肝素是一类结构复杂的高分子糖类,以N-硫酸、6-O硫酸氨基葡萄糖和2-O硫酸艾杜糖醛酸为主要成分组成二糖。常见的肝素注射液是以未分级肝素为原料纯化灭菌制备而来,在临床上使用更为广泛的是将肝素降解得到的低分子片段,低分子肝素保留了肝素糖链基本结构,但是不同制备工艺导致其具有不同的还原及非还原端。本研究以肝素类药物为研究对象,使用肝素裂解酶玉,域及芋将肝素注射液、低分子肝素注射液（达肝素、那屈肝素、依诺肝素）、化学合成的类肝素（磺达肝素）进行酶催化降解产生二糖,结合强阴离子交换色谱（ Strong anion exchange high performance liquid chromatography,SAX-HPLC）以及紫外检测器在线分析,使用市售肝素二糖标准品确定各种二糖结构。此外,使用反向离子对色谱和电喷雾质谱联用分析磺达肝素中的甲基二糖以及达肝素和那屈肝素中的脱氨二糖结构,为肝素以及类肝素药物的质量控制提供更为精确的结构信息。
Heparin and low molecular weight heparin have been widely used in clinical therapy as anticoagulants in cardiovascular disease and in hemodialysis. Crude heparin is usually prepared from porcine intestinal mucosa. Purified heparin is a mixture of polysaccharides consisting mainly of repeating GlcNS（6S）-IdoA2S disaccharides and other disaccharides with different GlcNAc/GlcNS±3S±6S-GlcA/IdoA±2S residues. Heparin injections are drugs prepared from heparin active pharmaceutical ingredient （ API ） that is prepared from crude heparin. Low molecular weight heparins are dominant heparin-based drugs used clinically, which are prepared by degrading heparin into smaller sizes. As a result, low molecular weight heparins are sharing the same major disaccharides but have different reducing and non-reducing ends. In current study, we focused on the disaccharide compositional analysis of clinically used heparin and heparin-based drugs. HeparinaseⅠ,II, and Ⅲ were used to degrade all heparin and heparin-based drugs including heparin sodium injection, Enoxaparin sodium injection, Nadroparin calcium injection, Dalteparin sodium injection, Fondaparinux sodium into disaccharides. All the degraded products were analyzed by strong anion high perforance liquid chromatography （ SAX-HPLC） coupled with an UV-detector. Commercially available unsaturated disaccharide standards were then used for structral identification. Furthermore, unusual disaccharides present in Nadroparin, Dalteparin, and Fondaparinux were confirmed by reversed-phase ion pair HPLC coupled with mass spectrometry. The developed method produced detailed structural information, which should be useful for quality control of heparin and heparin-based drugs.
Chinese Journal of Analytical Chemistry
Low molecular weight heparin