C was2.0-100.0μg/mL,2.0-100.0μg/mL and5.0-80.0μg/mL, respectively. The relative standard deviation for the determination of the three constituents in samples was less than5.0%, and the recovery ranged between95.1%and103.2%. Moreover, the dissociation constant of loureirin A and loureirin B was determined by the capillary zone electrophoresis as8.32and8.36, respectively.2. A sensitive MECC method with mixed micelles of SDS and Tween-20has been developed for the quantitative analysis of five coumarines (imperatorin, isoimperatorin, oxypeucedanin, byakangelicin and byakanegelicol) in Angelica dahurica with UV detection at211nm. By using a20mmol/L borax buffer (pH9.11) containing32.5mmol/L SDS,1.2%(v/v) Tween-20and30%(v/v) methanol as electrolytic solution, the analytes could be well resolved within30min at24kV separate voltage and25℃capillary temperature. The sample solution was dissolved with1:1(v/v) of methanol and water containing20mmol/L SDS and injected for5s with0.5psi. The linear range for the determination of imperatorin, isoimperatorin, oxypeucedanin, byakangelicin and byakanegelicol was3.0-90.0μg/mL,1.0～90.0μg/mL,1.5～110.0μg/mL,1.0～110.0μg/mL and2.0～100.0μg/mL, respectively. The contents of the five active compounds in Dracaena cochinchinensis (Lour.) S. C. Chen from different districts were determined with good repeatability (RSD<5.0%) and satisfactory recovery (95.9-104.4%).3. A MECC method modified by β-CD was performed for the analysis of five important pharmaceutical constituents included schisandrin, chisandrol B, deoxyschisandrin, γ-schisandrin and schisandrin C in the fruits of Schisandra chinensis (Turcz.) Baill. The simultaneous determination of these constituents was achieved using a running buffer (pH8.92) containing20mmol/L sodium borate,17.5mmol/L SDS,9mmol/L β-CD and14%(v/v) acetonitrile at the detector wavelength of220nm, separate voltage of24kV and capillary temperature of25℃. The sample solution was dissolved with methanol and water (v/v=1:1) containing10mmol/LSDS and injected for5s with0.5psi. The method with a linear range for the determination of schisandrin, chisandrol B, deoxyschisandrin, γ-schisandrin and schisandrin C as3.5-75.0μg/mL,1.0-75.0μg/mL,2.0-50.0μg/mL,1.5-75.0μg/mL and2.0～75.0μg/mL could be used successfully for the determination of these constituents in real samples with RSD of less than5.0%and recovery ranged between95.3%and103.2%.4. A reverse-flow micellar electrokinetic chromatographic (RF-MEKC) method was developed for the simultaneous quantitative determination of praeruptorin A, praeruptorin B and praeruptorin C. The influence of essential background electrolyte (BGE) components on the separation were investigated and the final BGE consisted of10mmol/L phosphoric acid,100mmol/L SDS and35%(v/v) acetonitrile (pH5.59) was used. Baseline separation was obtained in24min for the three analytes at reverse polarity with a voltage of-20kV and capillary temperature of25℃. The sample solution was dissolved with methanol and water (v/v=1:1) containing5mmol/L SDS and injected for5.0s with0.5psi. The results validated that the method was of wide linear range for the determination of praeruptorin A (3.0～90.0μg/mL), praeruptorin B (1.0～90.0μg/mL) and praeruptorin C (1.0～90.0μg/mL), respectively. The contents of analytes in Peucedanum praeruptorum Dunn collected from the different places were successfully determined by the developed method with RSD of less than5.0%and recovery ranged between94.5%and103.5%.