Sample preparation and GC-FID analysis

Lipid extraction from the brain tissue was carried out according to Bligh and Dyer method⁵². The hippocampus samples (15 mg) were homogenized in a glass potter and treated with 3 mL of CH₂Cl₂:CH₃OH mixture (1:2 v/v). Then 1 mL of CH₂Cl₂ and 1 mL of distilled water were added and the resulting mixture was stirred for 30 s. The organic layer was filtered using PTFE microfilters (pore size is 0.45 μm), and the solvent distilled off in vacuum to give a residue of about 2 mg for hippocampus. Finally, a weighted amount of octanoic acid C8:0 (internal standard, IS) was added to the extract. Samples suitable for the GC-FID analysis were prepared by converting the fatty acid (FA) into the corresponding methyl esters (FAME) by treatment of sample (2–15 mg) with 17 % BCl₃ hexane solution (1 mL) according to the method by Morrison and Smith⁵³. The hexane solution was left at 90 °C for 1 h, and then concentrated to a final volume of 0.2 mL before the GC-FID analysis.

The GC-FID analyses were carried out using a Thermo Scientific FOCUS GC equipped with a FID detector, split/splitless injector, and HP-Innowax capillary column (30 m × 0.25 mm I.D., 0.25 μm film thickness) from Agilent Technologies. Oven temperature was programmed with a ramp from 100 to 240 °C at 10 °C min⁻¹ and then at 240 °C for 20 min. Injector and detector temperatures were set at 240 and 250 °C, respectively. Helium was employed as carrier gas with a flow rate of 1.7 mL min⁻¹; 1 μL aliquots were injected and the split ratio of 1:10 was used. Data acquisition was carried out using Chrom-Card Data System. Identification of the chromatographic peaks was made by comparing the retention times of commercial standards.