Ikegami Tohru, Horie Kanta, Saad Nabil, Hosoya Ken, Fiehn Oliver, Tanaka Nobuo
Department of Biomolecular Engineering, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan.
Anal Bioanal Chem. 2008 Aug;391(7):2533-42. doi: 10.1007/s00216-008-2060-6. Epub 2008 Apr 16.
A polyacrylamide (PAAm)-modified monolithic silica capillary column of increased phase ratio, 200T-PAAm, for hydrophilic interaction liquid chromatography (HILIC) was prepared. The column showed high separation efficiency, with a theoretical plate height H = 7-20 microm at a linear velocity, u = 1-7 mm/s. From a kinetic plot analysis, it was expected that the monolithic column could provide three times faster separation than particle-packed HILIC columns under a pressure limit at 20 MPa. HILIC coupled with electrospray ionization (ESI)-mass spectrometry (HILIC-ESI-MS) using the 200T-PAAm column was employed for the analysis of underivatized carbohydrates to achieve fast and efficient separations of mixtures containing mono-, di-, and trisaccharides within 5 min. Under single MS full scan mode, 200 pg of oligosaccharides was detected by the system. The limit of detection (LOD) of the LC-ESI-MS/MS system was determined using selected reaction monitoring (SRM) to be as low as 3.2 ng/mL (attomol level) for nonreducing saccharides. The system was successfully applied to the detection of disaccharides in extracts of plant, such as corn, soybean, and Arabidopsis thaliana.
制备了一种用于亲水作用液相色谱（HILIC）的具有更高相比的聚丙烯酰胺（PAAm）修饰整体硅胶毛细管柱，即200T - PAAm柱。该柱显示出高分离效率，在1 - 7 mm/s的线速度u下，理论塔板高度H = 7 - 20微米。通过动力学曲线分析，预计在20 MPa的压力限制下，整体柱的分离速度比填充颗粒的HILIC柱快三倍。使用200T - PAAm柱的HILIC与电喷雾电离（ESI）-质谱联用（HILIC - ESI - MS）用于分析未衍生化的碳水化合物，以在5分钟内实现对含有单糖、二糖和三糖的混合物的快速高效分离。在单MS全扫描模式下，系统检测到200 pg的寡糖。使用选择反应监测（SRM）确定LC - ESI - MS/MS系统对非还原糖的检测限（LOD）低至3.2 ng/mL（阿托摩尔水平）。该系统成功应用于检测植物提取物中的二糖，如玉米、大豆和拟南芥中的二糖。
