2.2.2. Preparation of FAME and GC-FID Analysis of Fatty Acids

Analysis of the fatty acid methyl esters (FAMEs) was performed according to the AOAC method 969.33 [18]. FAMEs were obtained through trans-methylation, which was performed by suspending 200 mg of the Soxhlet-extracted lipid fraction in 2 mL of 1.25 M HCl/CH3OH solution in Pyrex test tubes with screw caps and incubated in a water bath at 90 °C for 60 min. After the addition of 2 mL of deionized water, FAMEs were extracted with n-hexane and filtered using Millex 0.45 µm PVDF disposable syringe filters (EDM Millipore Corp., Billerica, MA, USA). GC analyses were performed using a 7890A gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) equipped with a flame ionization detector (FID), using an SP-2380, 100 m × 0.20 mm capillary column (Supelco-Sigma-Aldrich, St. Louis, MO, USA). FAME extracts (1 µL) were introduced through a split–splitless injection system of the autosampler in split mode (ratio 1:100) at 260 °C. The oven temperature program started at 140 °C (held for 5 min) and linearly increased to 260 °C (4 °C min⁻¹) up to the end of the analysis. The FID temperature was 260 °C. The FA composition of samples was obtained by comparing with the retention time of the standard mixture FAME 37 components (Sigma-Aldrich). Relative abundance was obtained by software-assisted peak integration (Chemstation vers. B04.03, Agilent, Santa Clara, CA, USA) and was expressed as percentage area.