3.2.2. Fatty Acid Composition

The fatty acid (FA) composition was evaluated using gas chromatography with flame ionization detector (GC-FID). To carry this out, the lipid-rich extracts obtained by the Soxhlet unit (Section 2.2) were subjected to a derivatization procedure to obtain volatile fatty acid methyl esters (FAMEs). Briefly, 100 μL of lipid extracts were mixed with 2 mL of heptadecanoic acid (HAD) and 4 mL of n-hexane. The mixture was stirred for 1 min and then 0.5 mL of sulfuric acid (2% in methanol) was added. The resulting mixture was stirred for 5 more minutes and then, centrifuged at 3000 rpm for 5 min. Next, 2 mL of supernatant holding FAMEs were mixed with 5 mL of n-hexane and stored until analysis.

FAMEs-containing samples were analyzed using a gas chromatograph (Chromatographic System Agilent 7820A) equipped with a column Agilent HP-88 (60 m × 250 µm × 0.25 µm) and a flame ionization detector. The chromatographic run was performed using two temperature programs. The first one consisted of a ramp of 5 °C/min until reaching 220 °C and 15 min of holding time, while the second one used a temperature ramp of 40 °C/min until reaching 250 °C with a holding time of 2 min. Helium was used as carrier gas and was injected at 1 mL/min. Finally, FA identification was conducted using external standards, and their calibration curve was used to determine FA composition. Results were reported as % of total fatty acid content.