2.3. Lipid extraction and fatty acid methyl ester preparation

Approximately 50 mg of seeds of each sample (n = four independent replicates) were placed in a hexane-washed, hand-held, group-glass homogenizer and boiled in 1 mL of isopropanol (80 °C) for 10 min. The seeds were then cooled on ice for 5 min. Thereafter, 1 mL of hexane and 2 mL of 3:2 hexane:isopropanol (HIP) was added and the seeds were homogenized until completely pulverized. An additional 2 mL of 3:2 HIP was added and grinding continued. The homogenate was transferred to a screw-capped glass tube, and 2 mL of 3.3% (W/V) Na₂SO₄ was added, capped, and shaken for 2 min. The tubes were spun at 555 g for 2 min, and the upper organic phase was transferred to a new hexane-washed screw-capped tube. The aqueous phase was re-extracted with 4 mL of 7:2 HIP, capped, and shaken for 2 min. The tubes were spun again at 555 g for 2 min, and the upper organic phase added with the first extracted organic phase. The combined organic phases were evaporated to dryness in a heating block (37 °C) under a gentle N₂ stream. To isolate fatty acid methyl esters (FAMEs), 1.2 mL of HCl-methanol (1.5 M HCl in methanol made fresh) was added to the dried lipid and incubated at 100 °C for 1 h. Then, 1 mL of double distilled water was added to quench the transesterification reaction. The FAMEs were then extracted with 2 mL of hexane. The samples were centrifuged as above and the upper organic phase containing the FAMEs transferred to a clean hexane-washed test tube. The aqueous phase was re-extracted with an additional 2 mL of hexane and centrifuged and the resulting upper phase transferred and combined with the previously collected organic phase. The combined organic phases containing the FAMEs were then dried down completely in 1 mL of hexane and transferred to gas chromatography vials and capped.