Development Of Capillary Electrophoresis Methods For The Analysis Of Glycosaminoglycans And Its Application

Glycosaminoglycans are a family of linear polysaccharides composed of disaccharide repeating units, and they have the property of highdispersion and microheterogeneity. Glycosaminoglycans can becategorized into six groups:heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, hyaluronan andkeratan sulfate. Since glycosaminoglycans display a variety of important biological roles, they are widely used in pharmaceutical industry. Since the structures of glycosaminoglycans are quite complex and their separation are very difficult, obtaining the absolutely pure glycosaminoglycans is very challenging. The difference in structure leads to the variety in their pharmacological effects, and the side effects of heparin make the authority pay more attention to the analysis and determination of glycosaminoglycans. So, the analysis and determination of glycosaminoglycans are of vital important for the quality control of their related products. Chondroitin sulfate is widely used in the treatment of osteoarthritis, but the variety in its structure can lead to the difference in its activity, so the establishment of a fast method for the identification of the source of chondroitin sulfate is very important. Glycosaminoglycans are a kind of endogenous macromolecules and their in vivo concentration is closely related to the health status. Now it is administered orally or by injection as health care products and medicine, but its in vivo existence status is controversial now. The pharmacokinetic parameters of glycosaminoglycans are mostly obtained by the analysis of their disaccharides, which cannot reveal the concentration level of their intact molecules, so the development of a method for the determination of the glycosaminoglycans is quite meaningful for the diagnosis of disease, monitoring of the therapeutic drugs and their pharmacokinetic study. In a word, now the analysis of glycosaminoglycans is mainly about the analysis of the disaccharide obtained from enzyme or chemical degradation, it is tedious and time costing; in addition, it cannot represent the concentration of intact glycosaminoglycan. So, this study focused on the development of fast and accurate methods for the analysis of glycosaminoglycans.1. In this work, a capillary electrophoresis method with bare gold nanorods based pseudostationary phase was developed and applied for the separation of chondroitin sulfate and dermatan sulfate based on the previous study. After investigation of different kinds of separation buffer, ethylenediamine phosphate buffer was finally chosen. The separation efficiency was investigated by varying the experimental parameters such as concentration and pH of the background electrolyte, separation voltage, different size and morphology of gold nanomaterials. Results showed that different size and morphology of gold nanomaterials had different effects on the separation of chondroitin sulfate and dermatan sulfate. The best separation of chondroitin sulfate and dermatan sulfate was achieved in the background electrolyte composed of 150 mmol/L ethylenediamine+20 mmol/L sodium dihydrogen phosphate+30%(v/v) gold nanorods, pH 4.5, at the separation voltage of-10 kV. Capillary was 59.2 cm in length (effective length 49 cm),50 μm I.D. capillary thermostated at 25 ℃. Capillary electrophoresis with bare gold nanorods used as pseudostationary phase was shown to be a suitable technique for the separation of chondroitin sulfate and dermatan sulfate mixture. RSD of migration time and peak area of chondroitin sulfate and dermatan sulfate were 0.13%,0.14% and 0.86%, 1.07%, respectively.2. In this study the dynamic simulation of sodium citrate and glucuronic acid with gold nanoparticles was accomplished by using Materials studio software. After equilibration of the system, the interaction energy and diffusion coefficient were calculated and the result demonstrated that interaction energy between gold nanoparticles and glucuronic acid was bigger than sodium citrate and the diffusion coefficient of glucuronic acid was bigger than that of sodium citrate, which indicate that in the competitive adsorption between sodium citrate and glucuronic acid, glucuronic acid was dominant. The results were in accordance with the reference and revealed that the system was suitable for the study of the interaction between carbohydrate and gold nanoparticles.3. An electrophoretically mediated microanalysis protocol for the determination of different chondroitin sulfate origins based on the difference in the content of unsaturated disaccharides produced by degradation with chondroitinase ABC was developed in this work. Separations were performed in an uncoated fused silica capillary (total length:60.2 cm; effective length:50 cm; 50 μm I.D.) at 37 ℃. The influences of various parameters, such as different kinds of separation buffers, substrate concentration and incubation time, on separation were investigated. The optimum conditions were as follows:separation buffer,25 mmol/L tetraborate buffer (pH 9.5); incubation buffer,50 mmol/L Tris-60 mmol/L acetate buffer (pH 8.0); incubation buffer (0.5 psi×5 s) was injected into the capillary, followed by enzyme (0.5 psi×10s) and subsequent injection of substrate solution (0.5 psi×10s) and incubation buffer (0.5 psi×5 s). Upon the application of-1 kV/+l kV/-1 kV/+1 kV each for 6 s, the enzyme and substrate solutions were mixed. The reaction was initiated by the application of+1 kV voltage for 8 min. Then a constant voltage of +20 kV was applied to separate the product and enzyme. The concentration of chondroitin sulfate for identification was 500 μg/mL. Nonsulfated, monosulfated, disulfated and trisulfated disaccharides were separated well under the above optimal conditions. The developed method was used to determine the contents of disaccharides in chondroitin sulfate from different sources and the results were compared with those obtained by offline analysis. The results indicated that the developed method could successfully distinguish chondroitin sulfate with minor differences and could obtain good coherence results compared with the traditional method.4. In this work a capillary electrophoresis method for the simultaneous separation of hyaluronan acid, chondroitin sulfate, dermatan sulfate and heparin was established. Different kinds of buffer systems were investigated in this study and finally diethylenetriamine phosphate buffer system was chosen for further study. The study investigated the effects of buffer pH and concentration, separation voltage and inner diameter of the capillary on separation. The effect of salt concentration on resolution and migration order was also investigated. Finally separation was performed in an uncoated fused silica capillary (total length:60.2 cm; effective length:50 cm; 50 μm I.D.) and separation buffer was 80 mmol/L diethylenetriamine phosphate buffer (pH 5.0). Detection wavelength was 200 nm. Separation tempature was 37℃ and sample storage temperature was 4℃. Separation voltage was -24 kV and sample was injected into the capillary at 0.5 psi for 5 s. Determination of hyaluronan acid, chondroitin sulfate and dermatan sulfate was fully validated and the results were shown that the established method can be used for the determination of the three glycosaminoglycans. The established method was successfully applied for the determination of hyaluronan acid, chondroitin sulfate, and dermatan sulfate in a glycosaminoglycan mixture sample.5. The developed separation method was applied to the simultaneous determination of hyaluronan acid and chondroitin sulfate in mouse plasma. Different extraction methods were investigated in this study and finally 4 mol/L NaCl, chloroform and ethanol were applied for the pretreatment. Plasma volume was also investigated to increase the sensitivity and 200 μL was chosen. The impact of hemolysis on determination was also investigated and the results revealed that it could interfere the detection of chondroitin sulfate. The method was validated over the concentration range from 50 to 600 μg/mL for hyaluronan acid and 500 to 6000 μg/mL for chondroitin sulfate in mouse plasma. Results from assay validations showed that the method was selective and robust.