| High performance capillary electropheresis (HPCE) is an analytical technique whose separation mechanism is distinctively different from high performance liquid chromatography (HPLC). The characteristics of HPCE include high separate efficiency, low solvent consumption and easily cleanable column system. It can be used as a useful supplement to HPLC. The Traditional Chinese medicines (TCM) has complicated composition, and are therefore difficult to analysis. This research explored the potential application of HPCE in TCM analysis using four important Chinese herbs as the target species. HPCE was used as a main instrument in order to supply related theory and application groundwork for the quality control of TCM. In this text, high performance liquid chromatography-electrospray-time-of-flight mass spectrometry has also been used to analyze active components in Radix Gentianae and Semen Nelumbinis. The results can be summarized as follows.1. A modern HPCE analysis method was developed for the determination of cyclic peptide Pseudostellarin B in Pseudostellaria heterophylla (Miq.) Pax samples. Separations and determinations were carried out by HPCE under the following conditions: bared fused silica capillary (50 cm×75μm i.d.), 20 mmol/L borate (pH=9.3)as buffer, the run voltage is +15 kV, detection length of UV at 203 nm, and column temperature of 20℃. The results indicated that the developed method was simple, accurate and reliable for the determination of Pseudostellarin B with a good linearity(Y=0.6357X+2.546, r=0.9985), and quantitative recovery ranging from 93.8%～105.6%. The HPCE fingerprints of Pseudostellaria heterophylla (Miq.) Pax were established using this method. The chromatographic fingerprints were compared by the software of"similarity evaluation system". The fingerprint congruence coefficients of 8 electropherograms in 10 samples were above 0.90. The HPCE fingerprinting method is thus reliable and accurate.2. Set up a method for the determination of chlorogenic acid and flavonoids in honeysuckle flower by HPCE, and for the study of their fingerprints. Separation and determination were carried out by HPCE under the following conditions: bared fused silica capillary (50 cm×75μm i.d.),buffer: 30 mmol/L borate (pH=10.2), run voltage: +18 kV, detection length: 238 nm, column temperature: 25℃. Under the optimized conditions, the determination of chlorogenic acid and flavonoids showed good linearity, and the recovery ranged from 95.2～104.4%. Based on this method, the HPCE fingerprints of Honeysuckle Flower were established.The fingerprints were compared by the software of the similarity evaluation system. The fingerprint congruence coefficients of 15 samples from the same group were above 0.93, and the congruence coefficients of samples from 7 different areas varied within 0.77-0.98. Honeysuckle Flower and Lonicera Confusa DC can be distinguished effectively by HPCE fingerprint. This method is simple, accurate, rapid and has a good reproducibility, so, it can be used to control the quality of Honeysuckle Flower.3. A method to enrich, separate and purify gentiopicroside and loganic acid from Radix Gentianae by macro porous absorbing resin was developed. The Radix Gentianae was extracted by ASE technique. The performance of four types of macro porous absorbing resin were compared, and D301 was chosen based on its performance. The operating conditions of HPLC was optimized. The best condition are as following: the sample concentration: 0.2 g/mL, the maximum capacity for medicinal materials was 0.25g medicinal material per 1g resin, the best absorbing time:8 hours, after absorbing ,wash down the gentiopicroside and loganic acid with 8% and 55% ethanol respectively. Condense and freeze-dry the washed solution resulted in the production of coarse product of gentiopicroside and loganic acid The respective purities are 74.3% and 80.9%, and the respective recoveries are 70.11% and 67.82%.4. A reliable and rapid HPCE method in MEKC and MEEKC modes was developed for the determination of gentiopicroside and loganic acid in the extracts of Radix Gentianae. The analyte was extracted from Radix Gentianae samples via accelerated solvent extraction, and the extraction conditions were optimized. Separation and determination were carried out by HPCE in a bared fused silica capillary(50 cm×75μm i.d.), with corresponding buffer . The run voltageof MEKC and MEEKC were 30 kV and 22KV respectively, the detection wavelength of DAD was 238 nm, and the column temperature was 25 oC. The developed HPCE method is simple and reliable for the determination of gentiopicroside and loganic acid in Radix Gentianae samples with a wide linear dynamic range, a recovery range of 96.3%～105.16%, and a detection limit of below 10 mg/L. The contents of gentiopicroside and loganic acid in six samples from different regions were determined by the developed method in two modes. T test result indicated that the measured contents of gentiopicroside and loganic acid by MEKC and MEEKC modes were consistent. The method is simple, accurate, rapid and with good reproducibility. It can be used to determine active components in Radix Gentianae.5. A reliable and rapid method by HPCE in MEKC, MEEKC and NACE modes was developed for the determination of alkaloids in extracts of Semen Nelumbinis. The analyte was extracted from Semen Nelumbinis samples via accelerated solvent extraction, and the extraction conditions were optimized. Separation and determination were carried out by HPCE in a bared fused silica capillary(50 cm×75μm i.d.), with corresponding buffer. The run voltage of MEKC, MEEKC and NACE are 30 kV, 22 kV and 30 kV respectively, the detection wavelength of DAD was at 282 nm and column temperature was 25 oC. The developed HPCE method is simple and reliable for the determination of alkaloids in Semen Nelumbinis samples with a wide linear dynamic range, a recovery range of 83%～104%, and a detection limit of below 10mg/L. The contents of liensinine, isoliensinine and neferine in 8 samples from different regions were determined by the developed method in three modes. The method is simple, accurate, rapid and with good reproducibility. It can be used to determine liensinine, isoliensinine and neferine in Semen Nelumbinis.6. An HPLC-DAD-ESI-TOF/MS analysis method was developed for the determination of gentiopicroside and loganic acid in Radix Gentianae samples. Fingerprinting technique for the herbs was also studied. The extract was separated into a water phase and a chloroform phase. Based on MS analysis of the chromatographic peaks and their comparison with standard references, three compounds were identified: gentiopicroside, asafetida acid and loganic acid. In the chloroform phas, DAD/MS data reveal 4 isomeric compounds. The compounds remain to be determined since no literature information is currently available. Based on this method, the HPLC fingerprints of Radix Gentianae were established. The fingerprints were compared by the software of the similarity evaluation system. The fingerprint congruence coefficients of ten samples from the same group were above 0.98, indicated that the method is reliable and accurate. The water phase fingerprint congruence coefficients of samples from six different areas are above 0.90. However, the chloroform phase fingerprint congruence coefficients varies from 0.62-0.99, indicating that Radix Gentianae from different producing area differ widely, and the diversities are embodied in the content of triterpenoids.7. An HPLC-DAD-ESI-TOF/MS method was developed for the analysis and identification of alkaloids in Semen Nelumbinis. The samples were extracted using ASE for 10 min under 100℃and 1400Psi. The extract was analyzed with HPLC-DAD-ESI-TOF/MS, and six compounds were identified based on MS analysis and reference compound comparison. The six compounds identified are pronucifefine, 1otusine, nuciferine, 1iensinine, isolisensinine, and neferine. Based on this method, the HPLC fingerprints of Semen Nelumbinis were established. The fingerprints were compared by the software of the similarity evaluation system for chromatographic fingerprint, the fingerprint congruence coefficients of samples from eight different areas varied in the range of 0.62-0.99, indicating that Semen Nelumbinis from different producing area differ widely.This method can be used for the determination of Alkaloids component in Semen Nelumbinis and its quality control.The establishment and application of those analytical methods demonstarted that the HPCE technology can be used for the quality control of TCMs through compositional or fingerprinting analysis. HPLC-ESI-TOF/MS is also shown to be a powerful tool for compounds identification in TCM. |
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