背景:非靶向代谢组学研究有望涵盖具有高度化学多样性和复杂性的广泛化合物类别。因此,优化(预)分析参数,如分析液相色谱(LC)柱是至关重要的,柱的选择主要取决于研究目的。
目的:本研究旨在比较六种不同的分析柱。首先,通过比较所选化合物的色谱分辨率。第二,关于使用汇集的人类肝微粒体(pHLM)的非靶向毒性代谢组学研究的结果,大鼠血浆,和大鼠尿液作为基质。
方法:使用三种不同的反相(苯基-己基,BEHC18和GoldC18),两种亲水相互作用色谱(HILIC)(磺酸铵和磺基甜菜碱),和一个多孔石墨碳(PGC)柱耦合到高分辨率质谱(HRMS)。它们的影响是根据色谱柱性能和特征计数的大小进行评估的,在其他人中。
结果:所有三个反相柱均显示出相似的性能,而PGC柱至少在极性化合物方面优于两个HILIC柱。比较所有数据集的特征计数的大小,使用苯基-己基或磺基甜菜碱柱检测到大多数特征。考虑到矩阵,使用磺基甜菜碱后在尿液和pHLM中以及使用铵磺酸柱后在血浆中检测到最重要的特征。
结论:结果强调,这种非靶向的毒性代谢组学研究LC-HRMS代谢组学研究的结果受到分析柱的高度影响,苯基-己基或磺基甜菜碱柱是最合适的。然而,柱的选择也可以取决于所研究的化合物以及所研究的基质。
BACKGROUND: Untargeted metabolomics studies are expected to cover a wide range of compound classes with high chemical diversity and complexity. Thus, optimizing (pre-)analytical parameters such as the analytical liquid chromatography (LC) column is crucial and the selection of the column depends primarily on the
study purpose.
OBJECTIVE: The current investigation aimed to compare six different analytical columns. First, by comparing the chromatographic resolution of selected compounds. Second, on the outcome of an untargeted toxicometabolomics
study using pooled human liver microsomes (pHLM), rat plasma, and rat urine as matrices.
METHODS: Separation and analysis were performed using three different reversed-phase (Phenyl-Hexyl, BEH C18, and Gold C18), two hydrophilic interaction chromatography (HILIC) (ammonium-sulfonic acid and sulfobetaine), and one porous graphitic carbon (PGC) columns coupled to high-resolution mass spectrometry (HRMS). Their impact was evaluated based on the column performance and the size of feature count, amongst others.
RESULTS: All three reversed-phase columns showed a similar performance, whereas the PGC column was superior to both HILIC columns at least for polar compounds. Comparing the size of feature count across all datasets, most features were detected using the Phenyl-Hexyl or sulfobetaine column. Considering the matrices, most significant features were detected in urine and pHLM after using the sulfobetaine and in plasma after using the ammonium-sulfonic acid column.
CONCLUSIONS: The results underline that the outcome of this untargeted toxicometabolomic
study LC-HRMS metabolomic
study was highly influenced by the analytical column, with the Phenyl-Hexyl or sulfobetaine column being the most suitable. However, column selection may also depend on the investigated compounds as well as on the investigated matrix.