2.13. Total Fatty Acid Analysis

Tridecanoic acid was added to TL extracts as internal standard. After methylation with 1M sulfuric acid in methanol for one hour at 85 °C [23], samples were analyzed by gas chromatograph (Shimadzu 2014, Kyoto, Japan) on Rtx 2330 columns (Restek, Bellefonte, PA, USA). Initial column temperature was set at 140 °C and hold for 5 min. Then temperature was increased to 220 °C by 3 °C/minute. Final column temperature was held for 20 min. Peaks of fatty acids (FA) in samples were identified by comparing the retention times with those obtained for FA standard mixtures PUFA-2 and Supelco37 Component FAME Mix (Sigma-Aldrich, Darmstadt, Germany). Quantity of identified FA in lipid extract was calculated by comparing their areas in chromatographs with internal standard peak area in sample.