Center for Systems Biology, University of Iceland, Sturlugata 8, Reykjavik, Iceland.
J Chromatogr B Analyt Technol Biomed Life Sci. 2012 Jun 1;898:111-20. doi: 10.1016/j.jchromb.2012.04.026. Epub 2012 Apr 30.
An extraction method for intracellular metabolite profiling should ideally be able to recover the broadest possible range of metabolites present in a sample. However, the development of such methods is hampered by the diversity of the physico-chemical properties of metabolites as well as by the specific characteristics of samples and cells. In this study, we report the optimization of an UPLC-MS method for the metabolite analysis of platelet samples. The optimal analytical protocol was determined by testing seven different extraction methods as well as by employing two different LC-MS methods, in which the metabolites were separated by using hydrophilic interaction liquid chromatography (HILIC) and reversed phase liquid chromatography (RPLC). The optimal conditions were selected using the coverage of the platelets' metabolome, the response of the identified metabolites, the reproducibility of the analytical method, and the time of the analysis as main evaluation criteria. Our results show that methanol-water (7:3) extraction coupled with HILIC-MS method provides the best compromise, allowing identification of 107 metabolites in a platelet cell extract sample, 91% of them with a RSD% lower than 20. A higher number of metabolites could be detected when analyzing the platelet samples with two different LC-MS methods or when using complementary extraction methods in parallel.
一种用于细胞内代谢物分析的提取方法，理想情况下应该能够从样品中尽可能广泛地回收存在的代谢物。然而，由于代谢物的物理化学性质的多样性以及样品和细胞的特定特征，此类方法的开发受到了阻碍。在本研究中，我们报告了一种优化的超高效液相色谱-质谱（UPLC-MS）方法，用于血小板样品的代谢物分析。通过测试七种不同的提取方法，并采用两种不同的 LC-MS 方法，即亲水相互作用液相色谱（HILIC）和反相液相色谱（RPLC）分离代谢物，确定了最佳分析方案。最佳条件的选择以血小板代谢组的覆盖范围、鉴定代谢物的响应、分析方法的重现性和分析时间为主要评价标准。我们的结果表明，甲醇-水（7:3）提取与 HILIC-MS 方法相结合提供了最佳的折衷方案，可在血小板细胞提取物样品中鉴定出 107 种代谢物，其中 91%的代谢物的相对标准偏差（RSD%）低于 20。当使用两种不同的 LC-MS 方法分析血小板样品或并行使用互补提取方法时，可以检测到更多的代谢物。
