Rtx 2330

Fatty acids from total plasma lipids were isolated as previously described^{47 ,48} . Fatty acids were directly trans-esterified with 3 N HCl in methanol, at 85 °C for 60 min. Fatty acid methyl esters were analyzed by gas chromatograph SHIMADZU 2014 which was equipped with capillary column RESTEK Rtx 2330. The temperature program was 140–210 °C for 3°/min. Individual FA was identified by comparison with retention time of FA methyl esters commercial standards PUFA-2 (Supelco, Inc., Bellefonte, Pennsylvania, USA). Methyl esters from oils were prepared in the same way. Oils were analyzed in triplicate. The results are presented as a percentage of the total FA.

The fatty acids were esterified with a mixture of 2 M KOH in methanol. Methyl esters of fatty acids were analyzed in a gas chromatograph (TRACE™ 1300, Thermo Scientific) equipped with a flame ionization detector. The separation process was carried out on a capillary column RTX-2330 (60 m × 0.25 mm × 0.2 μm, Restek). The oven temperature was set at 50 °C (3 min); the temperature increase rate was 3 °C/min up to 250 °C (5 min). Nitrogen (1.6 mL/min) was the carrier gas. The temperatures of the injector and detector were set at 230 °C and 260 °C, respectively. The fatty acids were identified on the basis of standard retention time (Nu-Chek Prep, Inc., USA).

Fatty acid composition of seeds oil was determined by GC, according to the American Oil Chemists’ Society Official Method Ce 1-62 [55 ]. Boron trifluoride in methanol was used as methylating agent. Fatty acid methyl esters (FAMEs) were analyzed by an Agilent 7820A gas chromatograph (Agilent Technologies, Santa Clara, CA, USA), equipped with a capillary column RTX-2330, 105 m length, 0.25 mm i.d., 0.2 μm film thickness (Restek, Bellefonte, PA, USA). Injector and detector (FID) temperatures were 260 °C and 280 °C, respectively. Column temperature was set to 200 °C for 21 min, then increased to 250 °C at a rate of 10 °C/min; the final temperature was held for 6 min. Helium was used as a carrier gas, at a linear flow rate of 35 cm/sec. Individual FAMEs were identified using the Certified Reference Material (CRM) 47885 (Supelco, Bellefonte, PA, USA). The following fatty acid combinations were calculated: total saturate fatty acids (SFA), total monounsaturated fatty acids (MUFA) and total polyunsaturated fatty acids (PUFA).

Lipids from muffins were extracted according to Folch, Less, and Sloane-Stanley [33 (link)], using a 2:1 chloroform/methanol (v/v) mixture. Fatty acid (FA) methyl esters (FAME) were prepared by transmethylation of fat samples using H₂SO₄ (95%) and methanol accordingly American Oil Chemists’ Society (AOCS, 2000) [34 ]. FAMEs were analysed by gas chromatography (GC) using an Agilent 6890N (USA) instrument equipped with flame ionisation detector (FID). FAMEs were separated on a capillary column Rtx 2330 with highly polar stationary phase (100 m × 0.25 mm I.D. × 0.1 µm thickness, Restek Corp., USA). The operating conditions and separation of FA methyl esters (FAME) were published in detail elsewhere [35 (link)]. The Supelco 37 standard (No. 47885-U, Sigma Aldrich) was applied to identify FAs. The FAs were quantified in relation to the internal standard, nonadecanoic FA (C19:0) (Sigma, Aldrich, USA) that was added before transesterification to lipid samples.

Permethylation was performed as previously described with slight modification^{54 (link)}. The glycan sample was purified to a monosaccharide content <1.0% using chromatography before permethylation. The glycan sample (500 µg) was dissolved in dimethyl sulfoxide (DMSO) for 30 min with gentle stirring. A freshly prepared sodium hydroxide suspension in DMSO was added, followed by a 10 min incubation. Iodomethane (100 µL) was added, followed by a 20 min incubation. A repeated round of sodium hydroxide and iodomethane treatment was performed for complete permethylation. The permethylated sample was extracted, washed with dichloromethane (DCM), and blow dried with nitrogen gas. The sample was hydrolyzed in 2 M trifluoroacetic acid (TFA) for 2 h, reduced overnight with sodium borodeuteride (10 mg mL⁻¹ in 1 M ammonia), and acetylated using acetic anhydride/TFA. The derivatized material was extracted, washed with DCM, and concentrated to 200 µL. Glycosyl linkage analysis was performed on an Agilent 7890 A GC equipped with a 5975 C MSD detector (EI mode with 70 eV), using a 30-meter RESTEK RTX®-2330 capillary column. The GC temperature program: 80 °C for 2 min, a ramp of 30 °C min⁻¹ to 170 °C, a ramp of 4 °C min⁻¹ to 245 °C, and a final holding time of 5 min. The helium flow rate was 1 mL min⁻¹, and the sample injection was 1 µL with a split ratio of 10:1.