| Biopharmaceutical analysis in one of subjects which study pharmic relationships and varieties between quantity and quality in organism using kind of analytical methods in order to discover pharmacokinetic parameters, metabolite modes and routes etc. The object of biopharmaceutical analysis could be any of body fluides, organes, tissues or egestas. In all of these objects, blood and pharmaceuticals in it are the most commonly researched. Whereas, it is difficult to study biopharmaceuticals for their low concentration and many interferes from endogenous ingredients. So, fussy pretreatments are commonly needed and sensitive detection are desired.In this paper, a simple in-line dialysis capillary electrophoresis (CE) method is established to eliminate the interferes from endogenous macromolecules and simplify pretreatment. Moreover, the sensitive chemiluminescence and electrochemical detections for capillary electrophoresis are also established. The characteristics of the methods are evaluated. Additionally, some applications of the the methods in biopharmaceutical analysis are realized.The main research and results are as below:1. A novel CE method with dialysis sampling function was established. The dialysis membrane is in situ prepared in the inlet end of the separation capillary by phase-inversion process, which endued the capillary with an additional function of clean-up sampling. The materials and crafts of membrane preparation are researched. The ability of membrane cutting off macromolecules and particles is also assayed qualitatively. The membrane prepared using membrane-casting solution with components of 22% w/v polysulfone and 8% w/v PEG 600 can effectively cut off bovine serum albumin ( MW 18 kDa), while the membrane prepared using solution with components of 25% w/v polysulfone can partly cut off lysozyme (MW 18 kDa).2. The performance of dialysis-capillary electrophoresis method are evaluated. As dialysis membrane is formed in situ in the inlet end of separation capillary, there is almost no dead volume, and the separation efficiency with little reduction is achieved. Using this capillary in electrophoresis, the RSDs of migration times and peak areas of chlorpheniramine were 0.8% and 4.6%, respectively. During continuous use of this capillary for 12 hours, the performance of the membrane changed little.3. Whether equilibrium dialysis injection is applicable to the dialysis-capillary elctrophoresis method is studied. The linear relationship between peak area and concentration answers true, which show that the method can be applied in in vivo analysis. Additionally, in order to improve the detection sensitivity, electrochemical detection method is developed, which will extends the application field to monitor trace pharmaceuticals in biomedia.4. The novel CE method is applied in biopharmaceutical analysis. The concentration of free caffeine in Coffee Milk is directly determined using the method without any sample pretreatment, which is 0.68 mmolL-1. The glucose in human blood is also evaluated using the method with electrochemical detection. Whole blood is diluted and injected directly without any other pretreatment. The concentration of blood glucose is 5.53 mmol·L-1,and the limit of detection is 0.1 mmol·L-1. Moreover, the method is applied to evaluate the interaction between promethazine hydrochloride and BSA, the measured binding constant is 1.47×104 L·mol-1.5. A sensitive CE-CL combined instrument is set up in the laboratory. The performance of combined method is evaluated. The active metabolite paracetamol of benorilate is analyzed using the combined method. The concentrations-time curve of paracetamol is achieved, and the peak concentration of paracetamol is 4.60×10-3 g·L-1, and peak time is about 4 h.6. The pharmacokinetic by injection Ephedrine to the ear edges of rabbit is researched, the plasma concentration of Ephedrine is determined using HPLC. The elimination rate constant is 0.299 h-1, and half time is 2.32 h.
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