LC-ESI-HRMS - lipidomics of phospholipids – Characterization of extraction, separation and detection parameters

29 August 2023, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


Lipids are a diverse class of molecules involved in all biological functions including cell signaling or cell membrane assembly. Owing to this relevance, LC-MS/MS based lipidomics emerged as a major field in modern analytical chemistry. Here, we thoroughly characterized the influence of MS and LC settings – of a Q Exactive HF operated in Full MS/data-dependent MS2 TOP N acquisition mode - in order to optimize the semi-quantification of polar lipids. Optimization of MS-source settings improved signal intensity by factor 3 compared to default settings. Polar lipids were separated on an Acquity Premier CSH C18 reversed-phase column (100 x 2.1 mm, 1.7 µm, 130 Å) during an elution window of 28 min, leading to sufficient number of both data points per peaks as well as MS2 spectra. Analysis was carried out in positive and negative ionization mode enabling detection of a broader spectrum of lipids and to support the characterization of lipids. Optimal sample preparation of biological samples was achieved by liquid-liquid extraction using MeOH/MTBE resulting in an excellent extraction efficiency of >85% with an intra-day and inter-day variability of <15%. The optimized method was applied on the investigation of changes in the phospholipid pattern in plasma from human subjects supplemented with n3-PUFA (20:5 and 22:6). The strongest increase was observed for lipids bearing 20:5 while 22:4 bearing lipids were lowered. Specifically, LPC 20:5_0:0 and PC 16:0_20:5 were found to be strongest elevated while PE 18:0_22:4 and PC 18:2_18:2 were decreased by n3-PUFA supplementation. These results were confirmed by targeted LC-MS/MS using commercially available phospholipids as standards.


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