Department of Analytical Chemistry, Faculty of Chemical Technology, University of Pardubice, Studentská 573, 53210 Pardubice, Czech Republic.
Department of Analytical Chemistry, Faculty of Chemical Technology, University of Pardubice, Studentská 573, 53210 Pardubice, Czech Republic.
J Chromatogr A. 2015 Jan 16;1377:85-91. doi: 10.1016/j.chroma.2014.12.023. Epub 2014 Dec 12.
A novel normal-phase (NP) ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC/MS) method is developed for a separation and quantitation of nonpolar lipid classes occurring in human plasma, erythrocytes and plasma lipoprotein fractions. The baseline class separation of cholesteryl esters (CE), cholesterol, triacylglycerols (TG), regioisomers of 1,2- and 1,3-diacylglycerols (DG) and 1-monoacylglycerols (1-MG) is achieved using an optimized hexane - 2-propanol-acetonitrile mobile phase within 18min for all nonpolar lipid classes or only 9min excluding monoacylglycerols not detected in studied samples. The determination of individual nonpolar lipid classes is performed by the response factor approach and the use of dioleoyl ethylene glycol as a single internal standard. Polar lipid classes, such as phosphatidylglycerols (PG), phosphatidylethanolamines (PE), phosphatidylcholines (PC), sphingomyelins (SM) and lysophosphatidylcholines (LPC), are separated by hydrophilic interaction liquid chromatography (HILIC) using 5mmol/L aqueous ammonium acetate-methanol-acetonitrile gradient within 13minutes. The quantitation of polar lipid classes is done by a similar approach as for nonpolar lipid classes, but a different internal standard (sphingosyl PE d17:1/12:0) is used. The complementary information on fatty acyl profiles after the transesterification of the total lipid extract is obtained by gas chromatography with flame ionization detection (GC/FID). The applicability of developed methodology for fast and comprehensive characterization of blood lipidome is illustrated on samples of human plasma, erythrocytes, high-density lipoprotein (HDL), low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) fractions.
一种新型的正相（NP）超高效液相色谱-质谱（UHPLC/MS）方法被开发出来，用于分离和定量人血浆、红细胞和血浆脂蛋白部分中存在的非极性脂质类别。使用优化的正己烷-2-丙醇-乙腈流动相，在 18 分钟内实现了胆固醇酯（CE）、胆固醇、三酰甘油（TG）、1,2-和 1,3-二酰甘油（DG）的区域异构体和 1-单酰甘油（1-MG）的基线分离，对于所有非极性脂质类别，或者仅排除在研究样品中未检测到的单酰甘油，时间为 9 分钟。通过响应因子法和使用二油酰基乙二醇作为单一内标来测定各个非极性脂质类别。使用亲水相互作用液相色谱（HILIC），在 13 分钟内，用 5mmol/L 水氨乙酸-甲醇-乙腈梯度分离极性脂质类别，如磷脂酰甘油（PG）、磷脂酰乙醇胺（PE）、磷脂酰胆碱（PC）、神经鞘磷脂（SM）和溶血磷脂酰胆碱（LPC）。通过类似的方法对极性脂质类别进行定量，但使用不同的内标（鞘氨醇 PE d17:1/12:0）。通过气相色谱-火焰离子化检测（GC/FID）获得总脂质提取物进行酯交换后的脂肪酸图谱的补充信息。所开发的方法学的适用性，用于快速和全面表征血液脂质组学，在人血浆、红细胞、高密度脂蛋白（HDL）、低密度脂蛋白（LDL）和极低密度脂蛋白（VLDL）部分的样品上得到了说明。
