Hp 88 column

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.

For FA analysis, approximately 100 mg of feed, liver, or breast muscle was weighed and hydrolyzed, extracted, saponified, and methylated. The FA methyl esters were subjected to Trace1310-ISQ gas chromatography and mass spectrometry (Thermos, Waltham, MA, USA) using an HP-88 column (100 m × 0.25 mm × 0.20 μm, Agilent, Santa Clara, CA, USA) for fatty acid separation. The column oven temperature was maintained at 100°C for 15 min after sample injection and was programmed to increase from 100 to 190°C at 15°C/min and maintained at 190°C for 25 min. Then the temperature was increased to 235°C at a rate of 2.5°C/min and held for 4 min. The injector and detector temperatures were 240 and 280°C, respectively. Helium was used as the carrier gas at a flow rate of 1.0 ml/min. Fatty acids were identified and quantified by comparing the retention times and peak areas with those of standard components (Sigma, Louis, MO, USA). The results were recorded as a percentage of fresh feed or breast muscle.

Total intracellular fatty acids were extracted from 60 mm culture dishes using 2 mL of sulfuric acid/methanol (2.5:1, v/v) and collected in an 8 mL glass tube for methyl esterification, as previously reported [55 (link)]. Methylated fatty acids were analyzed by gas chromatography (Agilent 7890B, Agilent Technologies Inc., Santa Clara, CA, USA) with HP-88 column (Agilent Technologies Inc., Santa Clara, CA, USA). Supelco 37 Component FAME Mix (CRM47885, Sigma) was used as external standard. Relative proportions of fatty acids in Intramuscular precursor adipocytes were determined as percentages of the total peak area.

For sterols analysis, the unsaponifiable matters of all canola oil samples and DD were extracted by the official AOCS method Ca 6a-40 [19 ]. After extraction, the unsaponifiable matters were dissolved in 5 mL of hexane. Then the sample solution was run on the GC-FID instrument (Agilent 7890 series) used for sterols analysis. For the separation of sterols, the HP-88 column (100 m, 0.25mm Agilent Technologies) was used. About 2 μL of an aliquot was injected in a splitless mode. As a carrier gas, helium (He) was used with a flow rate of 10.2 mL/min. The initial temperature programming of the oven was set to 100 °C and then increased to 10 °C /min to the final temperature of 295 °C and hold for 20 min. For the confirmation of the sterol peaks, the retention times of the authentic standards were compared. The peak areas under each sterol were determined as relative peak areas to the total peak area of all sterols.

To proceed with GC-MS assay, 5 mL of liquid coming from gastric resistance test chamber were added of 100 µL of metyl pentadecanoate solution (115 μg/mL) as internal standard. Extraction with ethyl acetate was performed three times. The obtained organic phases were pooled together, anhydrified with sodium sulfate and led to dryness. The extracts were added of 3 mL of methanol and 1 mL of dichloromethane and were acidified with concentrated sulfuric acid. Samples were heated under reflux for 40 min, then the solution were cooled down and diluted with water and diethyl ether. The etheric phase was investigated.
The standard was prepared mixing 100 mg of SRO fatty acids GC with 1 mL of metyl pentadecanoate solution (115 μg/mL) as internal standard, and adding 2 mL of dichloromethane, 10 mL of methanol and 0.2 mL of concentrated sulfuric acid. Standard sample was heated under reflux, then cooled down and diluted with water and diethyl ether. The etheric phase was investigated.
The GC-MS system was comprised of a Varian 3800 equipped with autosampler and coupled with a Varian Saturn 2100 MS/MS ion trap mass spectrometer. A HP-88 column (60 m × 0.25 mm) was used for separation (J and W Scientific, Agilent technologies Inc., Santa Clara, CA 95051, USA)

For sterols analysis, the unsaponifiable matters of all canola oil samples and DD were extracted by the official AOCS method Ca 6a-40 [19 ]. After extraction, the unsaponifiable matters were dissolved in 5 mL of hexane. Then the sample solution was run on the GC-FID instrument (Agilent 7890 series) used for sterols analysis. For the separation of sterols, the HP-88 column (100 m, 0.25mm Agilent Technologies) was used. About 2 μL of an aliquot was injected in a splitless mode. As a carrier gas, helium (He) was used with a flow rate of 10.2 mL/min. The initial temperature programming of the oven was set to 100 °C and then increased to 10 °C /min to the final temperature of 295 °C and hold for 20 min. For the confirmation of the sterol peaks, the retention times of the authentic standards were compared. The peak areas under each sterol were determined as relative peak areas to the total peak area of all sterols.