凝血酶滴定法测定云南菲牛蛭中水蛭素含量的改进研究

Improvement Research on Thrombin Titration of Hirudin Content of Yunnan Poecilobdella Manillensis

目的改进原云南省菲牛蛭质量标准中水蛭素的含量测定方法。方法考察白瓷板法代替原试管方法、两种浓度的凝血酶溶液、几种滴定体积对含量测定的影响;用原方法和改进后的方法分别对五批样品进行滴定比较。结果改进后,用白瓷板法测定,配制两种浓度的凝血酶溶液(40NIH·m L-1与20NIH·m L-1),采用几种滴定体积(1~5μL),每1min滴加1次,先滴加高浓度凝血酶溶液初测消耗的凝血酶溶液体积后,再用两种浓度的凝血酶溶液重新进行精确测定;增加空白对照实验,限定反应时间为10min;菲牛蛭样品浓度在0.00678~0.0597g·m L-1(r=0.9983)内与消耗的凝血酶单位量呈线性相关;五批样品的含量为156.68~234.25U·g-1,RSD均小于5%;含量供试品溶液在4℃下24h内稳定。结论改进后的方法简便、快速,重复性和准确度较原方法好,可作为一种新的云南菲牛蛭水蛭素含量测定方法。

Objective To improve using white porcelain plates method, two the Hirudin content determination method kinds of thrombin solution concentration, of Poecilobdella manillensis. Methods The effect of several kinds of titration volume on the results of content of Hirudin was studied ; The results of 5 batches of samples were compared by two different methods. Results The white porcelain method was used, using two kinds of thrombin solution （40 NIH·mL-1and 20 NIH·mL-1） and several kinds of titration volume （1～5 μL）, once every minute, the accurate consumption volume of the test sample was determined with two different concentration of thrombin solution, after preliminary tested with the higher one. The blank test was increased, response time was limited for 10 minutes; The linear range of PoecilobdeUa maniUensis was 0. 00678 ～ 0. 0597 （r=0. 9983） g·mL-1, The content rang of Hirudin in 5 batches of samples was 156. 68～234. 25U·g-1, the RSD of repetitiveness tests was not more than 5%; The sample solution was stable under 4℃ in 24 hours. Conclusion The improved method was simple and quick, more repeatability and accuracy than the original method, it can be used as a new content determination method of Yunnan Poecilobdella manillensis.