2.3.3. Oleic acid

Gas chromatography (GC) with a Flame Ionization Detector (FID) was used to measure the oleic acid content as a Fatty Acid Methyl Ester (FAME). Nonadecanoic acid (C19:0) triacylglycerol was used as an internal standard to check if the hydrolyses was complete. A stock solution of 1 mg ml⁻¹ was prepared. The previously extracted oil was mixed with the standard (0.1 ml), hexane (10 mg oil 0.9 ml hexane⁻¹), and 4.25 M potassium hydroxide in methanol (KOH/MeOH) solution (60 μl), and then incubated at 60 °C for 10 min. After centrifugation, the supernatant was analysed with the GC-FID (Agilent model 7890B) using a 30 m × 320 μm × 0.25 μm Agilent column (product DB-23). Hydrogen (H₂) was used as the carrier gas. A 1 μl sample was injected into the inlet, which was heated to 260 °C. The inlet pressure was 4.2566 psi (pound-force per square inch) and the split ratio was 1/50. The oven was held at 140 °C for 1 min before the temperature was increased at a rate of 4 °C min⁻¹ to 220 °C and held for 5 min. The chromatographic data were processed using MS ChemStation (Agilent Technologies, USA). The retention time of the FAMEs was compared with commercial standards for the identification of oleic acid. Oleic acid was quantified based on peak area ratios. Samples were analysed in duplicate.