New fractionation scheme for lipid classes based on “in-cell fractionation” using sequential pressurized liquid extraction

A new preparation scheme is proposed to fractionate neutral lipids (acylglycerines, sterol esters, long-chain free fatty acids) from polar phospholipids in biological matrices. This fractionation is mandatory in the microbial community, for the control of bioremediation processes, in the study of phytoplankton growth in lakes and rivers, and in the quality control of processed food. Basically, a two-step pressurized liquid extraction (PLE) scheme is combined with an “in-cell-fractionation” using silica-based sorbents placed at the outlet of the PLE cartridge. The optimized extraction scheme consists of n-hexane/acetone (9:1, v/v) extraction at 50 °C (2 cycles, 10 min each) to obtain neutral lipids followed by chloroform/methanol (1:4, v/v) extraction at 110 °C (2 cycles, 10 min each). Thermally pre-treated silicic acid and cyanoproyl-modified silica turned out to be appropriate sorbents to ensure clear-cut boundaries between neutral lipids and phospholipids. The proposed protocol is superior to commonly used approaches consisting of an exhaustive lipid extraction followed by off-line lipid fractionation using solid-phase extraction (SPE) regarding fractionation efficiency, time and solvent consumption. In this paper, it is also shown that the transmethylation using trimethylchlorosilane/methanol (1:9, v/v) provides a complete reaction to give methyl esters without artefact formation across the array of different lipid classes even with polyunsaturated fatty acid moieties.