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).