FATTY ACID COMPOSITION
The procedure used to analyze the fatty acid composition of phospholipids is similar for an aliquot of purified phospholipids and for isolated phospholipid classes but some differences in the derivatization time are observed.
14% BF3 solution in methanol
Aliquots of a phospholipid solution are evaporated in a screw-capped (Teflon-lined) tubes, 1 ml of BF3 solution is added and the tightly closed tubes are treated during 90 min at 100°C (water bath or thermoblock). After cooling to room temperature, the tubes are opened and after addition of 1 ml of water and 2 ml of pentane they are vortexed for 1 min. After a short centrifugation, the upper pentane layer is collected (without any trace of the lower layer) and evaporated in small glass tubes. The dry residue is dissolved in a small portion of hexane and the fatty acid methyl esters are analyzed by GLC on a polar capillary column.
Scraped silica gel spots are treated with the same procedure but the methylation time is adapted according to the chemical structure of the analyzed phospholipid. Glycerophospholipids are derivatized for only 15 min while 90 min are required for sphingomyelin (as for total phospholipid extracts which could contain this component). A rapid method for analysis of sphingomyelin was proposed using derivatisation with boron trifluoride in a microwave oven (Devle H et al., Eur J Lipid Sci Technol 2011, 113, 708). The reaction time has been reduced to only 27 min, with excellent yield.
A similar procedure has been proposed for the determination of fatty acid profile in plasma total phospholipids after solid-phase extraction an methylation with trimethylsulfonium hydroxide (Taylor L et al., Eur J Lipid Sci Technol 2009, 111, 912). These detailed analysis of fatty acid profiles in plasma phospholipids are valuable as marker for dietary habits or interventions.
As the fatty acid composition of plasma phospholipids is frequently useful to appreciate long term dietary intake, a simple and rapid method is necessary (Liu G et al., Prost Leukotr essent Fatty acids 2020, 57). This study reports a system capable of selectively separating plasma phospholipids from other lipid classes in just a few minutes in applying a drop of plasma to Whatman SG81 collection paper. Then, the neutral lipids are washed away using an organic solvent, and the bound phospholipids are directly transesterified into fatty acid methyl esters for analysis by gas chromatography. A strong agreement between the fatty acid profiles obtained from the proposed method and reference TLC method in 110 human subjects validates its potential clinical application and results can be achieved much more rapidly.