Fat extracted from all samples were derivatized using boron trichloride-methanol, 12% (w/w) according to the procedure provided by sigma-Aldrich(997).
Toluene(2 mL) was added to 50 mg of sample in a screw top test tube.
Subsequently, 2 mL of boron trichloride-methanol solution was added and the mix ture was flushed with nitrogen gas for 10 s and heated in a water bath at 60 oC for 10 min.
Once cooled, water(2 mL) and hexane(2 mL) were added into the test tube and shaken lightly to extract the fatty acid methly esters(FAME).
Anhydrous sodium sulfate was added to the hexane extracts to remove moisture then the an hydrous hexane extracts were transferred into a 10 mL volumetric flask and filled to volume with hexane.
The moisture removal step was carried out twice to ensure maximum extraction of FAMEs from the oil