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目的用己二酸二酰肼活化透明质酸,并用2,4,6-三硝基苯磺酸显色法测定活化后产物中活性氨基的含量。方法利用2,4,6-三硝基苯磺酸与活性氨基形成稳定的特征显色化合物的特性,采用L-缬氨酸作为活性氨基标准物质绘制了标准曲线,并测定了不同反应条件下制备的透明质酸-己二酸二酰肼中间体中活性氨基的量。结果绘制的标准曲线的线性及精密度较好,回归方程为:Y=4.8405X+0.0258(r2=0.9972);测得3种反应条件下制备的透明质酸-己二酸二酰肼中间体的活性氨基取代度分别为100.7、111.9、142.2μmol.g-1。结论所用方法合理准确,对活化反应的优化和进一步的接合反应提供了一定的定量依据。
Objective To activate hyaluronic acid with adipic acid dihydrazide and determine the content of active amino group in activated product with 2,4,6-trinitrobenzene sulfonic acid. Methods The characteristics of 2,4,6-trinitrobenzene sulfonic acid and active amino group were characterized by the formation of stable characteristic chromogenic compounds. The calibration curves were prepared by using L-valine as the active amino group reference standard. The reaction conditions were determined under different reaction conditions The amount of active amino groups in the prepared hyaluronic acid-adipic acid dihydrazide intermediate. Results The linearity and precision of the standard curve were good. The regression equation was: Y = 4.8405X + 0.0258 (r2 = 0.9972). The hyaluronic acid-adipic acid dihydrazide intermediate prepared under the three reaction conditions The degree of active amino substitution was 100.7, 111.9 and 142.2 μmol.g-1, respectively. Conclusion The method is reasonable and accurate, which provides some quantitative basis for the optimization of activation reaction and further conjugation reaction.