Bf3 meoh

For formation of FAMEs the NL and PL fractions were redissolved in 2 mL of heptane, before addition of 1.5 mL of 3.3 mg/mL sodium methoxide. The sodium methoxide solution was made by dissolving metallic sodium (Purum, Merck, Darmstadt, Germany) in methanol to a concentration of 3.3 mg/mL. The culture tubes were then shaken horizontally for 30 min at 350 rpm (Biosan Ltd., PSU-10i, Riga, Latvia) and left to settle vertically for 10 min before the heptane phases were transferred to vials for storage at −20°C prior to GC-MS analysis. The FFA fractions were redissolved in 1 mL BF₃-MeOH (14%, Sigma-Aldrich, St. Louis, MO, USA). The samples were heated for 5 min at 70°C in a water bath. After heating, 1 mL heptane was added to each sample tube before mixing on a vortex mixer. The heptane phases were transferred to vials and stored at −20°C prior to analysis by GC-MS.

Methanol, chloroform, dichloromethane of analytical standard grade and SPME fibers were purchased from Supelco (Bellefonte, PA, USA). The fibers of carboxen/polydimethylsiloxane (CAR/PDMS), polydimethylsiloxane (PDMS), carbowax/polydimethylsiloxane divinylbenzene (CAR/PDMS/DVB) were tested. BF₃-MeOH and tretramethylamonnium hydroxyde (TMAH) used for derivatization were purchased from Sigma-Aldrich (Darmstadt, Hessen, Germany). C₉–C₁₈ standard solution was obtained from separate compounds provided by Sigma Aldrich. Solutions were performed in methanol at 2 mM, mixed and further diluted before the experiment. Standards of low molecular weight compounds for SPME (certified reference material CRM46975) were purchased from Supelco.

Sodium methylate in methanol (CH3ONa, Merk) was used for the saponification of milk fat. Boron trifluoride solution in methanol (14% w/v) (BF3/MeOH, Sigma‐Aldrich) was used for the esterification of fatty acids, and n‐hexane (Karal) and sodium chloride (NaCl, Fermont) for the extraction of fatty acids. Anhydrous sodium sulfate (Na2SO4, J.T. Baker) was used as a desiccant. Hydrogen (INFRA) was used as a carrier gas for the gas chromatograph. As an internal standard, C13: 0 methyl ester (tridecanoic acid, Sigma) was used; while for the determination of the retention time of the fatty acids, the pure standards FAME Mix C4‐C24 (purity 98.7%–99.9%, Supelco), trans‐6‐Octadecenoic acid methyl ester (trans‐6‐Petroselaidic acid methyl ester, purity 99.0%, Supelco), linoleic Acid Methyl Ester Mix (cis/trans) (purity 98.0%, Supelco), and trans‐9‐Octadecenoic acid (elaidic acid, purity >99%, Supelco) were used.

The previously obtained HepG2 crude lipid extract was dissolved in 5 ml of 1 M potassium hydroxide in methanol (KOH/MeOH) solution and boiled with reflux for 15 min. Then, after cooling, 5 ml of distilled water was added, and the solution was extracted three times with 2 ml of hexane. The solution and hexane were then collected together and dried over anhydrous magnesium(II) sulfate(VI) (MgSO₄). Then, the solvent was removed on a rotary evaporator and 5 ml of 14% solution of boron trifluoride in methanol (BF₃/MeOH) (Sigma-Aldrich, Steinheim, Germany) was added for the methylation process for 15 min at 80 °C. After cooling, 2 mL of distilled water was added and the solution was extracted three times with 2 mL of hexane, which were again combined together. The hexane was washed with saturated NaCl solution and dried over anhydrous MgSO₄. In the last step, HepG2 FAMEs solution was densified to an approximate volume of 200 μL under nitrogen and transferred to a GC-MS vial with a glass insert.
After the GC-MS analysis, when the absence of the heptadecanoic acid methyl ester (Me. C17:0) (Sigma-Aldrich, Steinheim, Germany) was confirmed, the procedure was repeated with the addition of 20 μg of Me (C17:0 before dissolving the sample in KOH/MeOH solution, as per the internal standard (IS)). The preparations were made in triplicate.

Folin–Ciocalteu reagent, sodium carbonate, gallic acid, FRAP reagent, acetate buffer, acetic acid, 2,4,6-tripyridyl-1,3,5-triazine (TPTZ), 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), potassium persulfate, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), p-methoxyphenol, chloroform/(folsch), BF₃/MeOH, potassium hydroxide, hexane, sodium chloride, methanol, hydrochloric acid, magnesium sulfate, pyridine, and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) were purchased from Sigma-Aldrich (Steinheim, Germany).

Acetone, calcium carbonate, ethanol, potassium persulphate, sodium acetate, and sodium carbonate were purchased from IDALIA (Radom, Poland); azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS), diphenyl-2-picrylhydrazyl (DPPH), ferric-reducing antioxidant power (FRAP), Folin–Ciocalteu's phenol reagent, gallic acid, tripyridyl-S-triazine (TPTZ), and Trolox were purchased from Archem (Łany, Poland); acetic acid, activated carbon, ammonium metavanadate, ammonium molybdate tetrahydrate, ascorbic acid, barium chloride dihydrate, cyclohexane, magnesium nitrate, 65% nitric acid, oxalic acid, sodium bicarbonate, and sodium sulfate were purchased from CHEMPUR (Piekary Śląskie, Poland); 2,6-dichlorophenolindophenol sodium salt hydrate was purchased from ACROS ORGANICS (ARGENTA; Poznań, Poland); chloroform, hydrochloric acid (38%), and methanol were purchased from STANLAB (Lublin, Poland); standard solutions and Tween TM 80 were purchased from Merck (Darmnstadt, Germany); 2-undecanone and BF₃/MeOH were purchased from Sigma-Aldrich (Saint Louis, MO, USA); hexane and sodium bicarbonate were purchased from UQF (Wrocław, Poland); n-hexane (99%) was purchased from POCH Basic (Gliwice, Poland); helium was purchased from Air Products (Warsaw, Poland); and potassium hydroxide was purchased from Avantor (Gliwice, Poland).

For the fatty acid composition analysis, 10 g of each sample was mixed with 100 mL of a chloroform and methanol (2:1) solution, extracted at room temperature for 24 h, and then concentrated under reduced pressure, following the method described by Folch et al. [18 (link)]. After methyl esterification of the extracted lipids with a 14% boron trifluoride–methanol (BF₃-MeOH; Sigma-Aldrich) solution, they were analyzed with a GC-FID (Hewlett Packard) coupled to an HP-88 column (100 m × 0.25 mm, 0.2 μm; Agilent Technologies). The column temperature was maintained at 140 °C for 5 min, heated at 4 °C per min, and maintained at 240 °C for 20 min. The inlet temperature was maintained at 260 °C, and the Agilent Flame Ionization Detector (FID; HP 6890 Plus; Agilent Technologies) was kept at 270 °C. N₂ gas was used as the carrier gas at a flow rate of 1 mL/min, and the split ratio was adjusted to 1/50. A sample volume of 1 μL was injected, and the fatty acid peaks were confirmed by comparing the retention times of the methyl esters with those of a standard 37-component fatty acid methyl ester (FAME) mix (CRM47885; Supelco, St. Louis, MO, USA). The analyzed individual fatty acid contents were obtained by calculating the area ratio of each fatty acid to the total fatty acid area and expressed as a percentage of each fatty acid.