Southam Andrew D, Haglington Liam D, Najdekr Lukáš, Jankevics Andris, Weber Ralf J M, Dunn Warwick B
School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.
Analyst. 2020 Oct 21;145(20):6511-6523. doi: 10.1039/d0an01319f. Epub 2020 Aug 6.
Clinical metabolic phenotyping employs metabolomics and lipidomics to detect and measure hundreds to thousands of metabolites and lipids within human samples. This approach aims to identify metabolite and lipid changes between phenotypes (e.g. disease status) that aid understanding of biochemical mechanisms driving the phenotype. Sample preparation is a critical step in clinical metabolic phenotyping: it must be reproducible and give a high extraction yield of metabolites and lipids, and in high-throughput studies it needs to be rapid. Here, we assessed the extraction of polar metabolites from human urine and polar metabolites and lipids from human plasma for analysis by ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) metabolomics and lipidomics. We evaluated several monophasic (urine and plasma) and biphasic (plasma) extractions, and we also tested alterations to (a) solvent-biofluid incubation time and temperature during monophasic extraction, and (b) phase partitioning time during biphasic extraction. Extracts were analysed by three UHPLC-MS assays: (i) hydrophilic interaction chromatography (HILIC) for urine and plasma, (ii) C aqueous reversed phase for urine, and (iii) C reversed phase for plasma lipids, and the yield and reproducibility of each method was assessed. We measured UHPLC-MS injection reproducibility as well as sample preparation reproducibility to assess sample solvent composition compatibility with UHPLC-MS and to pinpoint the origin of variance within the methods. For HILIC UHPLC-MS plasma and urine analysis, monophasic 50 : 50 methanol : acetonitrile had the most detected putatively-identified polar metabolites with high method reproducibility. This method had the highest lipid yield for plasma extracts analysed by the HILIC method. If lipid removal from the plasma polar HILIC extract is required, then the biphasic methanol/chloroform/water method is recommended. For C (aqueous) UHPLC-MS urine analysis, 50 : 50 methanol : water had high reproducibility and yield. For C UHPLC-MS plasma lipidomics, monophasic 100% isopropanol had the highest detection response of all annotated lipid classes with high reproducibility. Increasing monophasic incubation time and temperature had little benefit on metabolite and lipid yield and reproducibility for all methods.
临床代谢表型分析采用代谢组学和脂质组学技术来检测和测量人体样本中的数百至数千种代谢物和脂质。这种方法旨在识别不同表型（如疾病状态）之间的代谢物和脂质变化，以帮助理解驱动该表型的生化机制。样本制备是临床代谢表型分析中的关键步骤：它必须具有可重复性，能够高效提取代谢物和脂质，并且在高通量研究中需要快速完成。在此，我们评估了从人尿液中提取极性代谢物以及从人血浆中提取极性代谢物和脂质的方法，以便通过超高效液相色谱 - 质谱联用（UHPLC - MS）代谢组学和脂质组学进行分析。我们评估了几种单相（尿液和血浆）和双相（血浆）提取方法，还测试了（a）单相提取过程中溶剂与生物流体的孵育时间和温度，以及（b）双相提取过程中的相分离时间的变化。提取物通过三种UHPLC - MS分析方法进行分析：（i）用于尿液和血浆的亲水相互作用色谱（HILIC），（ii）用于尿液的C18水相反相色谱，以及（iii）用于血浆脂质的C18反相色谱，并评估了每种方法的提取率和可重复性。我们测量了UHPLC - MS进样的可重复性以及样本制备的可重复性，以评估样本溶剂组成与UHPLC - MS的兼容性，并确定方法内差异的来源。对于HILIC UHPLC - MS血浆和尿液分析，单相50∶50甲醇∶乙腈检测到的推定鉴定的极性代谢物最多，且方法重现性高。该方法对通过HILIC方法分析的血浆提取物的脂质提取率最高。如果需要从血浆极性HILIC提取物中去除脂质，推荐使用双相甲醇/氯仿/水方法。对于C18（水相）UHPLC - MS尿液分析，50∶50甲醇∶水具有高重现性和提取率。对于C18 UHPLC - MS血浆脂质组学，单相100%异丙醇对所有注释脂质类别的检测响应最高，且重现性高。增加单相孵育时间和温度对所有方法的代谢物和脂质提取率及重现性几乎没有益处。
