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

Abstract

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 (DT CCSN2) 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 DT CCSN2 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.

Methods of the Analysis of Oxylipins in Biological Samples

“…This approach has been successfully used by Kyle et al [104] for separation of 42 isomer pairs or groups in either positive or negative mode, for example R and S isomers of HETEs; better separation was obtained for sodiated [M + Na] + ions rather than deprotonated species [M − H] − . A variation of IMS was also used by Hinz et al [105]. In this study ions were travelling through drift tube filled with low pressure nitrogen gas in which their drift time was affected by ion size and shape, which allowed for formation of different oxylipin conformers.…”

“…In this study ions were travelling through drift tube filled with low pressure nitrogen gas in which their drift time was affected by ion size and shape, which allowed for formation of different oxylipin conformers. However, the use of IMS requires building additional CSS libraries, use of either computationally generated standards, or literature CCS [105], which complicates implementation of this method.…”

Lipidomics from sample preparation to data analysis: a primer

“…For readers interested in the actual IMS workflows, Paglia et al provide very detailed protocols on various IMS-coupled mass spectrometry methods [69]. Another good example of the integration of IMS into LC-coupled systems is shown by Hinz et al, who separated oxylipins and fatty acids using both chromatography and IMS [70]. Since oxylipins show a particularly high degree of isomerism, the acquisition of drift times can be a helpful tool which may even enable the positions of hydroxy groups on the fatty acyl chain to be determined.…”

Non-targeted and targeted analysis of oxylipins in combination with charge-switch derivatization by ion mobility high-resolution mass spectrometry

“…Hinz et al [ 27 ] developed an analytical LC-IM-QTOF-MS method allowing the profiling and quantification of the oxylipin content in biological samples. Using ion mobility spectrometry, they were able to identify 57 features using an internal library of standards and quantify 16 analytes in thrombin-activated human platelets [ 30 ]. However, the application of ESI in the negative mode is in contradiction with the use of acidified eluents needed for the separation of oxylipin mixtures, thereby limiting the generation of negatively charged ions and ultimately reducing the sensitivity of the method.…”

“…Still, the low concentrations as well as the large number of structurally similar molecules, including regioisomers, constitute a challenge for the separation and quantification of oxylipins [5,7]. In recent years, ion mobility quadrupole time-of-flight mass spectrometry (IM-QTOF-MS) has evolved as a new tool for non-targeted analysis [29][30][31]. Ion mobility is offering an additional separation dimension and enhances the separation of isomers.…”