Novel reverse-phase ion pair-high performance liquid chromatography separation of heparin, heparan sulfate and low molecular weight-heparins disaccharides and oligosaccharides
Novel reverse-phase ion pair-high performance liquid chromatography separation of heparin, heparan sulfate and low molecular weight-heparins disaccharides and oligosaccharides
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DOI：
10.1016/j.chroma.2013.02.013
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发表时间：
2013-04-05
影响因子：
4.1
通讯作者：
Volpi, Nicola
中科院分区：
文献类型：
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作者：
Galeotti, Fabio;Volpi, Nicola
本研究采用硫酸四丁基铵作为离子配对试剂，分离出了所有由肝素酶作用产生的肝素/硫酸肝素双糖，以及在磺酰基氨基上具有3- o -硫酸盐基团且抗酶作用的主要肝素四糖。此外，这种新的高效液相色谱方法能够分离和定量低分子量肝素中不常见的双糖/低聚糖，这些低分子量肝素是由硝酸、达特帕林或苯基化、碱性水解、依诺肝素等化学处理产生的。此外，与传统的强阴离子交换高效液相色谱分离相比，该方法的灵敏度提高了4倍。这是通过在232 nm的普通紫外检测器获得的，避免了使用能够通过柱后衍生化增加灵敏度的复杂程序。最后，值得一提的是，HPLC和UV检测双糖/寡糖组成是质量控制实验室在提取和生产过程中评价肝素和低分子量肝素结构和质量的常用分析方法。这种简单的高效液相色谱方法为药物天然肝素及其衍生物的快速区分提供了高分辨率和灵敏度，也为生物来源的少量样品的几百纳克糖胺聚糖水平的成分分析提供了高分辨率和灵敏度。(C) 2013 Elsevier B.V.版权所有
In this study, by using tetrabutylammonium bisulfate as ion-pairing reagent, we were able to separate all the main heparin/heparan sulfate disaccharides generated by the action of heparinases along with the main Hep tetrasaccharide possessing a 3-O-sulfate group on the sulfoglucosamine unit and resistant to enzymatic action. Moreover, this novel HPLC method was able to separate and quantify uncommon disaccharides/oligosaccharides present in low molecular weight-heparins produced by chemical treatment with nitrous acid, dalteparin, or benzylation followed by alkaline hydrolysis, enoxaparin. Additionally, this procedure yields a sensitivity similar to 4-times higher compared to conventional strong-anion exchange-HPLC separation. This was obtained by a common UV detector at 232 nm avoiding the use of complex procedures capable of increasing sensitivity by post-column derivatization. Finally, it is worth mentioning that disaccharide/oligosaccharide composition by HPLC and UV detection is a common analytical approach in quality control laboratories to evaluate heparins and low molecular weight-heparins structure and quality during their extraction and production. This simple HPLC approach offers high resolution and sensitivity for the rapid differentiation of pharmaceutical native heparins and derivatives and for the compositional analysis of small amounts of samples derived from biological sources at a glycosaminoglycans level of a few hundred nanogram. (C) 2013 Elsevier B.V. All rights reserved.