Center for Research and Advanced Therapies. CITA-Alzheimer Foundation, Mikeletegi 71, 20009 Donostia-San Sebastian, Gipuzkoa, Spain.
Lipids & Liver Research Group, Department of Physiology, Faculty of Medicine and Nursing, University of the Basque Country (UPV/EHU), Barrio Sarriena s/n, 48940 Leioa, Bizkaia, Spain.
Talanta. 2019 Apr 1;195:619-627. doi: 10.1016/j.talanta.2018.11.101. Epub 2018 Nov 29.
The cerebrospinal fluid (CSF) lipidome is attracting increasing attention due to the importance of lipids in brain molecular signaling and their involvement in several neurological diseases. Different solvent systems have been used for the extraction of multiple lipid classes from CSF but no comparative study of the effectiveness of these protocols has been carried out. To optimize CSF lipid extraction for lipidomic measurements by untargeted ultra-high performance liquid chromatography - mass spectrometry, we evaluate and compare two sample preparation protocols, one involving protein precipitation with isopropanol (IPA) and other consisting of a liquid-liquid extraction with chloroform-methanol. For that purpose, human CSF from neurologically healthy and normolipidemic volunteers was used. The criteria established to compare these two methods were based on four critical aspects of sample preparation: simplicity, lipid coverage, reproducibility and recovery efficiencies. We found that both methods were highly reproducible techniques (>75% of the lipids with coefficient of variation (CV) <30%). In terms of recovery, the single-step IPA procedure yielded better values for most of the lipid classes and it was less toxic and simpler than the liquid-liquid extraction method. In relation to lipid coverage, variation in selectivity was observed between methods, providing evidence that IPA was more selective for polar lipids. Overall, IPA precipitation provides excellent results in terms of simplicity of execution, lipid coverage, reproducibility and recovery. We conclude that it is a choice procedure for large-scale, untargeted lipid profiling using UHPLC-MS in CSF analysis.
由于脂质在大脑分子信号转导中的重要性及其在几种神经疾病中的参与，脑脊液（CSF）脂质组学越来越受到关注。已经使用了不同的溶剂系统从 CSF 中提取多种脂质类，但尚未对这些方案的有效性进行比较研究。为了通过非靶向超高液相色谱-质谱法优化 CSF 脂质的脂质组学测量提取，我们评估并比较了两种样品制备方案，一种涉及异丙醇（IPA）的蛋白质沉淀，另一种涉及氯仿-甲醇的液-液萃取。为此，使用了来自神经健康和正常脂质志愿者的人 CSF。用于比较这两种方法的标准基于样品制备的四个关键方面：简单性、脂质覆盖率、重现性和回收率效率。我们发现这两种方法都是高度可重复的技术（>75%的脂质具有变异系数（CV）<30%）。就回收率而言，单步 IPA 程序对大多数脂质类的回收率更高，而且毒性和复杂性低于液-液萃取方法。关于脂质覆盖率，方法之间观察到选择性的变化，这表明 IPA 对极性脂质更具选择性。总体而言，IPA 沉淀在执行的简单性、脂质覆盖率、重现性和回收率方面提供了极好的结果。我们得出的结论是，它是使用 CSF 分析中的 UHPLC-MS 进行大规模、非靶向脂质分析的首选程序。
