Anal Chem. 2018 Nov 6;90(21):13073-13080. doi: 10.1021/acs.analchem.8b04124. Epub 2018 Oct 18.
The structural analysis of carbohydrates remains challenging mainly due to the lack of rapid analytical methods able to determine and quantitate glycosidic linkages between the diverse monosaccharides found in natural oligosaccharides and polysaccharides. In this research, we present the first liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method for the rapid and simultaneous relative quantitation of glycosidic linkages for oligosaccharide and polysaccharide characterization. The method developed employs ultrahigh-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC/QqQ-MS) analysis performed in multiple reaction monitoring (MRM) mode. A library of 22 glycosidic linkages was built using commercial oligosaccharide standards. Permethylation and hydrolysis conditions along with LC-MS/MS parameters were optimized resulting in a workflow requiring only 50 μg of substrate for the analysis. Samples were homogenized, permethylated, hydrolyzed, and then derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP) prior to analysis by UHPLC/MRM-MS. Separation by C18 reversed-phase UHPLC along with the simultaneous monitoring of derivatized terminal, linear, bisecting, and trisecting monosaccharide linkages by mass spectrometry is achieved within a 15 min run time. Reproducibility, efficacy, and robustness of the method was demonstrated with galactan ( Lupin) and polysaccharides within food such as whole carrots. The speed and specificity of the method enables its application toward the rapid glycosidic linkage analysis of oligosaccharides and polysaccharides.
碳水化合物的结构分析仍然具有挑战性，主要是因为缺乏能够快速分析和定量天然寡糖和多糖中不同单糖之间糖苷键的分析方法。在这项研究中，我们提出了第一个基于液相色谱-串联质谱（LC-MS/MS）的方法，用于快速同时相对定量寡糖和多糖特征的糖苷键。该方法采用超高效液相色谱与三重四极杆质谱（UHPLC/QqQ-MS）联用，在多反应监测（MRM）模式下进行分析。使用商业寡糖标准品建立了一个包含 22 个糖苷键的库。优化了全甲基化和水解条件以及 LC-MS/MS 参数，使该方法仅需 50 μg 底物即可进行分析。样品经均质化、全甲基化、水解和 1-苯基-3-甲基-5-吡唑啉酮（PMP）衍生化后，通过 C18 反相 UHPLC 进行分离，同时通过质谱监测衍生化的末端、线性、分支和三分叉单糖键。在 15 分钟的运行时间内实现了分离。该方法的重现性、有效性和稳健性通过食用中的半乳聚糖（羽扇豆）和多糖（如整个胡萝卜）得到了证明。该方法的速度和特异性使其能够应用于寡糖和多糖的快速糖苷键分析。
