Off-line mixed-mode liquid chromatography coupled with reversed phase high performance liquid chromatography-high resolution mass spectrometry to improve coverage in lipidomics analysis

Abstract

The confident identification and in-depth profiling of molecular lipid species remain to be a challenge in lipidomics analysis. In this work, an off-line two-dimensional mixed-mode and reversed-phase liquid chromatography (RPLC) method combined with high-field quadrupole orbitrap mass spectrometer (Q Exactive HF) was developed to profile lipids from complex biological samples. In the first dimension, 22 different lipid classes were separated on a monolithic silica column with elution order from neutral to polar lipids. A total of 13 fractions were collected and run on a RPLC C30 column in the second dimension for further separation of the lipid molecular species based on their hydrophobicity, with the elution order being determined by both the length and degree of unsaturation in the fatty-acyl chain. The method was applied to analyze lipids extracted from rat plasma and rat liver. Fatty acid methyl ester analysis by gas chromatography-mass spectrometry was used to identify the fatty acyls from total lipid extracts, which provided a more confident identification of the lipid species present in these samples. More than 800 lipids were identified in each sample and their molecular structures were confidentially confirmed using tandem mass spectrometry (MS/MS). The number of lipid molecular species identified in both rat plasma and rat liver by this off-line two-dimensional method is approximately twice of that by one-dimensional RPLC-MS/MS employing a C30 column. This off-line two-dimensional mixed-mode LC-RPLC-MS/MS method is a promising technique for comprehensive lipid profiling in complex biological matrices.

Evaluation of LC-MS and LC×LC-MS in analysis of zebrafish embryo samples for comprehensive lipid profiling

“…It has been reported by Wang et al [28] that 372 lipids in plasma were identified by a stop-flow two-dimensional LC-MS method within 130 min, while the number of lipids identified was 284 using 1 D RPLC analysis. Narváez-Rivas et al [44] identified about 800 lipid species in rat plasma and rat liver by an offline two-dimensional mix-mode LC-RPLC-MS/ MS method about 7 h, and approximate 400 lipids by RPLC-MS method within 31 min [17]. However, in this study, there were over 1000 lipid species annotated using the HILIC×RPLC compared with RPLC-MS of about 400 annotated lipids.…”

Common cases of improper lipid annotation using high-resolution tandem mass spectrometry data and corresponding limitations in biological interpretation

“…Despite the proposed shorthand notation, there has been a mix of annotation styles present in literature. For example, of the lipidomics research articles published in 2017 determined on Science Direct (accessed 02/09/2017), 3 articles [29,32,33] incorrectly used “/”, 3 articles [34–36] used “-”, and1article [37] used “_” between fatty acyl constituents, when positional isomers were not identified. One potential source of confusion in annotating lipids is that current lipid identification software, for example LipidSearch, MS-DIAL [38], LipidBlast [39], and Greazy [40] all employ “/”when fatty acyl positions are not known.…”

Comprehensive analysis of oxylipins in human plasma using reversed-phase liquid chromatography-triple quadrupole mass spectrometry with heatmap-assisted selection of transitions

“…Most lipid mediator-related studies employ C18-based columns to perform the separation; however, C30 column also showed great potential in separation of lipid molecular species [32,33] and could be used as an alternative for analyzing oxylipins. We compared the chromatographic performances of core-shell C30 and HSS T3 C18 column.…”