Fine Structure Analysis Of Low Molecular Weight Heparin

Heparins are a linear polymer made up of 1â†'4 linked disaccharide repeating units, which is consisted of a hexuronic acid, L-iduronic or D-glucuronic acid and a D-glucosamine. Heparins play key roles in many physiological processes, such as cell differentiation, cell migration, inflamation and so on. As the composition of low molecule weight sulfated polysaccharides is very complex, quantitative analysis of their compositions are very important for understanding their bioactivity and the purpose of the quality control. Low molecular weight heparins (LMWHs) are widely used as the anticoagulant drug to prevent and treat the thromboembolic disease. The structure of LMWH is much more complex than its parent heparin because the reaction of chemical depolymerization (β-elimination under basic conditions) will lead to unusual chain cleavage and appearance of the 1,6-anhydro derivatives, which are the characterized structure of LMWHs. Therefore, fine structure analysis of LMWHs represents a challenge analytical work for quality control of this drug.This paper is mainly divided into three parts.The results and conclusions of the studies are the following:1. Herein, we report a new method for fine structure analysis of low molecular weight heparins with capillary electrophoresis. With the acidic background electrolyte composed of 120 mmol/L formic acid buffer (pH 3.4) most of the building block oligosaccharides of low molecular weight heparins obtained by complete digestion with haparinaseâ… ,â…¡,â…¢can be effectively separated. Addition of 1% (w/v) PEG 10000 and 2 mmol/L CaCl2 in the buffer is necessary for resolution of some co-migrated oligosaccharides which have almost the same mass-to charge ratio. Thus, almost all the building blocks of low molecular weight heparins can be quantitatively measured. Moreover, it was found that the electrolytic mobilities of oligosaccharides displayed a linear relationship to their charge-to-mass ratio. This can be used for assignment the peaks in the electropherogram according to their migration time. The method was validated in terms of the reproducibility and the quantitative parameters. The quantitative measurement data are comparable with those obtained by SAX chromatography method. But capillary electrophoresis based method can provide higher resolution than the SAX chromatography method for fine structure analysis of low molecular weight heparins.2. A method used for separation and quantitative analysis of the composition of low molecular weight heparins was developed. Effect of parameters, such as constitution, ionic strength, pH of the mobile phase, column length and temperature, flow rate of the mobile phase on the separation were systematically investigated. The obtained optimal conditions were as follows: two coupled TSK-GEL G2000SWxl columns (7.8 mm×300 mm); mobile phase,100 mmol/L NH4Ac (pH=7.0), flow rate,0.5 mL/min; column temperature,35℃, injection volumn,5μL; sample concentration,10 mg/mL. The method was validated in terms of its reproducibility and robustness. Under the optimized conditions, each component of a sample of low molecular weight heparins can be clearly separated, and their distribution ratios were quantifitively measured. With this method, the composition profiling between samples from USP, a commercial available sample and two Hangzhou JiuYuan gene Co., LTD samples were quantitatively compared. It was established that the method can be used for quality control of the low molecular weight heparins based drugs.3. This part based on the composition distribution of low molecular weight heparins to recognize each chromatographic peak.Polymerization degree is decasaccharide to preparation from low molecular weight heparin.After dividing salt and purification gets relative high purity decasaccharide.