2.4. Determination of Fatty Acids

Fatty acid methyl esters (FAME) were prepared according to Garcia, Hernandez, and Lozano [20]. Fifty milligrams of ground, freeze-dried (48 h) sample was measured into a 4 mL amber vial. A 10 µL aliquot of tridecanoic acid (2 g/L) was added as an internal standard. Toluene (490 µL) and freshly prepared 5% methanolic HCl (750 µL) were then added before filling the vial with nitrogen. After incubation in the water bath at 70 °C (2 h), vials were cooled to room temperature before 6% aqueous K₂CO₃ (1 mL) and toluene (500 µL) were added. After centrifugation at 1100× g for 5 min, the top layer was removed using a glass Pasteur pipette for FAME analysis.

Derivatized methyl esters of fatty acids were separated and quantified using a Shimadzu GC 2010 with a Flame Ionisation Detector (FID) (Kyoto, Japan). The column used was a 0.25 mm × 30 m × 0.25 µm film thickness, fused-silica column (ZB-WAX, Phenomenex, CA, USA). Nitrogen was used as the carrier gas, with a split ratio of 50, a head pressure of 8.7 PSI, and 1 mL/min column flow. The injector temperature was set at 250 °C. The initial oven temperature was programmed at 140 °C, then increased to 245 °C at 5 °C/min, and held at 245 °C for 15 min. Thirty-seven FAME standards (Supelco product 47885-U) obtained from Sigma-Aldrich (Sydney, Australia) were serially diluted to six concentrations (from 10 to 0.3125 g.L⁻¹), and the standard calibration curve constructed was used for quantitative analysis.