Toida T, Huang Y, Washio Y, Maruyama T, Toyoda H, Imanari T, Linhardt R J
Faculty of Pharmaceutical Sciences, Chiba University, Yayoi, Inage, Chiba, 263, Japan.
Anal Biochem. 1997 Sep 5;251(2):219-26. doi: 10.1006/abio.1997.2277.
A new, simple, and highly sensitive method for the determination of heparin has been established. Heparin was first converted into unsaturated disaccharides through the action of heparin lyases I, II, and III. A major trisulfated unsaturated disaccharide product results, consistent with structural analysis of a number of pharmaceutical heparins using one- and two-dimensional 1H NMR spectroscopy. This disaccharide was analyzed by HPLC using fluorometric postcolumn derivatization. The correlation between the amount of this trisulfated unsaturated disaccharide and anticoagulant activity of heparin as measured by anti-IIa was determined. The analysis of these pharmaceutical heparins showed a linear correlation between both HPLC and bioassay. This HPLC method was then applied to a pharmacokinetic study of heparin intravenously administered to rabbits.
已建立一种测定肝素的新的、简单且高度灵敏的方法。肝素首先通过肝素裂解酶I、II和III的作用转化为不饱和二糖。产生了一种主要的三硫酸化不饱和二糖产物，这与使用一维和二维1H NMR光谱对多种药用肝素进行的结构分析一致。该二糖通过使用荧光柱后衍生化的HPLC进行分析。测定了这种三硫酸化不饱和二糖的量与通过抗IIa测定的肝素抗凝活性之间的相关性。对这些药用肝素的分析表明，HPLC和生物测定之间存在线性相关性。然后将该HPLC方法应用于对兔静脉注射肝素的药代动力学研究。
