J w db 23 capillary column

10 mL of cell culture was washed with ddH₂O three times and dried overnight at 104 °C. Dry cell weight was measured using an analytical balance. To identify and quantify lipids in cell biomass, lipids were transesterified to FAMEs as described previously [17 (link)]. Briefly, 1-mL cell culture was washed with ddH₂O. 100 μL glyceryl triheptadecanoate (2 mg μL⁻¹ methanol) was added to the cell pellet and internal standard. Lipids were transesterified to FAMEs with 500 μL of 0.5 N sodium methoxide (20 g L⁻¹ sodium hydroxide in methanol) followed by 40 min of vortexing at 2000 rpm. The solution was then neutralized with 40 μL sulfuric acid. FAMEs were extracted by adding 850 μL hexane followed by 20 min vortexing at 2000 rpm. The mixture was centrifuged for 1 min at 8000 rpm and the hexane layer (750 μL) was collected for GC–FID analysis (Agilent 7890B). Samples were injected with an injection volume of 1 μL, split ratio of 10, and injector temperature of 250 °C. FAME species were separated on an Agilent J&W DB-23 capillary column (30 m × 0.25 mm × 0.15 μm), with helium carrier gas at a flow rate of 1 mL min⁻¹. The temperature of the oven started from 175 °C, held for 1 min, and then ramped to 200 °C within 5 min. The FID was operated at a temperature of 280 °C with a helium make up gas flow of 25 mL min⁻¹, hydrogen flow of 30 mL min⁻¹, and air flow of 300 mL min⁻¹.

Fatty acid analyses were carried out as described (Broadwater et al., 2002). For total FA analysis, lipids were extracted in methanol/chloroform (2 : 1) from 100 seeds and 5 mg heptadecanoic acid (17 : 0) was added as an internal standard. Total seed lipids were converted into fatty acid methyl esters (FAMEs) in 1% sodium methoxide at 50°C for 1 h and extracted with hexane. FAMEs from single seeds were prepared by incubating the seed with 50 μL 0.2 m trimethylsulfonium hydroxide in methanol (Butte et al., 1982). Lipid profiles and acyl group identification were analysed on a Hewlett‐Packard 6890 gas chromatograph equipped with a 5973 mass selective detector and Agilent J&W DB 23 capillary column (30 m × 0.25 μm × 0.25 μm). The injector was held at 225°C and the oven temperature was increased from 100 to 160°C at 25°C/min, then to 240°C at 10°C/min. The FA percentage values were presented as a mean of at least three biological replicates.

Sea buckthorn seed oil was isolated using a methanol-chloroform extraction procedure^{65 (link),66 (link)}. Seed sample powder (300 mg) was homogenised in methanol (2 ml) for 1 min, and after adding chloroform (4 ml) homogenization was continued for a further 2 min. The mixtures were sonicated in an ultrasonic bath for 30 min, centrifuged and filtered. The solid residues were re-suspended in chloroform/methanol (2:1, v/v, 4 ml) and homogenised again for 3 min and filtered. The volume of 1 ml of 0.88% KCl solution was added to the combined filtrates, and the mixtures were mixed thoroughly by vortexing and then centrifuged. The lower phase containing the purified oils was collected and evaporated to dryness under nitrogen.
Fatty acid composition was determined as fatty acid methyl esters (FAMEs) based on the boron trifluoride in methanol catalysis^{67 (link)}. GC–TOF/MS analysis of FAMEs was performed on a Clarus 680 GC coupled with AxION iQT TOF/MS system (PerkinElmer, Shelton, USA). The system was equipped with Agilent J&W DB-23 capillary column (60 m × 0.25 mm × 0.25 μm). Fatty acid composition was measured and expressed as weight percentage of each fatty acid to the total fatty acids according to our previous study⁴⁵ . The analyses were conducted in three replicates.

Plasma lipids from stored frozen plasma aliquots were extracted by the Bligh and Dyer method [19 (link)]. Fatty acid methyl esters for gas chtomatography with flame ionization detector (GC-FID) analysis were prepared by a saponification step (methanolic NaOH) followed by acid esterification with BF₃ in methanol, according to ISO 5509:2000 [20 ], and heptadecanoic acid was used as internal standard. A liquid–liquid extraction was carried out with isooctane containing BHT (5 µg/mL) as preservative, and the samples were stored at −20 °C, protected from light until further analysis. PUFA analysis was performed using a Thermo Scientific TRACE GC Ultra (Thermo Scientific, Milano, Italy) GC-FID. The separation of sample components was achieved using a J&W DB-23 capillary column (Agilent Technologies, Inc., Santa Clara, CA, USA), 60 m × 0.25 mm internal diameter and 0.25 μm phase thickness. GC-FID analysis conditions and fatty acids identification were described previously [21 (link)] and results are expressed as mg·L⁻¹ of plasma.