National Glycoengineering Research Center, Shandong University, Jinan 250100, China.
Division of Chemical Metrology and Analytical Science, National Institute of Metrology, Beijing 100029, China, China.
Carbohydr Polym. 2018 Mar 1;183:81-90. doi: 10.1016/j.carbpol.2017.11.039. Epub 2017 Nov 13.
Low molecular weight heparins (LMWHs) are widely used anticoagulant drugs. The composition and sequence of LMWH oligosaccharides determine their safety and efficacy. The short oligosaccharide pool in LMWHs undergoes more depolymerization reactions than the longer chains and is the most sensitive indicator of the manufacturing process. Electrospray ionization tandem mass spectrometry (ESI-MS/MS) has been demonstrated as a powerful tool to sequence synthetic heparin oligosaccharide but never been applied to analyze complicated mixture like LMWHs. We established an offline strong anion exchange (SAX)-high performance liquid chromatography (HPLC) and ESI-MS/MS approach to sequence the short oligosaccharides of dalteparin sodium. With the help of in-house developed MS/MS interpretation software, the sequences of 18 representative species ranging from tetrasaccharide to octasaccharide were obtained. Interestingly, we found a novel 2,3-disulfated hexauronic acid structure and reconfirmed it by complementary heparinase digestion and LC-MS/MS analysis. This approach provides straightforward and in-depth insight to the structure of LMWHs and the reaction mechanism of heparin depolymerization.
低分子量肝素（LMWHs）是广泛使用的抗凝药物。LMWH 寡糖的组成和序列决定了它们的安全性和疗效。LMWHs 中短的寡糖池比长链经历更多的解聚反应，是制造过程最敏感的指标。电喷雾串联质谱（ESI-MS/MS）已被证明是一种对合成肝素寡糖进行测序的强大工具，但从未应用于分析像 LMWHs 这样复杂的混合物。我们建立了一种离线强阴离子交换（SAX）-高效液相色谱（HPLC）和 ESI-MS/MS 方法来对达肝素钠的短寡糖进行测序。借助内部开发的 MS/MS 解释软件，我们获得了 18 种代表性物质的序列，范围从四糖到八糖。有趣的是，我们发现了一种新型的 2,3-二硫酸化六糖结构，并通过补充肝素酶消化和 LC-MS/MS 分析对其进行了重新确认。该方法为 LMWHs 的结构和肝素解聚反应机制提供了直接而深入的见解。
