Institute of Chemistry of Renewable Resources, Department of Chemistry, University of Natural Resources and Life Sciences Vienna, Konrad-Lorenz-Straße 24, 3430 Tulln an der Donau, Austria; Department of Marine, Faculty of Fisheries and Marine, Universitas Airlangga, Mulyorejo, Surabaya 60115, Indonesia.
Institute of Food Chemistry, Faculty of Life Science, Technische Universität Braunschweig, Schleinitzstr. 20, 38106 Braunschweig, Germany.
Carbohydr Polym. 2024 Oct 15;342:122302. doi: 10.1016/j.carbpol.2024.122302. Epub 2024 May 31.
Methylation followed by depolymerization and gas chromatography (GC) is an effective methodology for the linkage analysis of polysaccharides, including fucoidan, a sulphated algal polysaccharide. However, this sample material demands attention to experimental details to prevent aberrations in the analytical result. The use of deficient bases for methylation, the presence of water, analyte degradation during hydrolysis, and coelution of the target analytes during gas chromatography create doubts about published results. We therefore investigated critical parameters of the method and carefully optimized the steps of the protocol to ensure the integrity of the results for the fucose monomers. Fucoidan from Cladosiphon okamuranus was used as reference sample to determine the glycosidic bonds, and sulphate positions in the monomer. Fucoidan in protonated form was methylated in a strictly water-free environment using lithium dimsyl as base and methyl iodide for methylation. The methylated polymer was isolated by solid phase extraction, which was crucial to recover also the highly sulfated fraction. Hydrolysis was conducted with trifluoroacetic acid. To separate all target analytes in GC-FID/MS, a stationary phase with high cyanopropyl content (HP-88) was required, as the commonly employed phenyl siloxane phases result in co-elution, which distorts the result severely.
甲基化后解聚和气相色谱（GC）是一种有效的多糖连接分析方法，包括褐藻糖胶，一种硫酸化海藻多糖。然而，这种样品材料需要注意实验细节，以防止分析结果出现偏差。甲基化使用不充分的碱基、水的存在、水解过程中分析物的降解以及气相色谱中目标分析物的共洗脱都会对已发表的结果产生怀疑。因此，我们研究了该方法的关键参数，并仔细优化了该方案的步骤，以确保结果的完整性。我们使用 Okamuranus 角叉菜的褐藻糖胶作为参考样品来确定糖苷键和单体中的硫酸盐位置。在严格无水的环境中，使用亚甲基锂作为碱和碘甲烷对质子化形式的褐藻糖胶进行甲基化。将甲基化聚合物通过固相萃取进行分离，这对于回收高度硫酸化的部分至关重要。使用三氟乙酸进行水解。为了在 GC-FID/MS 中分离所有目标分析物，需要使用具有高氰丙基含量（HP-88）的固定相，因为常用的苯基硅氧烷相导致共洗脱，这会严重扭曲结果。
