Preparation of fatty acid methyl esters (FAME)

Lipid extract from each sample was dissolved in 1000 μl chloroform. According to sample’s wet weight, 100 to 800 μl of this lipid extract and 50 nmol pentadecanoic acid (C15:0, as an internal standard) were placed in a screw-cap tube labelled for each sample. The solvent was completely evaporated; 1 ml of 3 M methanolic hydrochloric acid was added, bubbled with nitrogen gas, and the sample was heated at 78 °C for 30 min. Then 2 ml water and 2 ml hexane:chloroform (4:1, v/v) were added, vortexed 30 s and centrifuged at least 5 min to separate the phases. The upper phase (hexane:chloroform) was transferred to a clean tube. Two (2) ml hexane:chloroform was added to the remaining aqueous phase, which was vortexed and centrifuged. The upper phase was removed and combined with previous upper phase. After solvent was evaporated, the extract was dissolved in a little amount of hexane and transferred to a GC vial with insert for total fatty acid analysis.