| Traditional Chinese Medicine (TCM) has several thousand years in clinical practice. As drugs effectiveness and safety being understood deeply by public, the quality of TCM is became a new focus. The quality control of TCM also becomes one of the important factors in the development of the TCM industry and one of the important content in the modernization of TCM. Because of the complexity, diversity and limitations in material foundation study of TCM, the development of TCM quality control technology becomes the key. At present, chromatographic methods, one of the analysis methods of TCM quality control, from the traditional thin layer chromatography (TLC), high performance liquid chromatography (HPLC), gas chromatography (GC) to the capillary electrophoresis technology and spectrometry technology, can be adapted to the new concept and requirements undoubtedly. They show respective advantages and solve many problems in the complex composition analysis of TCM. Moreover, HPLC including the features of multiple-choice of chromatographic column, various detector and versatile mobile phase is suitable for the most of the compounds analysis test and to be widely applied and the most promising method undoubtedly.Microemulsion liquid chromatography (MELC) is a novel high performance liquid chromatography which uses microemulsion as mobile phase. Microemulsions are dispersion of nanometer-sized droplets of an immiscible liquid with another liquid, they are transparent or subtransparent, low viscosity, isotropic and thermodynamically-stable mixtures, including oil, surfactant, co-surfactant and water. Its particle size is less than100nm. Compared to conventional HPLC, MELC has many advantages, such as, high separation efficiency and fast separation speed, be used for the separation of the complex samples, directly analysis of serum, urine samples after simple dilution without pretreatment and the advantage in the gradient elution and low UV wavelength (190nm) test.In the present work, the MELC was explored to investigate the separating effect of fat-soluble and water-soluble composition in the TCM and the feasibility of gradient elution and directly determination of plasma sample. In addition, the composition and proportion of each mobile phase were optimized. Finally, the MELC methods were compared with conventional HPLC. And the W/O MELC was preliminarily studied.1. The analysis of the fat-soluble constituents in TCM using MELC methodRheum and Jiyipaishi Granule were selected to investigate the separating effect of fat-soluble constituents in TCM with MELC method. The factors of separation and retention time were discussed.(1) A method for the determination of aloe-emodim, rhein, emodin, chrysophanol and physcione in rheum by MELC was established. The method was also validated. The optimized conditions for the determination were as follows: mobile phase:2.5%(w/v) sodium dodecyl sulfate-0.1%(v/v) n-octane-8.0%(v/v) n-butanol-0.5%triethylamine (PH=3.00); analytical column:Hypersil ODS2(4.6mm×250mm,5μm); temperature:28℃; flow rate:1mL/min; detection wavelength:254nm. The results show that:1) The five anthraquinone derivatives in rheum were to be baseline separation, precision was between0.48%～1.29%, and accuracy was high, recovery was between97.53%～101.37%, and it also had a wide linear range and good linear relationship (r between0.9987and1.0000). The method can be used to determine the five anthraquinone derivatives in rheum.2) Using the same instrument, conventional HPLC methods, were also adapted to compare with MELC. Using methanol-0.05%phosphoric acid solution (75:25)[28] as mobile phase showed that the five anthraquinone had been eluted, the average retention time was within39.93min, but the peaks were poor. Using methanol-acetonitrile-0.01%phosphoric acid solution (3:5:2)[27] as mobile phase showed a poor separation between aloe-emodim and rhein and the peak of physcione was collapsed, but the average retention time was within15min. Using methanol-0.1%phosphoric acid solution (85:15)[29] as moble phase showed the retention time was within17min, but aloe-emodim and impurity and rhein in sample had poor separations. Though the retention time was within36min when using MELC method, the separation and the peaks were proved to be better.(2) On the base of the above mobile phase, a method for the determination of chrysophanol in Jiyipaishi Granule by MELC was established. The method was also validated. After further optimization, the final mobile phase was2.5%(w/v) sodium dodecyl sulfate-0.1%(v/v) n-octane-10.0%(v/v) n-butanol-0.5%triethylamine (v/v), adjusting pH=3.0with phosphoric acid. The analytical column was Hypersil ODS2(4.6mm×250mm,5μm), with the column temperature of28℃, flow rate was1mL·min-1and detection wavelength was254nm. The results show that:1) Increasing the concentration of n-butanol to10%to shorten the retention time, the content of chrysophanol in Jiyipaishi Granule was successfully determined. The calibration curve was linear in the range of3.9～39μg·mL-1for chrysophanol with r=0.9998, the average spiked recovery and RSD value (n=6) were99.66%and0.64%. It showed the MELC method can be used to determine chrysophanol in Jiyipaishi Granule.2) Compared to conventional HPLC methods that were reported by literature, methanol-0.1%phosphoric acid solution (85:15)[30] and methanol-0.1%phosphoric acid solution (65:35)[31] were used as mobile phase respectively to determine the chrysophanol in Jiyipaishi Granule. The results showed that the peak was poor in the former and was not appeared in the latter. However, the MELC method showed a good separation.(3) In a word, MELC method can separate many kinds of complex and similar nature ingredients (fat-soluble constituents) in TCM and its preparations. Compared with conventional HPLC, the effect is better. So the MELC method can be a new choice in the field of TCM quality control methods. 2. The analysis of the water-soluble constituents in TCM using MELC methodShuanghuanglian injection was selected to investigate the separating effect of water-soluble constituents in TCM with MELC method. A method for the determination of chlorogenic acid, forsythin and baicalin in Shuanghuanglian injection by MELC was established and its influential factors were discussed. The final mobile phase was microemulsion consisting of2.0%(v/v) SDS-0.4%(v/v) hexane-6.0%(v/v) ethanol-water, adjusting pH=3.0with phosphate acid, the analytical column was Hypersil BDS C18(4.6mm×150mm,5μm), with column temperature of30℃, flow rate was0.8ml/min and detection wavelength was278nm and326nm. The results show that:1) The calibration curves were linear in the range of192.0μg/mL～0.8μg/mL with r=0.9998;104.0μg/mL～0.4μg/mL with r=0.9999;230.0μg/mL～0.9μg/mL with r=0.9989for Chlorogenic Acid, Forsythin and baicalin respectively. The average recoveries and RSD were98.97%,0.20%;97.40%,1.09%and100.98%,1.11%. It investigated that the method can be used for the determination of chlorogenic acid, forsythin and baicalin in Shuanghuanglian injection.2) There also has a good separation for the water-soluble constituents when using MELC method. So the MELC method can be use for analyse highly polar, good water-soluble components.3. The analysis of the fat-soluble and water-soluble constituents in TCM using MELC method with gradient elutionPolygonum cuspidatum was selected to investigate the separating effect of fat-soluble and water-soluble constituents in TCM using MELC method with gradient elution. A method for the determination of polydatin, resveratrol, emodin and physcion in polygonum cuspidatum by MELC with gradient elution was established and its influential factors were discussed. The optimal microemulsion were2.5%(w/v) SDS-0.1%(v/v) n-heptane-6.0%(v/v) isopropanol-0.5%TEA (pH was adjusted by phosphoric acid to3.0)(A) and2.5%(w/v)SDS-0.1%(v/v)octane-6.0%(v/v)n-amyl alcohol-0.5%TEA(pH was adjusted by phosphoric acid to3.0)(B), gradient started at100%A for13min, ramped up to100%B over7min, and then held it for18min. Agilent Eclipse XDB-C18column (4.6mm×250mm,5μm) was selected, with the column temperature of25℃. The contents of four ingredients in polygonum cuspidatum were determined simultaneously at a flow rate of0.8mL·min-1and the detection wavelength was299nm. The results show that:1) The calibration curve were linear with r=0.9998in the range of9.13～91.3μg·mL-1for polydatin, r=0.9999in the range of1.72～17.2μg·mL-1for resveratrol, r=0.9998in the range of8.12～81.2μg·mL-1for emodin and r=0.9997in the range of5.0～50μg·mL-1for physcion, the average spiked recoveries and RSD value (n=6) were98.42%and0.87%,98.91%and0.59%,99.88%and1.06%,99.72%and1.26%, respectively. It investigated that the method can be used for the determination of the four ingredients in polygonum cuspidatum.2) Compared to isocratic elution, using A as mobile phase showed that the four ingredients had a good separation, but the retention time was within52min. Using B as mobile phase showed the retention time was within26min, but the separation of polydatin and resveratrol were poor. It showed that A condition also can be used for the determination of four ingredients in polygonum cuspidatum.2) Compared to conventional HPLC methods that were reported by literature [36,37], the mobile phase, acetonitrile-0.1%phosphoric acid, was applied. And there have two ways of gradient elution. The first way showed a good separation for the four ingredients in polygonum cuspidatum, retention time was within39.0min. While using MELC method, the retention time was within37.0min. The other way also showed a good separation for the four ingredients, but the retention time was within53.0min. Compared to the HPLC methods, the effect of MELC was equal.4) This study further investigated the separation effect of small polar, poor water-soluble components is better than that of highly polar, good water-soluble components when using MELC method. For example, when the cosurfactant, butanol was substituted for propanol, isopropanol and n-amyl alcohol, the retention time of polydatin and resveratrol basically unchanged, but it had great effect on the retention time of emodin and physcion. It showed there has better selectivity for the fat-soluble constituents using O/W microemulsion.2) In a word, The MELC method with microemulsion-microemulsion gradient elution was first studied in the whole research. It showed stable baseline and it can be used to separate the complex component in TCM.4. Directly injection in MELC methodHow to simplify the biological sample pretreatment process has been a hot spot in the analysis work. Paclitaxel was selected, and a microemulsion liquid chromatography system with direct sample loading for determining the plasma level of paclitaxel in human was established. The mobile phase was microemulsion that was optimized by inspecting the influence factors of separation and retention time. The optimized microemulsion was2.0%(w/v) sodium dodecyl sulfate-0.4%(v/v) n-octane-10.0%(v/v) n-butanol-89.6%water (pH was adjusted by phosphoric acid to4.0). Hypersil ODS2Chromatography columns (4.6mm×250mm,5μm) was selected, with the column temperature of25℃. The flow rate was1mL·min-1and the detection wavelength was227nm. The plasma was dilution1:1with the mobile phase and then centrifugation, and injected directly into the HPLC system after filtration. The results show that:1) The calibration curve was linear with r=0.9993in the range of0.064～1.96μg·mL-1for paclitaxel. The RSD of intra-and inter-day precision were not exceed5%(n=5), and the lower limit of quantitation (LLOQ) was0.064μig·mL-It investigated the directly injection of human plasma is feasible.2) As solvent is the unique property of microemlsion. It can dissolve protein. The property makes the biological sample be directly injected into HPLC without tedious pretreatment. and then reduces some errors.3) In addition, directly injection has great effect on the column. In order to ensure the reproducibility and extend the life of chromatographic column, the column should be flushed for2～3h by water.5. The analysis of the water-soluble constituents in TCM using W/O MELC methodAbove research, O/W microemulsions were used, while W/O microemulsions were applied to analyze the water-soluble constituents in TCM in this part. A method for the determination of glycyrrhizic acid in liquorice root by MELC using water-in-oil microemlsion as eluent was established. The mobile phase was water-in-oil microemulsion that was optimized by inspecting the influence factors of separation. The optimized microemulsion was8.33%(w/v) sodium dodecyl sulfate-16.6%(v/v) n-pentanol-78.4%(v/v)(70mM) sodium acetate (pH was adjusted by lmol/L hydrochloric acid to7.5). Normal phase column, spherisorb SiO2Chromatography columns (4mm×200mm,5μm) was selected, with the column temperature of25℃. The content of glycyrrhizic acid in liquorice root was determined at a flow rate of0.8mL·min-1and the detection wavelength was250nm. The results show that:1) The calibration curve was linear with r=0.9998in the range of18.3～91.5μg·mL-1for glycyrrhizic acid. And the average spiked recoveries and RSD value (n=6) were99.58%and0.96%. It investigated that the method can be used for the determination of glycyrrhizic acid in liquorice root.2) The same instrument and chromatographic condition were applied to separate chlorogenic acid, forsythin and baicalin, polydatin and resveratrol, matrine and ligustrazine. The result showed there had strong elutingpower. There may have better chromatographic condition and W/O MELC method should be further researched.Summary:1. MELC method can be used for the determination of the complicated component in TCM and its preparations. There have a good separation for both fat-soluble constituents and water-soluble constituents. But there has better separation operation for fat-soluble constituents when using O/W MELC method. The method validation showed they can satisfy the quantitative requirements.2. Fat-soluble constituents and water-soluble constituents can be separated simultaneously, but there has better selectivity for the fat-soluble constituents.3. MELC method with gradient elution can be used for the analysis of different composition, and the base line is stable.4. Directly injection can be achieved when MELC method is used for the determination of the plasma sample. It simplifies pretreatment and reduces the errors. The quantitative requirements can be satisfied.5. Compared to conventional HPLC methods, there have the same or better separation effect and analysis efficiency for the MELC method. In addition, O/W microemulsion greatly reduced the use of organic solvent, and reduced the organic pollution and cost analysis.6. Normal phase MELC method (W/O MELC method) can be used for the analysis of the TCM, but the analytical characteristics should be further studied. |
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