3.5.7. Fatty Acid Analysis

The analyses were carried in an Agilent 7890B gas chromatograph (Santa Clara, CA, USA) coupled to a flame ionization detector (FID) (Santa Clara, CA, USA) and a 7963 auto sampler (Santa Clara, CA, USA). Injector temperature was maintained at 260 °C. The injection mode used was split 50:1 at 280 °C with a pressure pulse of 30.9 psi. Hydrogen was used (analytical grade 5.0) as the carrier gas at a constant flow of 1 mL/min during the entire analysis. The chromatographic column used was a 30.0 m TRBWAXOMEGA with 0.25 mm inner diameter and 0.25 µm film thicknesses (Barcelona, Spain). A temperature gradient carried out the separation, the initial temperature of the chromatographic oven was 225 °C which was held for 5 min; then, the temperature was increased at a rate of 15 °C/min for 2 min and posteriorly up to 240 °C at 10 °C/min for 12 min. Sample preparation 500 mg of CLCs were derivatized with boron trifluoride in methanol to convert the fatty acids in their respective methyl esters following the AOAC official method 969.33 [28].

Gas chromatographic peaks of FAME (Fatty acids methyl esters) were identified by comparing the retention time of certified standards of their ester form by derivatization (C:12 lauric acid, C:16 palmitic acid, C16:1 palmitoleic acid, C18:0 stearic acid, C18:1n9c oleic acid, C18:2n6c linoleic acid, C20:0 arachidonic acid, C18:3n3 α-linolenic acid, C23:0 tricosanoic acid). Peak areas were used for the quantification of fatty acids.