One hundred microliters of methylene chloride was then added, along with 1 mL of NaOH 0.5 N in methanol. Once nitrogen was added and the tube was sealed, it was heated to 90C for 10 minutes. The reaction tube was then cooled and 1 mL of 14% BF3 in methanol was added. After nitrogen addition, heating was ensued at the same temperature for the next 10 minutes. Afterward, the reaction tube was cooled to ambient temperature, and 1 mL of water and 200e500 mL of hexane were added. The mixture was then vortexed for 1 minute to extract the fatty acid's methyl ester. After centrifugation, the upper layer of sample was ready for GC analysis.