2.2. Lipid Extraction and Fatty Acid Ester Preparation (Blubber, Muscle, Liver)

After homogenization of slightly thawed samples and the addition of nonadecanoic acid (C19:0) as an internal standard, total lipids were extracted in duplicate using chloroform/methanol (2:1, v/v) and the Ultra Turrax T25 (IKA, Staufen, Germany) at 3 × 15 s, 15,777 g, and room temperature. For lipid extraction, approximately 2 g of muscle, 2 g of liver, and 1 g of blubber were used. The detailed sample preparation procedure of animal tissues was previously described [38]. Briefly, the final extraction mixtures were stored at 5 °C for 18 h in the dark and subsequently washed with 0.02% CaCl₂ solution. All of the solvents contained 0.005% (weight/volume (w/v)) of t-butylhydroxytoluene to prevent the oxidation of PUFAs. The organic phase was separated and dried with a mixture of Na₂SO₄ and K₂CO₃ (10:1, weight/weight (w/w)), and the solvent was subsequently removed under gentle nitrogen at room temperature. The lipid extracts were redissolved in 300 μL of toluene, and a 25-mg aliquot was used for methyl ester preparation. Total lipids were stored at −18 °C until transmethylation of fatty acids. For transmethylation, 2 mL of 0.5 M sodium methoxide in methanol were added to the lipid extracts, which were shaken in a 60 °C water bath for 10 min. Subsequently, 1 mL of 14% boron trifluoride in methanol was added to the mixture, which was then shaken for an additional 10 min at 60 °C. Saturated NaHCO₃ solution (2 mL) was added, and the fatty acid methyl esters (FAMEs) were extracted twice with 2 mL of n-hexane. The n-hexane extracts were dried with a mixture of Na₂SO₄ and K₂CO₃ (10:1, w/w). After filtration, the extracts were reduced to dryness using a vacuum centrifuge (438 g, 30 °C, 30 min). The FAMEs were resuspended in 100 µL of n-hexane and stored at −18 °C until use for high-resolution gas chromatography (HR-GC) analysis.