Fatty acid determination by GC/MS
The fatty acid composition of the oilswas determined by GC/MS analysis, after transesterification. The GC analyses were performed on Agilent 7890A GC system (Agilent Technologies, California, USA) equipped with a 5975C MSD and FID detector, using a DB-23
column (60m250mm0.25mm). Helium was used as the carrier gas at a flow rate of 0.9 ml/min. The column temperature was linearly programmed in the range of 175–235 C at the rate 4 C/min and held at 235 C for 5 min. The transfer line was heated at 240 C. The FID detector temperature was 300 C. The EI mass spectra (70 eV) were acquired in the m/z range 40–500. The fatty acid methyl esters were prepared in the following manner: 30.0mg
of oil and a drop of concentrated sulfuric acid were added in 6ml of methanol and the mixture was refluxed for 2 h with constant stirring. Acid was neutralized by addition of saturated potassium hydrogen carbonate upon completion of the transesterification
process (the change in pH value is monitored by a universal pH indicator (Sigma–Aldrich, Missouri, USA)). Following neutralization, the mixturewas extracted with hexane (36 ml). The hexane solution was dried over anhydrous sodium sulfate, filtered and
evaporated to dryness. The obtained transesterification oil was used for the preparation of an oil solution, in the concentration of about 1 mg/ml, which was then analyzed by GC/MS.