Simultaneous non-polar and polar lipid analysis by on-line combination of HILIC, RP and high resolution MS

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.

The Power of LC-MS Based Multiomics: Exploring Adipogenic Differentiation of Human Mesenchymal Stem/Stromal Cells

“…For targeted analysis, we employed isotopically 13 C-labeled metabolite and lipid standards produced in our laboratory for standardization [15,30,31] to follow fat cell differentiation of MSCs. Absolute compound-specific quantification (n = 3) of 12 lipids and 54 metabolites (compound- specific quantification using endogenous/ 13 C-labeled pairs) was performed using external calibration (8 standards within the calibration range of 0.1–25 µM), with internal standardization by in-house produced 13 C-labeled standards and protein normalization.…”

Shared reference materials harmonize lipidomics across MS-based detection platforms and laboratories

“…HILIC, similar to (38), was performed on a Waters BEH HILIC (100 × 2.1 mm; particle size, 1.7 μm), thermostatted to 40°C in a Thermo Vanquish UHPLC system. Mobile phase A was 40 mM aqueous ammonium formate, adjusted to pH 4 with formic acid, and mobile phase B was acetonitrile.…”

Merging metabolomics and lipidomics into one analytical run

“…[20][21][22][23] Rampler et al recently introduced an on-line combination of HILIC and reversed phase chromatography for increased coverage of hydrophilic and hydrophobic lipids. 24 However, the majority of in-depth lipid profiling is carried out by applying reversed phase chromatography high-resolution mass spectrometry (HRMS) only using run times of 1-2 hours. 4,6 To date, comprehensive two-dimensional separations are not widely spread in both fields.…”