Comprehensive lipid profiling of Microchloropsis gaditana by liquid chromatography - (tandem) mass spectrometry: Bead milling and extraction solvent effects

Abstract

Liquid chromatography - mass spectrometry (LC-MS) experiments were used to qualitatively and semi-quantitatively monitor the impact of bead milling and the influence of the nature of the extraction solvents, Bligh and Dyer (B&D) versus n-heptane, on the composition of the structural lipids extracted from the microalgae Microchloropsis gaditana (M. gaditana).Our data reveal that lipid extraction with n-heptane as the solvent becomes as efficient as the standard Bligh and Dyer method when a milling step is included before the extraction. Indeed, lipid extraction yields amount to ~1% (n-heptane on intact cells), ~5% (B&D on intact cells), ~5% (n-heptane on milled cells) and ~6% (B&D on milled cells). In all the extracts, using liquid chromatography separation combined to accurate mass measurements and tandem mass spectrometry experiments, we identified a huge number of lipids belonging to different structural lipid families, including LPC (lyso-phosphatidylcholine), PC (phosphatidylcholine), DGTS (diacylglyceryl-N-trimethylhomoserine), MGTS (monoacylglyceryl-N-trimethylhomoserine), DGDG (digalactosyldiacylglycerol), DGMG (digalactosylmonoacylglycerol), MGDG (monogalactosyldiacylglycerol), PG (phosphatidylglycerol), PI (phosphatidylinositol), SQDG (sulfoquinovosyldiacylglycerol), SGMG (sulfoquinovosylmonoacylglycerol) and free fatty acids (FFA). We further demonstrated that the milling step induces strong modifications in the lipid composition due to extensive lipid degradation into free fatty acids - probably by enzymatic processes, even when the milled cells are conserved at −20 °C.From a method development perspective, this work represents to the best of our knowledge the most complete structural analysis investigation of lipids extracted from intact and milled microalgae, including the use of a sector diagram representation that could be relevant for all lipidomic investigations. One of the main outcomes of the study is the confirmation that liquid chromatography - tandem mass spectrometry (LC-MS/MS) experiments are essential when lipid extraction procedures from any microalgae biomass have to be optimized. The analytical protocol developed in the present study certainly deserves to be extended to other microalgae.