Application Of Capillary Electrophoresis Enrichment Techniques In Analysis Of Traditional Chinese Medicine

Capillary electrophoresis（CE）has attracted more and more attention in the field of food,drug,environment and life science for its advantages of simple operation,quick analysis,high efficiency,low solvent consumption and several separation modes.However,the poor sensitivity caused by short optical path and small inner diameter of capillary restricted its capability in analyzing trace analytes.Therefore,on-line and off-line sample enrichment techniques were developed quickly in the recent years.In this work,the combination of on-line sample enrichment techniques as well as combination of on-line and off-line sample enrichment techniques were applied to improve the CE detection sensitivity by selecting the complex system and effective components of Traditional Chinese Medicine as the research object and analyte models.The main contents can be seen as follows:1.Discussion on the novel combination mode of on-line sample enrichment techniques.（1）A novel on-line proconcentration strategy coupling FASS and MCDS for determination of two alkaloids（matrine and oxymatrine）in complicated matrix samples has been proposed.The stacking mechanism of this method has been discussed in the article.Parameters affecting stacking effect such as SDS concentration in BGE,organic modifier in BGE,type of CD solution,amount of CD injected,and sample martix have been studied,the optimized conditions were as follows:BGE:60 mmol/L NH₄AC with75 mmol/L H₃PO₄.Sample matrix:15 mmol/L SDBS.CD solution:50 mmol/L hydroxypropyl-β-CD solution prepared in BGE.Sample injection was at 50 mbar for180 s followed by injection at 50 mbar for 60 s of CD solution.Under these conditions,an approving correlation coefficient（r≥0.9990）was obtained for each analyte,LODs were 0.01-0.02μg/m L,the SEFs were 169-218 folds compared with typical injection method.Finally,the proposed method was successfully applied to real samples.The method is simple,efficient,sensitive and environmental friendly,which could be a good alternative choice for the determination of alkaloids in complex sample matrix about Chinese medicine.（2）Based on the above research,a strategy for analyzing neutral analytes by combination of FESI and MCDS in RMM-MEKC mode has been first established to determine jujuboside A and B in complicated matrix samples.In the research,stacking mechanism was discussed and affecting factors of the method,for example,micelle concentration and organic solvent in BGE,injection time of CD solution,and sample matrix,were investigated.The optimized conditions were as follows:The BGE was a mixture of 100 mmol/L SDS,10 mmol/L Na H₂PO₄,50 mmol/L H₃PO₄,and 30%Methanol.The CD solution was 50 mmol/L hydroxypropyl-β-cyclodextrin prepared in100 mmol/L H₃PO₄.The sample matrix is 20 mmol/L SDS.The CD solution（50 mbar,180 s）and pure water（50 mbar,2 s）were successively introduced into capillary followed with the sample solution（-8 kv,180 s）.Under the optimized conditions,calibration curves with good correlation coefficient（r≥0.9990）were obtained for each analyte,LODs were 0.2-0.3μg/m L,the SEFs were 140-152 folds compared with typical injection method.In addition,the developed strategy was successfully applied in real sample analysis.This multistacking is a simple,reproducible and sensitive method.Compared to HPLC,it has advantages of quick analysis,superb separation efficiency,and reduced sample requirement.Thus,it provides an option for the determination of trace neutral analytes in Traditional Chinese Medicine.2.Discussion on the stratrgy of combination on-line and off-line enrichment techniques.（1）Off-line sample enrichment technique MSPD followed by on-line sample enrichment technique FESI-MCDS was developed to analysis coumarins in the complicated sample matrix about Chinese medicine.Parameters such as organic solvent in BGE,injection time of CD solution and sample,were optimized.For FESI-MCDS,the optimum conditions were as follows:BGE:100 mmol/L SDS,10 mmol/L Na H₂PO₄,50 mmol/L H₃PO₄,and 30%methanol.Sample matrix:20 mmol/L SDS.CD solution:50 mmol/L hydroxypropyl-β-CD solution in 100 mmol/L H₃PO₄.The CD solution（50mbar,120 s）and pure water（50 mbar,2 s）were successively introduced into capillary followed with the sample solution（-8 kv,150 s）.Parameters affecting extraction effiency including type of dispersant,mass ratio of sample to dispersant,grinding time,type of elution solvent and volume of elution solvent were also optimized.For MSPD,the optimum conditions were as follows:Mass ratio of sample（angelica dahurica）to dispersant:1:2.Grinding time:150 s.Elution:Me OH.Volume of elution solvent:500μL.The results show that proposed method not only simplified the extraction process,but also further improved the detection sensitivity of neutral analytes by combination of off-line sample enrichment technique and on-line sample enrichment technique.Compared with the conventional method,the SEF is 283-302 folds.（2）A simple,environment friendly and sensitive MSPD-FASS method has been established and successfully applied to the determination of organic acids in Traditional Chinese Medicine.MCM-48 was employed as the dispersive agent in MSPD.Parameters that affect extraction efficiency of MSPD were optimized,the optimum conditions were as follows:Mass ratio of sample（Fufang Danshen tablets）to dispersant:1:2.Grinding time:150 s.Elution:50%Me OH.Volume of elution solvent:250μL.Compared with the conventional extraction method,MSPD shows great advantages in sample pretreatment,such as small amount of sample,low consumption of organic solvent and decreased extraction time.In addition,the combination of MSPD and FASS has further improved the detection sensitivity of organic acids.To sum up,this method could be an alternative of choice for extraction and detection of organic acids in complex matrix about Chinese medicine.