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-质谱联用法（LC/MS）分析氧脂素（oxylipins）颇具挑战，原因在于这一多样的脂质家族占据的质量范围狭窄、存在大量结构异构体，且在生物样本中丰度极低。尽管高灵敏度的液相色谱-串联质谱（LC/MS/MS）方法应用广泛，但借助漂移管离子迁移谱与高分辨质谱联用（DTIM-MS）技术可实现进一步分离。本研究提出了一种用于氧脂素与脂肪酸鉴定的分析与计算联用方法。我们采用反相液相色谱-漂移管离子迁移谱-高分辨质谱联用（RP-LC/DTIM-MS）工作流程，可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，同时获取全扫描谱图与产物离子谱图。借助自主开发的处理工具，我们将检测到的物质的精确质量、氮气中碰撞截面积（DTCCSN2）以及保留时间等信息，与内部脂质标准品数据库以及脂质图谱结构数据库（LIPID MAPS Structure Database）进行比对。在分析标准品的DTCCSN2与质荷比（m/z）范围内检测到的特征信号将被标记为类氧脂素物质，可通过DTIM-MS特有的产物离子与前体离子漂移时间对齐技术进行进一步表征。此举不仅可通过减少脂质图谱结构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导。将该方法应用于肠炎沙门氏菌鼠伤寒血清型感染的小鼠骨髓源巨噬细胞以及凝血酶激活的人血小板样本中，所得结果与文献报道一致。该工作流程还将部分特征信号注释为潜在新型氧脂素，证实了其可为生物样本脂质分析提供更深层次见解的能力。