Application On Determination Of Drugs And Separation Of Proteins With Novel Capillary Electrophoresis Method

Capillary electrophoresis is an analytical method which has advantages of high efficiency, little time-consumption, low consumption of samples, low instrumental cost, seldom pollutions, automated-operation and robust. It is widely used for research, such as the analysis of drugs and proteins, etc. Therefore, it has been one of the most significant separation and analysis technologies in current life science. The aim of this work is to solve the problems in CE, for example, lack of sensitivity, non-specific adsorption of analytes in capillaries wall, and so on. We employed on-line preconcentration to improve the sensitivity of drug analysis in CE and surfactant coatings to suppress the adsorption of proteins in capillaries wall and to improve the efficiency of separation. In this study, we developed our work mainly around the subjects, which have been summarized as follows:(1) On-line determination of the anti-tumor drug 5-fluorouracil (5-FU) and its prodrug, tegafur (TF) was achieved for the first time by capillary electrophoresis with large-volume sample stacking (CE–LVSS). The optimal electrophoretic buffer consisted of 30 mM phosphate buffer at pH8.0. Without the LVSS procedure, the limits of detection (LOD) were 600.5 ng/mL and 771.4 ng/mL for 5-FU and TF, respectively. With the LVSS procedure, the sensitivity was significantly improved by about two orders of magnitude (the LODs of 5-FU and TF were decreased to 7.9 ng/mL and 6.5 ng/mL, respectively). The %RSD was less than 5%. This method compared favorably with other reported techniques and was applied successfully to the quantitative analysis of anti-tumor drugs in commercial injection preparations. The results show that the method is simple, fast (less than 3 min), highly selective, and sensitive.(2) Semipermanent surfactant coatings are effective for the prevention of wall adsorption of proteins in CE. However, they often suffer from their unsatisfactory coating stability as they essentially degrade from the capillary walls after the surfactants are removed from the buffer. We proposed a facile and universal method to improve the stability of semipermanent surfactant coatings based on addition of an oppositely charged surfactant into the coating. Didodecyldimethylammonium bromide (DDAB) and a gemini surfactant, 18-6-18, were used as the model semipermanent coatings, and sodium dodecyl sulfate (SDS) was chosen as their oppositely charged surfactant. SDS can strongly alter the packing parameter P of the cationic surfactants, and consequently improves the coating stability. With the increase of SDS concentration in coating, the coating stability first dramatically increases due to the enlarged P, and then decreases due to the weakness of electrostatic interaction between the capillary wall and surfactant coating. At the proper SDS concentration, very stable coatings can be obtained even after rinsing under 138 kPa for 60 min, the reversed electroosmotic flow (EOF) only decreases by 3.6%. These SDS-enhanced coatings show excellent stability and reproducibility in protein separation (RSD of migration time <1.1% for run-to-run assay, n=9). Also, the high separation efficiency (>500000 plates/m) and fine recovery of tested proteins indicate that these coatings are powerful in wall adsorption suppression. Finally, we found that the separation efficiency of protein was a more exact indicator for the coating stability than the traditional EOF magnitude.(3) We proposed a facile method to simultaneously separate acidic and basic proteins based on the giant micelle network formed by cationic surfactant as pseudostationary phases (PSPs). Cetyltrimethyl ammonium bromide (CTAB) was used as the model surfactant micelle, and ten typical acidic and basic proteins were chosen as model proteins. As the concentration of CTAB increased, the morphology of surfactant aggregates changed from spherical micelles to a giant micelle network. This surfactant giant micelle network is able to provide more hydrophobic space to facilitate the interaction of proteins with the PSP. Therefore, it is able to effectively reduce the interaction of proteins with the capillary wall and suppress the adsorption of proteins on capillary wall, thus yielding a simultaneous separation of acidic and basic proteins. This surfactant giant micelle network PSP showed excellent stability and reproducibility in protein separation (RSD <1.22% for run-to-run assay, n=9).