A Comprehensive UHPLC Ion Mobility Quadrupole Time-of-Flight Method for Profiling and Quantification of Eicosanoids, Other Oxylipins, and Fatty Acids

Analysis
of oxylipins by liquid chromatography mass spectrometry
(LC/MS) is challenging because of the small mass range occupied by
this diverse lipid class, the presence of numerous structural isomers,
and their low abundance in biological samples. Although highly sensitive
LC/MS/MS methods are commonly used, further separation is achievable
by using drift tube ion mobility coupled with high-resolution mass
spectrometry (DTIM-MS). Herein, we present a combined analytical and
computational method for the identification of oxylipins and fatty
acids. We use a reversed-phase LC/DTIM-MS workflow able to profile
and quantify (based on chromatographic peak area) the oxylipin and
fatty acid content of biological samples while simultaneously acquiring
full scan and product ion spectra. The information regarding accurate
mass, collision-cross-section values in nitrogen (DTCCSN2), and retention times of the species found are compared
to an internal library of lipid standards as well as the LIPID MAPS
Structure Database by using specifically developed processing tools.
Features detected within the DTCCSN2 and m/z ranges of the analyzed standards are
flagged as oxylipin-like species, which can be further characterized
using drift-time alignment of product and precursor ions distinctive
of DTIM-MS. This not only helps identification by reducing the number
of annotations from LIPID MAPS but also guides discovery studies of
potentially novel species. Testing the methodology on Salmonella
enterica serovar Typhimurium-infected murine bone-marrow-derived
macrophages and thrombin activated human platelets yields results
in agreement with literature. This workflow has also annotated features
as potentially novel oxylipins, confirming its ability in providing
further insights into lipid analysis of biological samples.

采用液相色谱-质谱联用（LC/MS）技术对氧脂素（oxylipins）进行分析颇具挑战，原因在于这类多样化的脂质类群占据的质量范围较窄，存在大量结构异构体，且在生物样本中丰度较低。尽管目前常用高灵敏度的LC/MS/MS方法，但通过将漂移管离子迁移谱与高分辨质谱联用（DTIM-MS），可实现进一步的分离效果。本文报道了一种用于氧脂素与脂肪酸鉴定的分析与计算联用方法。本研究采用反相LC/DTIM-MS工作流程，可在同步采集全扫描谱与产物离子谱的同时，基于色谱峰面积对生物样本中的氧脂素与脂肪酸含量进行定性与定量分析。借助专门开发的处理工具，将所检测到的物质的精确质量、氮气环境下的碰撞截面值（DTCCS_{N2}）以及保留时间，与内置的脂质标准品库以及LIPID MAPS结构数据库进行比对。在分析标准品的DTCCS_{N2}与m/z范围内检测到的特征峰将被标记为类氧脂素物质，这类物质可通过DTIM-MS特有的产物离子与前驱离子的漂移时间对齐进行进一步表征。这不仅通过减少LIPID MAPS数据库的注释数量助力物质鉴定，还可为潜在新型物质的发现研究提供指引。将该方法应用于肠炎沙门氏菌血清型鼠伤寒沙门氏菌（Salmonella enterica serovar Typhimurium）感染的鼠源骨髓源性巨噬细胞以及凝血酶激活的人血小板样本中，所得结果与已有文献报道一致。该工作流程还将部分特征峰注释为潜在新型氧脂素，证实了其可为生物样本的脂质分析提供更深层次见解的能力。