一种低分子肝素完全降解产物的色谱质谱联用检测方法

发布时间：2018-02-13 10:04

本文关键词： 低分子肝素二极管阵列检测器 2-氨基吖啶酮电喷雾电离质谱组成分析　出处：《山东大学》2014年硕士论文　论文类型：学位论文

【摘要】：肝素是一类带有大量负电荷,异质性的线性糖胺聚糖,主要存在于细胞表面和细胞外基质中。肝素是由重复的二糖单元组成的线性多糖,重复的二糖单元由己糖醛酸(HexA)和D-葡萄糖胺(G1cN)通过1-4糖苷键连接形成。二糖单元中的己糖醛酸可能是p-D-葡萄糖醛酸或者是a-L-艾杜糖醛酸。己糖醛酸可能是2-O-硫酸化,葡萄糖胺可能是6-0-硫酸化,N-硫酸化,N-乙酰化或3-O-硫酸化。肝素作为临床抗凝血药物广泛用于血栓性疾病的预防和治疗。然而,肝素作为抗凝血药物具有一定的副作用,如引起出血现象,血小板减少症和骨质疏松症等,因此限制了其临床应用。低分子量肝素(Low molecular weight heparin, LMWH)是由肝素多糖制备得到的一类新型抗凝血药物。LMWH具有分子量低、抗血栓作用强、生物利用度高和血浆半衰期长等特点,被广泛用于抗凝血药物。随着LMWH仿制药的出现,对LMWH结构和组成分析以及精细结构的分析鉴定是必不可少的。我们建立了一种新的基于反相(RP)-高效液相色谱(HPLC)-二极管阵列检测器(DAD)-电喷雾电离质谱(ESI-MS)分析方法用于LMWH精细结构组成分析。本论文中两种低分子量肝素达肝素钠和依诺肝素钠用肝素酶完全降解后通过柱前衍生方法与衍生试剂2-氨基吖啶酮(AMAC)结合,应用RP-HPLC-DAD-MS方法分析达肝素钠和依诺肝素钠两种低分子肝素完全降解组分,成功分离并鉴定到17种达肝素钠完全降解组分和15种依诺肝素钠完全降解组分,通过该实验方法各组分能实现高效分离,同时实现了对所有组分的定性和相对定量分析。达肝素钠和依诺肝素钠完全降解组分中除了8种常见的肝素二糖,还含有特征性的末端结构如还原端和非还原端结构,3-O-硫酸四糖和三糖,这些结构根据色谱的保留时间和质谱的质荷比(m/z)已经被准确分析鉴定。与传统的肝素组成分析方法相比,该方法几乎能检测LMWH完全降解产物中的所有组分。
[Abstract]:Heparin is a class of linear glycosaminoglycans with a large negative charge and heterogeneity. Heparin is a linear polysaccharide consisting of repeated disaccharide units. The repeated disaccharide units are linked by hexuronic acid (HexA) and D-glucosamine (G1cN) via 1-4 glycoside bonds. The hexuronic acid in the disaccharide unit may be p-D- glucuronic acid or a-L-Alduronic acid. Hexuronic acid may be 2-O- sulfated. Glucosamine may be 6-0-sulfated N-sulfated N-acetylation or 3-O- sulfated. Heparin is widely used as a clinical anticoagulant in the prevention and treatment of thrombotic diseases. However, heparin has some side effects as anticoagulant drugs. Such as bleeding, thrombocytopenia, osteoporosis and so on, which limits its clinical application. Low molecular weight heparin (LMWHs) is a new anticoagulant. LMWH prepared from heparin polysaccharide has the characteristics of low molecular weight, strong antithrombotic effect, high bioavailability and long plasma half-life. It is widely used in anticoagulant drugs. With the appearance of LMWH generic drugs, it is necessary to analyze the structure and composition of LMWH and identify the fine structure. We have developed a new method for the analysis of fine structure of LMWH based on RP-RPP-HPLCG-diode array detector (DADI-MS). In this paper, two kinds of low-molecular weight heparin (LMWH) have been used to analyze the fine structure of LMWH. Sodium and enoxaparin sodium were completely degraded by heparinase and then combined with 2-aminoacridine ketone AMAC by pre-column derivatization. Two kinds of low molecular weight heparin (LMWH) completely degraded components were analyzed by RP-HPLC-DAD-MS method. 17 fully degraded components and 15 completely degraded components of enoxaparin sodium were successfully isolated and identified. Through this method, the components can be separated efficiently, and the qualitative and relative quantitative analysis of all the components are realized. Except for 8 kinds of heparin disaccharide, the total degradation of heparin sodium and enoxaparin sodium can be achieved. They also contain characteristic terminal structures such as reductive and non-reductive terminal structures such as tetrasugar 3-Osulfate and triose, which have been accurately analyzed and identified according to the retention time of chromatography and mass charge ratio of mass mass spectrometry (MS), compared with traditional methods for the analysis of heparin composition. This method can be used to detect almost all the components in the completely degraded products of LMWH.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R973

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