Fragment profiling of low molecular weight heparins using reversed phase ion pair liquid chromatography-electrospray mass spectrometry
Fragment profiling of low molecular weight heparins using reversed phase ion pair liquid chromatography-electrospray mass spectrometry
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使用反相离子对液相色谱-电喷雾质谱法对低分子量肝素进行片段分析
DOI：
10.1016/j.carres.2015.01.016
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发表时间：
2015-04-30
影响因子：
3.1
通讯作者：
Chi, Lianli
中科院分区：
文献类型：
--
作者：
Xu, Xiaohui;Li, Daoyuan;Chi, Lianli
低分子肝素(LMWHs)是由肝素化学或酶解聚而成的直线型高电荷碳水化合物聚合物。与普通肝素(UFH)相比，低分子肝素因其较低的副作用和较好的生物利用度而被广泛用作临床抗凝药物。本文的工作为低分子肝素的结构表征提供了一种快速而有效的片段图谱方法。用肝素酶I(Hep I，Ease Commission(EC)#4.2.2.7)、肝素酶II(Hep II，未指定EC#)和肝素酶III(Hep III，EC#4.2.2.8)通过受控酶消化产生两种低分子肝素的链片段，即依诺肝素和那屈肝素。采用反相离子对高效液相色谱(RPIP-HPLC)与电喷雾离子陷阱飞行时间质谱仪(ESI-IT-TOF-MS)联用，分析了从二糖到十糖的低聚糖链。建立了一个包含所有理论结构组成的数据库，以帮助进行质谱学解释。来自两种低分子肝素的三种酶产生的六种酶具有明显的指纹图谱。共检测到94个依诺肝素片段和109个那屈肝素片段。除了常见的低分子量低聚糖外，还发现了许多含有特征低分子量低聚糖结构的成分，如饱和L-吡喃糖苷酸、2，5-脱水-D-甘露醇、1，6-脱水-D-氨基吡喃糖以及奇数低聚糖。对创新产品和非专利产品中的主要成分进行了定量比较。这种分析低分子肝素片段的方法为评估这类抗凝药物的质量或比较来自不同来源的样品之间的结构相似性提供了一个高度可重复性、高分辨率和信息丰富的工具。(C)2015爱思唯尔有限公司。保留所有权利。
Low molecular weight heparins (LMWHs) are linear and highly charged carbohydrate polymers prepared by chemical or enzymatic depolymerization of heparin. Compared to unfractionated heparin (UFH), LMWHs are prevalently used as clinical anticoagulant drugs due to their lower side effects and better bioavailability. The work presented herein provides a rapid and powerful fragment mapping method for structural characterization of LMWHs. The chain fragments of two types of LMWHs, enoxaparin and nadroparin, were generated by controlled enzymatic digestion with each of heparinase I (Hep I, Enzyme Commission (EC) # 4.2.2.7), heparinase II (Hep II, no EC # assigned) and heparinase III (Hep III, EC # 4.2.2.8). Reversed phase ion pair high performance liquid chromatography (RPIP-HPLC) coupled with electrospray ion trap time-of-flight mass spectrometry (ESI-IT-TOF-MS) was used to profile the oligosaccharide chains ranging from disaccharides to decasaccharides. A database containing all theoretical structural compositions was established to assist the mass spectra interpretation. The six digests derived by three enzymes from two types of LMWHs exhibited distinguishable fingerprinting patterns. And a total of 94 enoxaparin fragments and 109 nadroparin fragments were detected and identified. Besides the common LMWH oligosaccharides, many components containing characteristic LMWH structures such as saturated L-idopyranosuronic acid, 2,5-anhydro-D-mannitol, 1,6-anhydro-D-aminopyranose, as well as odd number oligosaccharides were also revealed. Quantitative comparison of major components derived from innovator and generic nadroparin products was presented. This approach to profile LMWHs' fragments offers a highly reproducible, high resolution and information-rich tool for evaluating the quality of this category of anticoagulant drugs or comparing structural similarities among samples from various sources. (C) 2015 Elsevier Ltd. All rights reserved.