Strathclyde Institute of Pharmacy and Biomedical Sciences, 161, Cathedral Street, Glasgow, Scotland, G4 0RE, UK; Institute of Clinical Pharmacology, Guangzhou University of Chinese Medicine, No. 12 Jichang Road, Guangzhou, 510405 China.
Strathclyde Institute of Pharmacy and Biomedical Sciences, 161, Cathedral Street, Glasgow, Scotland, G4 0RE, UK.
J Chromatogr A. 2014 Oct 3;1362:168-79. doi: 10.1016/j.chroma.2014.08.039. Epub 2014 Aug 17.
It has been reported that HILIC column chemistry has a great effect on the number of detected metabolites in LC-HRMS-based untargeted metabolite profiling studies. However, no systematic investigation has been carried out with regard to the optimisation of mobile phase characteristics. In this study using 223 metabolite standards, we explored the retention mechanisms on three zwitterionic columns with varied mobile phase composition, demonstrated the interference from poor chromatographic peak shapes on the output of data extraction, and assessed the quality of chromatographic signals and the separation of isomers under each LC condition. As expected, on the ZIC-cHILIC column the acidic metabolites showed improved chromatographic performance at low pH which can be attributed to the opposite arrangement of the permanently charged groups on this column in comparison with the ZIC-HILIC column. Using extracts from the protozoan parasite Leishmania, we compared the numbers of repeatedly detected LC-HRMS features under different LC conditions with putative identification of metabolites not amongst the standards being based on accurate mass (±3ppm). Besides column chemistry, the pH of the mobile phase plays a key role in not only determining the retention mechanisms of solutes but also the output of the LC-HRMS data processing. Fast evaporation of ammonium carbonate produced less ion suppression in ESI source and consequently improved the detectability of the metabolites in low abundance in comparison with other ammonium salts. Our results show that the combination of a ZIC-pHILIC column with an ammonium carbonate mobile phase, pH 9.2, at 20mM in the aqueous phase or 10mM in both aqueous and organic mobile phase components, provided the most suitable LC conditions for LC-HRMS-based untargeted metabolite profiling of Leishmania parasite extracts. The signal reliability of the mass spectrometer used in this study (Exactive Orbitrap) was also investigated.
据报道，亲水作用色谱柱化学对基于 LC-HRMS 的非靶向代谢组学研究中检测到的代谢物数量有很大影响。然而，对于优化流动相特性还没有进行系统的研究。在这项使用 223 种代谢物标准品的研究中，我们探索了三种带有不同流动相组成的两性离子柱上的保留机制，展示了色谱峰形状不佳对数据提取结果的干扰，并评估了每种 LC 条件下色谱信号的质量和异构体的分离情况。正如预期的那样，在 ZIC-cHILIC 柱上，在低 pH 值下，酸性代谢物的色谱性能得到了改善，这可以归因于与 ZIC-HILIC 柱相比，该柱上的永久带电基团的排列相反。使用原生动物寄生虫利什曼原虫的提取物，我们比较了不同 LC 条件下重复检测到的 LC-HRMS 特征的数量，并根据精确质量（±3ppm）对未包含在标准品中的代谢物进行了假定鉴定。除了色谱柱化学，流动相的 pH 值不仅在决定溶质的保留机制方面起着关键作用，而且在 LC-HRMS 数据处理的结果中也起着关键作用。碳酸铵的快速蒸发在电喷雾源中产生的离子抑制作用较小，因此与其他铵盐相比，提高了低丰度代谢物的检测能力。我们的结果表明，ZIC-pHILIC 柱与碳酸铵流动相（pH 9.2，水相 20mM 或水相和有机相成分各 10mM）的组合为基于 LC-HRMS 的利什曼原虫提取物非靶向代谢组学研究提供了最合适的 LC 条件。本研究中使用的质谱仪（Exactive Orbitrap）的信号可靠性也进行了研究。
