Several oxylipins including hydroxy- and epoxy-polyunsaturated fatty acids act as lipid mediators. In biological samples they can be present as non-esterified form, but the major part occurs esterified in phospholipids (PL) or other lipids. Esterified oxylipins are quantified indirectly after alkaline hydrolysis as non-esterified oxylipins. However, in this indirect analysis the information in which lipid class oxylipins are bound is lost. In this work, an untargeted liquid chromatography high-resolution mass spectrometry (LC-HRMS) method for the direct analysis of PL bearing oxylipins was developed. Optimized reversed-phase LC separation achieved a sufficient separation of isobaric and isomeric PL from different lipid classes bearing oxylipin positional isomers. Individual PL species bearing oxylipins were identified based on retention time, precursor ion and characteristic product ions. The bound oxylipin could be characterized based on product ions resulting from the α-cleavage occurring at the hydroxy/epoxy group. PL -1/-2 isomers were identified based on the neutral loss of the fatty acyl in the -2 position. A total of 422 individual oxPL species from 7 different lipid classes i.e., PI, PS, PC, PE, PC-P, PC-O, and PE-P were detected in human serum and cells. This method enabled to determine in which PL class supplemented oxylipins are incorporated in HEK293 cells: 20:4;15OH, 20:4;14Ep, and 20:5;14Ep were mostly bound to PI. 20:4;8Ep and 20:5;8Ep were esterified to PC and PE while other oxylipins were mainly found in PC. The developed LC-HRMS method enables the comprehensive detection as well as the semi-quantification of isobaric and isomeric PL species bearing oxylipins. With this method, we show that the position of the oxidation has a great impact and directs the incorporation of oxylipins into the different PL classes in human cells.

中文翻译：

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含氧磷脂的磷脂的 LC-HRMS 分析


包括羟基和环氧多不饱和脂肪酸在内的几种氧脂素充当脂质介质。在生物样品中，它们可以以非酯化形式存在，但大部分以磷脂 (PL) 或其他脂质的酯化形式存在。酯化氧脂质在碱性​​水解后间接定量为非酯化氧脂质。然而，在这种间接分析中，脂质类氧脂质结合的信息丢失了。在这项工作中，开发了一种用于直接分析带有 PL 的氧脂质的非靶向液相色谱高分辨率质谱 (LC-HRMS) 方法。优化的反相 LC 分离实现了同量异位和异构 PL 与带有氧脂质位置异构体的不同脂质类别的充分分离。根据保留时间、前体离子和特征产物离子，鉴定了带有氧脂素的各个 PL 种类。结合的氧脂质可以根据羟基/环氧基处发生的 α-裂解产生的产物离子来表征。 PL -1/-2 异构体是根据-2 位脂肪酰基的中性缺失来鉴定的。在人血清和细胞中检测到来自 7 种不同脂质类别（即 PI、PS、PC、PE、PC-P、PC-O 和 PE-P）的总共 422 种 oxPL 物种。该方法能够确定 HEK293 细胞中掺入了哪种 PL 类补充氧脂质：20:4;15OH、20:4;14Ep 和 20:5;14Ep 大多数与 PI 结合。 20:4;8Ep和20:5;8Ep被酯化为PC和PE，而其他氧脂质主要存在于PC中。开发的 LC-HRMS 方法能够对含有氧脂素的同量异位和同分异构 PL 物质进行全面检测和半定量。 通过这种方法，我们表明氧化的位置具有很大的影响，并指导氧脂质掺入人体细胞中的不同 PL 类别。