Fine Structure Analysis Of Enoxaparin Sodium Base On Capillary Electrophoresis

Enoxaparin sodium is a low molecular weight heparin marketed as Lovenox or Clexane. It is a new antithrombotic drug. Currently, high performance liquid chromatography (HPLC) analysis is only one method for the quality control used in the manufacturing process of Enoxaparin sodium, unfortunely, but the HPLC method must be used under the patent constraints. Based on caplillary electrophoresis, the paper developed a new practical method of determination to multi-component fine structure of Enoxaparin sodium.Firstly, Depolymerized enoxaparin sodium with a mixture of heparinase I, heparinase II and heparinase III, and then a capillary electrophoresis method was established for the analysis of oligosaccharides obtained by enzymatic depolymerization of Enoxaparin sodium. This paper mainly discussed capillary electrophoretic separation conditions. By one-factor analysis, the effect of the components, pH value and concentrarion of running buffer, separation voltage, injection volume, caplillary column length and internal diameter, addition of organic solvents and metal ions and the application of a pressure gradient were researched in detail. The optimum electrophoretic separation conditions were as follows:capillary column diameter 50μm, total length 75 cm and effective length 66.5 cm; A 120 mmol·L-1 formic acid at pH 3.5 was selected as running buffer, adding 2 mmol·L-1 BaCl2 when prepared, adding 10% methanol before use; injection volume is 50 mbar×15 s; an applied voltage of -20 kv (reversed polarity); an overimposed pressure gradient (20 mbar) for 5 min from inlet to outlet starting at 30 min; column temperature is 25℃; UV detection at 232 nm.Secondly, under the optimized caplillary electrophoresis separation conditions, using the new method,7 elementary disaccharides, oligosaccharides obtained by enzymatic depolymerization of heparin sodium, oligosaccharides obtained by enzymatic depolymerization of Enoxaparin sodium were identified one by one. A qualitative method based on electrophoretic mobility for disaccharides, triaccharides, tetrasaccharides and 1,6-anhydro derivatives were established. And it is there possible to quantify the amount of every component by peak areas in the electropherograms and by weight of all the oligosaccharides of the depolymerized mixture. The percentage of oligosaccharide chains ending with a 1,6-anhydro ring was also obtained.The analysis method was proved to be simple, accurate, and sensitivity and the analytical results have good reproducibility, which is possible to be used for the quality control during the process for preparing Enoxaparin sodium.