4.7. GC–MS Analysis of Lipid Extracts

The total fatty acid composition of organic extracts was determined using GC–MS on the corresponding fatty acid methyl esters (FAMEs) obtained after saponification of lipid extracts with Na₂CO₃ in methanol at 40 °C for 4 h. The reaction mixture was diluted with milliQ water (to dissolve completely Na₂CO₃), neutralized with HCl 1M, and extracted with diethylether three times. Combined organic extracts were dried under a nitrogen stream, dissolved in MeOH at a final concentration of 1 µg µL⁻¹ and analyzed using GC–MS (Thermo Focus GC Polaris Q) equipped with an ion-trap, EI (70 eV), a 5% diphenyl column, an injector temperature of 210 °C, a transfer line temperature of 280 °C. Elution of free fatty acid methyl esters required an increasing gradient of temperature according to the following method: 160 °C for 3 min, increase by 3 °C/min up to 260 °C, increase by 30 °C/min up to 310 °C, 7 min at 310 °C. FAMEs have been identified by the comparation of retention time and mass spectra with FAMEs’ standard mixture (Marine source analytical standards, Sigma Aldrich, Milan, Italy). Fatty acid content was expressed as a percentage of total fatty acids, according to Equation (3), as follows: