4.5. Gas Chromatography Mass Spectrometry (GC-MS) Analysis of Fatty Acid Methyl Esters

For GC-MS analysis of the fatty acids content, ML-SCO₂ and MLW-SCO₂ were derivatized with MeOH in the presence of H₂SO₄, leading to the esterification of fatty acids to fatty acid methyl esters, which offer excellent stability for GC analysis. Then, 150 mg of each extract was added to 15 mL of MeOH, 1 mL of CH2Cl2, 3 drops of H₂SO₄ and 25.7 mg of methylpentadecanoate (Sigma Aldrich, St. Louis, MO, USA), used as internal standard. The mixture was heated under reflux for 1 h and then cooled in an ice bath. A liquid/liquid partition was performed with 10 mL of water and 5 mL of diethyl ether, then the organic phase was collected and dried. The residue was re-dissolved with 1.5 mL of diethyl ether and put in a vial. GC-MS analysis was performed with an Agilent 7820A coupled to an Agilent 5977B MSD single quadrupole mass spectrometer, using an HP88 (60 m × 0.25 mm, 0.2 µm film thickness) as the stationary phase. Helium was the carrier gas with a column head pressure of 14.1 psi. The flow rate through the column was 1.19 mL/min. The injector was set at 300 °C with a split ratio of 20:1, the split flow was 23.9 mL/min and 1 µL injections were made. The temperature gradient started with an initial temperature of 120 °C before a linear increase to 240 °C at 3 °C/min. The total run time was 55 min. MS spectra were recorded in the range of m/z 40–650 using an EI ion source operating in positive ion mode.