*4.4. Fatty Acid Profiling*

Fatty acid methyl esters (FAMEs) were prepared using a methanolic solution of potassium hydroxide (2.0 M) as described by Melo et al. [67]. Subsequently, 2.0 μL of a hexane solution containing FAMEs were analyzed by GC–MS on an Agilent Technologies 6890 N Network (Santa Clara, CA, USA) equipped with a DB–FFAP column. The column was 30 m long, had 0.32 mm of internal diameter, and a film thickness of 0.25 μm (123-3232, J&W Scientific, Folsom, CA, USA). The GC equipment was connected to a Mass Selective Detector (Agilent 5973 Network) operating with an electron impact mode at 70 eV and scanning the range m/z 50–550 in a 1 s cycle in a full scan mode acquisition. The carrier gas Helium was used at a flow rate of 1.4 mL min−1. The elution relied on an increasing temperature gradient: 80 ◦C for 3 min, a linear increase to 160 ◦C at 25 ◦C min<sup>−</sup>1, followed by a linear increase at 2 ◦C min−<sup>1</sup> to 210 ◦C, then at 30 ◦C min−<sup>1</sup> to 250 ◦C, standing at 250 ◦C for 10 min Identification of FAs was performed considering retention times and mass spectrometry spectra of FA standards (Supelco 37 Component Fame Mix, Sigma-Aldrich, St. Louis, MO, USA), as well as through mass spectrum comparison with those in Wiley 275 library and AOCS Lipid Library. The relative amounts of FAs were calculated by the percent area method with proper normalization, considering the sum of all areas of the identified FAs. The results were expressed as means ± SD.
