4.2. Sample Preparation for Fatty Acid Analysis—Blood Plasma

EDTA blood samples were centrifuged at 2000× g for 15 min at 4 °C to separate plasma for the following fatty acid analysis. Plasma samples, approximately 1.5 g, were dropwise added to 8 mL of chloroform/methanol (2:1, v/v) at room temperature. The solution contained C19:0 as an internal standard. The detailed sample preparation procedure has been recently described [28]. Briefly, all the solvents contained 0.005% (w/v) of t-butylhydroxytoluene (BHT) to prevent the oxidation of PUFAs. The extraction mixtures were stirred two times for 15 min and stored at 5 °C for 18 h in the dark and subsequently washed with 0.02% CaCl₂ solution. After centrifugation (2500 rpm, 5 min), the organic phase was dried with Na₂SO₄ and K₂CO₃ (10:1, w/w), and the solvent was subsequently removed under gentle nitrogen stream at room temperature. The total lipid contents were stored at −18 °C until transmethylation of fatty acids. Sodium methoxide in methanol was added to the extracts, which were shaken in a 60 °C water bath for 10 min. Subsequently, 1 mL of 14% boron trifluoride (BF₃) in methanol was added to the mixture, which was then shaken for an additional 10 min at 60 °C. Finally, the fatty acid methyl esters (FAME) were resuspended in 500 µL of n-heptane and stored at −18 °C until use for gas chromatography (GC) analysis.