4.4. Extraction of Crude Lipid and Determination of Fatty Acids Content

Portions of 500 mg each of powdered G. corticata and G. edulis were mixed with 5 mL of a chloroform:methanol solution (2:1 v/v), tightly covered with aluminum foil and kept at room temperature for 24 h. After this period, solutions were filtered through 11 µm Whatman No. 1 filter paper, and the filtered extract was placed in a pre-weighed and oven-dried beaker. The beaker was weighed with lipids, and the difference in weight was taken as total lipid content and expressed as a percentage [43]. Afterwards, an aliquot of the total lipids of each sample was used to determine the fatty acids content, based on a method published [6]. For this purpose, 0.45 g was introduced into a 10 mL volumetric flask, dissolved in hexane containing 50 mg of butylated hydroxytoluene per L and diluted to 10 mL with the same solvent. Afterwards, 2 mL of the solution was transferred into a quartz tube and evaporated by means of a nitrogen flow. Further, 1.5 mL of a 20 g/L solution of sodium hydroxide in methanol, covered with nitrogen, was added, capped tightly with a polytetrafluoroethylene-lined cap, mixed and heated in a water bath for 7 min. After the water bath, samples were cooled at room temperature, and 2 mL of boron trichloride-methanol solution was added; then, they were blanketed with nitrogen, capped tightly, mixed and heated in a water bath for 30 min. After this period, samples were cooled to 40–50 °C, and 1 mL of trimethylpentane was added; then, they were capped and shaken vigorously for at least 30 s. Immediately, 5 mL of saturated sodium chloride solution was added, then the samples were covered with nitrogen, capped and vortexed or shaken thoroughly for at least 15 s. The upper layer was allowed to become clear and then transferred to a separate tube. In the separate tube, the methanol layer was shaken once more with 1 mL of trimethylpentane and combined with trimethylpentane extracts. The organic solvent was then removed, and FAME were subjected to gas chromatography (GC), performed on a Perkin Elmer Clarus 580 gas chromatograph (Perkin Elmer, Gaithersburg, MD, USA) equipped with a flame ionization detector and an HP-5 capillary column (30 m × 0.25 mm). Initial temperature was maintained at 70 °C, then increased to 250 °C (10 °C/min); the injection temperature employed was 225 °C. Helium was used as carrier gas, with a flow rate of 1 μL/min. FAME peaks were identified by comparison of their retention times and quantified by comparison with individual calibration curves performed with a standard FAME mix (Supelco, Sigma-Aldrich, St Louis, MO, USA). Tricosanoic acid (C23; Sigma-Aldrich) was used as internal standard. Fatty acid composition was determined in triplicate for each seaweed and was expressed as g FAME/100 g total fat.