Abstract
Given the chemical diversity of lipids and their biological relevance, suitable methods for lipid profiling and quantification are demanded to reduce sample complexity and analysis times. In this work, we present a novel on-line chromatographic method coupling hydrophilic interaction liquid chromatography (HILIC) dedicated to class-specific separation of polar lipid to reversed-phase chromatography (RP) for non-polar lipid analysis. More specifically, the void volume of the HILIC separation-consisting of non-polar lipids- is transferred to the orthogonal RP column enabling the on-line combination of HILIC with RP without any dilution in the second dimension. In this setup the orthogonal HILIC and RP separations were performed in parallel and the effluents of both columns were combined prior to high-resolution MS detection, offering the full separation space in one analytical run. Rapid separation for both polar and non-polar lipids within only 15 min (including reequilibration time) was enabled using sub-2 μm particles and UHPLC. The method proved to be robust with excellent retention time stability (RSDs < 1%) and LODs in the fmol to pmol (absolute on column) range even in the presence of complex biological matrix such as human plasma. The presented high-resolution LC-MS/MS method leads to class-specific separation of polar lipids and separation of non-polar lipids which is lost in conventional HILIC separations. HILIC-RP-MS is a promising tool for targeted and untargeted lipidomics workflows as three interesting features are combined namely (1) the decreased run time of state of the art shotgun MS methods, (2) the elevated linear dynamic range inherent to chromatographic separation and (3) increased level of identification by separation of polar and non-polar lipid classes.
Highlights
Lipids can be classified into categories by their chemical and structural similarity,[1] they can be grouped into polar and nonpolar lipids based on their overall hydrophobicity or categorized by their molecular building blocks.[2]
We present a novel strategy for polar and non-polar lipid analysis based on an on-line combination of hydrophilic interaction liquid chromatography (HILIC) and reversed-phase chromatography (RP) chromatography to enable HILIC-RP-High-resolution mass spectrometry (HRMS)
A C18 column (Acquity UHPLC HSS T3) with sub-2 μm particle and UHPLC capabilities was chosen with a commonly used eluent system for lipids consisting of isopropanol, acetonitrile, water, ammonium formate and formic acid.[14,22,27,34]
Summary
Lipids can be classified into categories by their chemical and structural similarity,[1] they can be grouped into polar and nonpolar lipids based on their overall hydrophobicity or categorized by their molecular building blocks.[2]. Class-specific coelution of all lipids occurs in HILIC and was successfully applied for polar lipid analysis e.g. glycerophospholipids, glycosphingolipids and phosphosphingolipids.[16,23,28,30,31,32] HILIC and RP chromatography as well as their combinations proved to be extremely powerful for lipid analysis in many previous studies.[13,14,22,27,28,30,31,32,33,34,35,36] With the advent of the UHPLC technology and the downscaling of chromatographic filling material by sub-2 μm particles or fused-core particles, increased plate numbers and enhanced chromatographic speed are enabled.[37,38,39,40,41] implementation of orthogonal RP and HILIC into 2D approaches is challenging due to incompatible eluent systems Solutions for this problem are (1) dilution of the incompatible mobile phase, (2) use of a trapping column (3) minimizing the transferred volume between both dimensions or (4) offline combinations of HILIC and RP.[21,28,32,36,42,43,44,45] In this work a novel UHPLC based workflow for the direct combination of a short HILIC column (sub[2] μm) in the first dimension with a RP column (sub-2 μm) in the second dimension is tested for the simultaneous analysis of polar and non-polar lipids. The HILIC-RP-HRMS workflow is tested for untargeted and targeted lipidomics applications
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