3.3. Sample Preparation

Aliquots of 100 µL plasma were transferred to Eppendorf tubes and mixed with 10 µL of internal standard mixture containing 10 µg/mL DHA-d5, ARA-d8, and EPA-d5, and 10 µg/mL LA-d4 and ALA-d1. The lipids were then extracted with hexane/isopropanol, 3:2 v/v at a 1:10 sample/solvent ratio. The tubes were vortexed and maintained at −20 °C for 10 min, then centrifuged at 14,000 g at 4 °C for 5 min. The supernatant was collected, transferred to glass tubes, and dried under nitrogen flow. Then, 1 mL of 80% methanol was added, and the tubes were thoroughly mixed. Of this, a volume of 100 µL was transferred into an autosampler vial for free fatty acids analysis. The remaining aliquot was subjected to alkaline hydrolysis.

The Bligh–Dyer method [25] (Supplementary Material 1) was also used for comparison.

A volume of 100 µL of a solution of 0.3 M KOH in 80% methanol was added to the lipid extract. The mixture was incubated at 80 °C for 30 min. The tubes were allowed to cool, and then 10 µL of formic acid was added to neutralize the pH. For separating the fatty acids, 1 mL hexane was added, and the tubes were placed on a rotary mixer for 5 min. The tubes were briefly centrifuged at 1000× g, and then the top hexane layer was transferred into a clean glass tube and dried under nitrogen flow. The sediment was reconstituted in 1 mL of 80% methanol, of which 100 µL was transferred into an autosampler vial for analysis.