Division of Metabolic and Nutritional Medicine, Dr. von Hauner Children's Hospital, University of Munich, Medical Center, Munich, Germany.
J Chromatogr B Analyt Technol Biomed Life Sci. 2011 Nov 15;879(30):3556-64. doi: 10.1016/j.jchromb.2011.09.043. Epub 2011 Sep 29.
The relationship between lipid status and metabolism, infant development and health has widely been studied, but the importance of individual glycerophospholipid species for biological functions in infants has hardly been considered. We developed a method for quantitative analyses of plasma glycerophospholipids from small sample volume. Proteins were precipitated with methanol, which eliminated further sample preparation. The supernatant was analysed by reversed-phase HPLC using a gradient of water, methanol and isopropanol as mobile phase. Electrospray ionisation in negative mode in combination with tandem mass spectrometry enabled detection of specific fatty acids as fragments of glycerophospholipid species. With this combination of chromatography and mass spectrometry, PC, lyso-PC, PE and lyso-PE species and their relevant isobaric compounds were quantified. Method validation showed a linear working range between 0.05 μmol/L and 10 μmol/L in diluted plasma samples. The intra-assay coefficients of variation (n=6) ranged from 1.1% to 13.9%. Results were comparable with data of the human metabolome database and gas chromatographic fatty acid analyses. All quantitatively important PE and PC species are covered. The method can be applied for investigating dietary effects on plasma GP composition from small plasma volumes.
脂质状态与代谢、婴儿发育和健康之间的关系已被广泛研究，但个体甘油磷脂种类对婴儿生物学功能的重要性几乎没有被考虑。我们开发了一种从小体积样本中定量分析血浆甘油磷脂的方法。用甲醇沉淀蛋白质，从而省去了进一步的样品制备步骤。用反相高效液相色谱法，以水、甲醇和异丙醇的梯度作为流动相，对上清液进行分析。电喷雾离子化在负离子模式下与串联质谱联用，使甘油磷脂种类的特定脂肪酸作为片段得以检测。通过这种色谱和质谱的组合，可以定量分析 PC、溶血磷脂酰胆碱、PE 和溶血磷脂酰乙醇胺及其相关等排化合物。方法验证表明，在稀释后的血浆样品中，线性工作范围在 0.05 μmol/L 至 10 μmol/L 之间。在 6 次重复实验中，日内变异系数（n=6）的范围为 1.1%至 13.9%。结果与人类代谢组数据库和气相色谱脂肪酸分析的数据相媲美。涵盖了所有定量上重要的 PE 和 PC 种类。该方法可应用于从小体积血浆中研究饮食对血浆 GP 组成的影响。
