Fatty Acid Profiling by GC-MS

The fatty acid profile was determined upon conversion of fatty acids to FAME, as previously described by Pinheiro et al. [17 (link)]. Briefly, to 300 mg of sample, 1 mL of the internal standard C19:0 (Thermo Fisher Scientific, Kandel, Germany) at 2 mg mL⁻¹ in methanol, 0.7 mL of KOH (10 N) in water and 5.3 mL of methanol were added. The tubes were incubated for 1.5 h at 55 °C. Afterwards, 0.58 mL of H₂SO₄ (24 N) was added and these were placed at the same temperature for another 1.5 h. Finally, 3 mL of hexane was added, followed by mixing and centrifugation. The hexane fraction was recovered and injected on a gas chromatograph mass spectrometer GCMS-QP2010 (Shimadzu, Kyoto, Japan) equipped with an AOC-20i auto-injector and a DB-5 ms column (30 m × 0.25 mm diameter, 0.25 µm thickness). The equipment operated under the following conditions: initial temperature, 70 °C for 5 min; temperature gradient, 4 °C min⁻¹; final temperature, 250 °C; temperature gradient, 2 °C min⁻¹; final temperature, 300 °C for 5 min; injection temperature, 320 °C; split ratio, 100:0. Identification of FAME was obtained by co-chromatography with authentic commercially available FAME standards. Total FAME content was quantified by comparison with the internal standard.