Study And Application Of Novel Method On Deteramination Of Active Compound In Herbal Medicine By Flow Injection-Capillary Electrophoresis

The hybrid techniques of the analysis instruments are one of the trends of the development of analytical chemistry. The combination of flow injection with capillary electrophoresis (FI-CE) is representative of the trend. The combined technique that integrates the essential favorable merits of FI and CE can significantly expand the application scope of CE by exploring the various on-line sample pretreatments, and overcomes the shortcoming of discontiguous injection of CE. However, hitherto, the ability of on-line sample pretreatments of FI is not widely applied, and many theories and applications need to deeply study in separation and determination of active compound in herbal medicine by FI-CE. Additional, the poor concentration sensitivity is one of the reasons of confined development of FI-CE. To exert the advantages of FI-CE and overcome the shortcoming brought by electrokinetic means in CE, the following major innovative work are carried out in this dissertation on the basis of the previous literatures:1. A Novel method on separation and determination of active compound in Juemingzi, Rhubarb and related herbal preparations by FI-capillary zone electrophoresis (FI-CZE) was developed for the first time, and the effect of matrix was discussed in detail.2. The changeful alpinetin and cardamonin in Alpinia Katsumadai were firstly separated and determined by FI-micellar electrokinetic chromatography (FI-MEKC).3. In order to improve the repeatability of the combination of FI-MEKC, the internal method was used, and a new method for the separation and determination of tetrandrine and fangchinoline was founded.4. A head-column field amplified sample injection (HC-FASI) in FI-MEKC was developed using electroosmotic flow to introduce a short water plug. Tetrandrine and fangchinoline were selected as model compound to demonstrate this stacking method. This dissertation consists of seven chapters.In chapter 1, the combination of FI and CE was discussed from the necessity of the combined technique, the principle of this technique, split-flow interfaces and the basic FI-CE system setup, as well as novel applications to a variety of analytical problems, and so on. More importantly, the methods of stacking in CE and FI-CE were reviewed in detail.In chapter 2, an unmodified effective length of 4.8 cm fused-silica capillary was used as separation capillary. A novel method for the separation and determination of physcion, chrysophanol, aloe-emodin and emodin in Rhubarb, Juemingzi and Chinese herbal preparations was developed by FI-CZE for the first time. The sample solvent was consisted of NaOH (100 mmol/L) and acetonitrile (ACN) (1:1 v/v), and a running buffer was composed of 15 mmol/L sodium borate -12.5 mmol/L sodium dihydrogen phosphate-42% (v/v) ACN . Under the optimized conditions, separation of the compounds could be achieved within 6 min. The sample throughput rate could reach up to 12 h^-1. The repeatability (defined as relative standard deviation, RSD) was 4.45%, 4.44%, 4.34%, 0.61% with peak height evaluation and 1.62%, 0.89%, 2.49%, 2.19% with peak area evaluation for physcion, chrysophanol, aloe-emodin and emodin, respectively.In chapter 3, a 50μm I.D. and unmodified effective length of 10.3 cm fused-silica was used as separation capillary. The background electrolyte (BGE) containing 4 mM sodium borate-8 mM NaH₂PO₄ (pH 8.1)-8 mM Sodium dodecyl sulfate (SDS)-19% (v/v) ethanol was used. A novel method for the separation and determination of alpinetin and cardamonin in Alpinia Katsumadai Hayata was developed by FI-MEKC for the first time. Under the optimized conditions, the sample throughput rate could reach up to 12 h^-1. The repeatabilities (n = 4) reached RSD of 3.0 and 2.5% for the peak areas and 2.5 and 3.1% for peak heights of alpinetin and cardamonin, respectively. Regression equations revealed linear relationships (r: 0.9996-0.9997) between the peak area of each analyte and the concentration. Recoveries were in the range 90-92% and 99-105% for alpinetin and cardamonin, respectively.In chapter 4, a FASI was first successfully combined with the FI-MEKC system. Fangchinoline and tetrandrine were selected as model mixture to demonstrate this stacking method. The parameters influencing the separation and stacking were studied. Under the optimum conditions, 6.8- to 8.9-fold improvement in concentration sensitivity relative to conventional FI-CE methods was achieved, and sample throughput rate can reach up to 50 h^-1 . The repeatability (RSD) was 4.8, 4.4% with peak height evaluation and 3.6, 0.94% with peak area evaluation for tetrandrine and fangchinoline, respectively.In chapter 5, a 50μm I.D. and unmodified effective length of 4.5 cm fused-silica was used as separation capillary. A novel method for the separation and determination of honokiol and magnolol in Magnolia officinalis and related herbal medicines was developed by combination of FI-CZE. The sample solvent consisted of 150 mM NaOH, a running buffer composed of 10 mM sodium tetraborate-10 mM sodium dihydrogenphosphate (NaH₂PO₄) at pH 12 was applied for the separation of the analytes. Under the optimized conditions, regression equations revealed linear relationships (r: 0.9991-0.9998) between the peak area of each analyte and the concentration. The repeatability (RSD) was 2.0%, 1.6% with peak area evaluation, 3.6%, 2.0% with peak height evaluation and 2.0%, 1.4% with migration time evaluation for honokiol and magnolol, respectively. The separation could be achieved within 5 min and sample throughput rate can reach up to 28 h^-1.In chapter 6, a novel method for the separation and determination of fangchinoline and tetrandrine was developed by combination of FI-MEKC system with trimethoprim as internal standard. A running buffer composed of 15 mM acetic acid-15 mM sodium acetate-3% (v/v) Tween 20-5% (v/v) methanol at pH 5.5 was applied for the separation of the analytes. Under the optimized conditions, the method resulted in excellent linearity with correlation coefficient of regression equation of 0.9996 and 0.9991 for TET and FAN, respectively. The real sample throughput was 19-40 samples per hour. The method was simple, rapid, reproducible, and has been successfully used for the determination of fangchinoline and tetrandrine in various herbal medicines. Recoveries were in the range 95-109% and 92-106% for tetrandrine and fangchinoline, respectively.In chapter 7, a novel method for the separation and determination of berberine, palmatine and jatrorrhizine in Coptidis Rhizoma and related herbal medicines was developed in this paper by combination of HC-FASI-FI-MEKC. The effect ionic strength of BGE and volume of sample loop were studied. Based on the characteristic of the 16-way auto-switching valve, a short water plug was introduced by the electroosmotic flow (EOF) at the capillary inlet end prior to sample introduction. The BGE containing 240 mM ammonium acetate (pH 4.7)-30 % (v/v) ethanol-2 % (v/v) Tween 20 was used. Under the optimized conditions, 64 to 86-fold improvements in the detection sensitivity was obtained for the analytes and the sample throughput can reach up to 24 h^-1. The limit of detection (LOD) for the berberine, palmatine and jatrorrhizine was found to be 27, 26, 22 ng mL^-1 (S/N = 3). The repeatabilities (n = 4) reached RSDs of 1.2,2.7 and 3.1 % for the peak areas and 1.6, 3.3 and 3.8 % for peak heights of berberine, palmatine and jatrorrhizine, respectively.