Fatty Acids Methyl Esters (FAME) Analysis
The methyl esters of lipid from the samples were prepared by trans methylation according to the method described by Ichihara et al. (1996) with a minor modification. Briefly, 25 mg of extracted oil was dissolved in 2 mL isooctane, followed by 4 mL of 2 M KOH (in methanol) addition. Then, the tube was vortexed for 2 min at room temperature. After centrifugation at 4000 rpm for 10 min, the isooctane layer was taken for Gas chromatography analysis.
Gas Chromatography (GC) Conditions: The fatty acid methyl esters were analyzed using Gas chromatograph of Agilent Technologies model 7820 equipped with a flame ionization detector (FID) and fitted with a HP-88 capillary column (60 m x 0.25 mm x 0.25 µm thickness). Helium was used as the carrier gas at a constant pressure of 16 psi. Injection port was maintained at 220 °C, and the sample was injected in split mode with a split ratio of 50:1. Detector temperature was 280 °C. Column temperature was started at 175 °C, and then programmed at 3 °C/min to 220 °C, ramped at 1 °C/min to 220 °C, and held for 10 min. The total running time was 26 minutes. Helium was used as the makeup gas at a constant flow of 40 mL/min, and hydrogen and dry air were used as detector gases. Identification of fatty acids was carried out by comparing sample FAME peak relative retention times with those obtained for Supelco standards (Supelco 37 Compounds FAME mix 10 mg/mL in CH2 Cl2–47885 U, Supelco 1819-1 Ampule FAME mix C4-C24). Results of each fatty acids were expressed as FID response area relative percentages of the total fatty acids determined (ISO, 1990).