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The fatty acid analysis was performed using capillary GC with a CP-Sil 88 CB column (100 m × 0.25 mm, Agilent, Santa Clara, CA, USA) that was installed in a PerkinElmer gas chromatograph CLARUS 680 with a flame ionization detector and split injection (PerkinElmer Instruments, Shelton, CT, USA). The detailed GC conditions were recently described [29 (link)]. Briefly, the initial oven temperature was 150 °C, which was held for 5 min; subsequently, the temperature was increased to 175 °C and then to 200 °C at a rate of 2 °C min⁻¹ and held for 10 min. Finally, the temperature was increased to 225 °C at a rate of 1.5 °C min⁻¹ and held for 25 min. Hydrogen was used as the carrier gas at a flow rate of 1 mL min⁻¹. The split ratio was 1:20, and the injector and detector were set at 260 and 280 °C, respectively. The quantification of fatty acids was done by the use of C19:0 as the internal standard. For the calibration procedure, the reference standard mixture “Sigma FAME” (Sigma-Aldrich, Deisenhofen, Germany), the methyl ester of C18:1cis-11, C22:5n-3, C18:2cis-9, trans-11 (Matreya, State College, PA, USA), C22:4n-6 (Sigma-Aldrich, Deisenhofen, Germany) and C18:4n-3 (Larodan, Limhamn, Sweden) were used. The five-point calibration of single fatty acids ranged between 16 and 415 mg/mL and was checked after GC analysis of five samples.

The fatty acid analysis of all sample extracts was performed using capillary GC with a CP-Sil 88 CB column (100 m × 0.25 mm, Agilent, Santa Clara, CA, USA) installed in a PerkinElmer gas chromatograph CLARUS 680 with a flame ionization detector and split injection (PerkinElmer Instruments, Waltham, MA, USA) as described earlier [50 (link)]. Briefly, hydrogen was used as the carrier gas at a flow rate of 1 mL × min⁻¹, while the split ratio was 1:20, with the injector and detector set at 260 and 280 °C, respectively. The GC oven temperature program was 150 °C for 5 min, followed by a heating rate of 2°/min until 200 °C and then being kept for 10 min, followed by a final heating rate of 1°/min until 225 °C and then being kept for 20 min. For the calibration, the reference standard mixture “Sigma FAME” (Sigma–Aldrich, Deisenhofen, Germany), the methyl ester of C18:1cis-11, C22:5n-3, and C18:2cis-9,trans-11 (Matreya, State College, PA, USA), C22:4n-6 (Sigma–Aldrich, Deisenhofen, Germany), and C18:4n-3 (Larodan, Limhamn, Sweden) were used. The five-point calibration of single fatty acids ranged between 16 and 415 µg/mL and was assessed after GC analysis of five samples. Fatty acid proportions were displayed as individual percent of total fatty acids.

Dried lipid extracts were redissolved in chloroform/methanol (2:1, v/v) and a solution containing nonadecanoic acid (19:0) as the internal standard was added. A detailed sample preparation protocol for fatty acid (FA) derivatization was previously published [24 (link)]. FA analysis was performed using capillary GC with a CP-Sil 88 for FAME column (100 m × 0.25 mm, Agilent, Santa Clara, CA, USA) and a PerkinElmer gas chromatograph CLARUS 680 with a flame ionization detector and split injection (PerkinElmer Instruments, Waltham, MA, USA). The detailed GC conditions were described only recently [25 (link)]. The calibration was performed with the help of the reference standard mixture ‘Sigma FAME’ (Sigma-Aldrich, Deisenhofen, Germany) and the methyl esters of 18:1cis-11, 22:5n-3 and 18:2cis-9, trans-11 (Matreya LLC, State College, PA, USA), 20:3n-9, 22:3n-3 and 22:4n-6 (Sigma-Aldrich), and 18:4n-3 (Larodan, Limhamn, Sweden). The five-point calibration of single FAs ranged between 16 and 415 µg/mL and was checked after GC analysis of five samples.