Study On Analytical Method For Active Components In Silybum Marianum And Establishment Of The Platform Of Pressurized Capillary Electrochromatography Coupled With Mass Spectrometry

Silymarin（Silybum marianum（L.）Gaertn.）is a ripe fruit of Silybum marianum（L.）Gaertn.It can be used for the relieve of fever and for treatment of the disease of liver and gallbladder,hypochondriac pain,jaundice and other diseases.Silymarin mainly contains Silybin A/B,Isosilybin A/B,Silychristin A/B,and Silydianin.Wherein each of the isomeric monomers has different bio-pharmacological activity,respectively.Therefore,it is of great significance to study the separation method of these seven monomers in silymarin.The column packed with core-shell packing materials has drawn attention of chromatographers because of its high column efficiency,fast separation,low backpressure,good durability and so on.Pressurized capillary electrochromatography（p CEC）has the dual separation mechanism of chromatography and electrophoresis,which can meet the needs of complex sample analysis and miniaturization of analytical instruments.Since there is no back pressure in p CEC,it also provides a good platform for the utility of column packed with smaller particle size.Mass spectrometry is a widely used detector,which has high sensitivity,accuracy,sensitivity and provides information on molecular mass and structure.It is a powerful tool for qualitative and quantitative analysis of complex samples..This thesis includes six chapters,the main contents include the followings:In chapter 1,the active ingredients and their pharmacological effects of silymarin were introduced.The current status and research progress of separation and analysis of silymarin such as analytical instrumentations,and the packing materials were introduced.In chapter 2,the component of silymarin and its separation methods were investigated.The structure of seven diastereomers in silymarin was identified by ion trap mass spectrometry.The cleavage of the ions was studied by CID-MS/MS analysis.Based on the different structures of the isomers,as well as the difference between the ion fragment and the abundance of the ions to identified seven diastereomers in silymarin.In chapter 3,seven kinds of diastereomers in silymarin were separated and analyzed by using Halo C₁₈ column in high performance liquid chromatography（HPLC）,and the analysis time was shortened.Good peak shape and resolution were obtained.The method has been applied to detect four kinds of actual Milk Thistle preparation samples,which fully shows the advantage of rapid separation and high efficiency separation based on the core-shell columns.In chapter 4,separation of 7 diastereomers in silymarin was performed on a capillary column packed with 1.8μm particles on the p CEC platform.The results showed that the resolution in p CEC using the capillary column packed with 1.8μm small particles could be improved greatly,fully reflects the advantages with small particle size capillary column.By using the unique separation mechanism of p CEC from both HPLC and electrophoresis,the resolution of the chromatographic peak was improved significantly.In chapter 5,a p CEC coupled with Nano ESI-MS on-line was set up.According to the separation principle of p CEC and the Nano ESI of mass spectrometry,the interface was designed and compared to minimize the dead volume,improve the separation and sensitivity,and reduce the external environmental influences.The interference has good stability and reproducibility.By the optimization of experimental conditions,the double separation mechanism of p CEC was proved to be able to separate the sample selectively,and also to improve the separation and analysis time.The combined system is stable and sensitive,combining the advantages of p CEC and mass spectrometry with Nano ESI ion source.In chapter 6,the research is summarized and the future perspective was discussed.