| It is difficult to analyze traditional Chinese medicine directly due to the complex matrixof traditional Chinese medicine and the diversity of composition in its preparations. As a newsimple and effective sample pretreatment technique, acetonitrile-water system phaseseparation technique has some outstanding features, such as simple operation steps, goodeffect for matrix purification and low consumption of organic solvent and sample solution.Besides, after phase separation, the acetonitrile phase can be directly injected into the reversedphase liquid chromatography for analysis. The purpose and significance of this study was toapply acetonitrile-water system phase separation technique for the analysis of traditionalChinese medicine preparations (Gegen Qinlian Decoction and Huoxiang-Zhengqi peroralliquid were chose as the research object). This paper is divided into four chapters, and themain contents are as follows:Chapter1: Several kinds of pretreatment technology for the analysis of traditionalChinese medicine preparation were briefly summarized. A new acetonitrile-water systemphase separation technique and its main applications were introduced. The background andpurpose of this study was also presented in this chapter.Chapter2: A high performance liquid chromatographic method was established for thesimultaneous determination of14compounds in Gegen Qinlian Tablet. The14compoundswere separated on a Dikma C18column (150mm×4.6mm i.d.,5.0μm) by gradient elution.The mobile phase consisted of ammonium formate buffer solution (20.0mmol·L-1, pH3.2,adjusted by formic acid)(A) and acetonitrile (B) at a flow rate of1.0mL min-1. Theultraviolet detection was accomplished at254nm and275nm. Ethanol-water (60:40, v/v) waschosen as extractant and the fourteen compounds were detected within46min. So thismethod is effective, simple, environmentally friendly and being well suitable for the qualityevaluation of Gegen Qinlian tablet.Chapter3: Based on the fact that acetonitrile-water system can form phase separation atsubzero temperature, a subzero-temperature liquid-liquid extraction coupled with ultraperformance liquid chromatography tandem quadrupole mass spectrometry (UPLC-MS/MS)method was developed for the simultaneous determination of12bioactive components inGegen-Qinlian decoction. After optimization, the extraction conditions were set as follows:3.0mL of aqueous sample solution (pH5.9) was extracted with2mL of acetonitrile at -35oC for35min. The separated acetonitrile phase was diluted10-fold with water beforeUPLC-MS/MS analysis. Separation was performed on a Waters ACQUITY UPLC○RBEH C18column (2.1×100mm.i.d.,1.7μm) with ammonium formate buffer solution (20.0mmol L-1,pH3.2, adjusted by formic acid) and acetonitrile as mobile phase under gradient conditions.The12target components could be separated within10min and quantified in multiplereaction monitoring (MRM) mode, both positive and negative ionization modes wereemployed. This is the first report on the combination of subzero-temperature liquid-liquidextraction with UPLC-MS/MS. UPLC is a high-speed separation technique and can solve thetime-consuming problem of HPLC. The established method provides a simple and effectiveframework for the quality control of Gegen-Qinlian decoction and related traditional Chinesemedicinal preparations.Chapter4: In this paper, based on the phenomenon that the acetonitrile-water system canform phase separation in the presence of sugar, a sugaring-out assisted liquid/liquid extractioncoupled with HPLC protocol was proposed for the determination of honokiol and magnolol intraditional Chinese herbal formula Huoxiang-Zhengqi peroral liquid. Extraction conditionssuch as the type and concentration of sugaring-out agent, the volume of acetonitrile, pH value,temperature and vortex time were investigated. Separation was performed on a Diamonsil C18column (150×4.6mm i.d.,5μm) and the column temperature was controlled at30oC. Themobile phase consisted of acetonitrile and0.05%formic acid (60:40, v/v) and the flow ratewas0.8mL·min-1. The column effluents were monitored at254nm. Compared withsubzero-temperature liquid/liquid extraction, sugaring-out assisted liquid/liquid extractiondoes not need special low-temperature equipment, while compared with salting-out assistedliquid/liquid extraction, the addition of salt is avoided. Compared with the ChinesePharmacopoeia method, the established method was simple, fast and greatly reduced theconsumption of toxic organic solvent. Statistical test showed that the analytical results for thedetermination of the same sample by the two methods had no significant difference. Theproposed method has been successfully applied to the determination of honokiol andmagnolol in Huoxiang-Zhengqi peroral liquid produced by different manufacturers. |
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