Cpsil 88 column

Total lipids were extracted from freeze-dried muscle samples (0.5~1 g) using Folch liquid (chloroform:methanol = 2:1 by vol.). Then, methanolic KOH was added, and the samples were blow dried with nitrogen before boron trifluoride and normal heptane were added sequentially. After the addition of saturated salt water, the supernatant was taken, and anhydrous sodium sulfate was added. Fatty acid methyl eaters were subjected to analysis using gas chromatography (GC-14B, Shimadzu, Suzhou, Jiangsu, China) with CP-Sil 88 column (0.25 mm × 50 m × 0.20 μm, Agilent Technologies, Beijing, China). A standard (47015-U, Supelco, Shanghai, China) was used to identify potential fatty acid components in each sample. Fatty acid composition (%) = 100 × (peak area of a certain fatty acid/peak area of total fatty acid).

Plasma total fatty acids were analyzed for controls (baseline [0 weeks], 10 samples) and in mice fed with corn oil 30 µL/mouse/day for 12–16 weeks (22 samples) or mineral oil 30 µL/mouse/day for 12–16 weeks (26 samples). The plasma samples were spiked with tricosanoic acid (C23:0) and extracted using the BUME method (Löfgren et al., 2012 (link)). Fatty acid methyl esters were produced by acid-catalyzed transesterification and were analyzed using an Agilent 7890A gas chromatograph coupled to an Agilent 5975C mass spectrometer. For separation, an Agilent DB-23 column was used. In total, 17 fatty acids were analyzed and quantified against the C23:0 fatty acid internal standard.
Liver fatty acids were extracted at 16 weeks from homogenized liver samples (500 µg protein per sample) by a Bligh and Dyer protocol (Bligh and Dyer, 1959 (link)). Approximately 10–20 mg samples of the different diets were used for dietary fatty acid composition analyses. After derivatization to fatty acid methyl esters, both liver and dietary fatty acids were analyzed by gas chromatography using a CP-Sil 88 column (Agilent Technologies Inc., Santa Clara, CA, United States) as described previously (Princen et al., 1995 (link)). In total, 17 fatty acids were analyzed.

The fatty acid (FA) profile and tocopherols content were analysed in both the dehulled hempseeds (from which oil was extracted by the Soxhlet method) and in the oil extracted by SSP. The FA profile was determined according to the AOAC official method 41.1.30 [31 ], using an Agilent-6890N gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with a CP-SIL88 Column (100 m × 0.2 mm i.d. × 0.36 μm) (Agilent Technologies, Santa Clara, CA, USA) following the method described by Rebolleda et al. (2012) [32 (link)]. An internal standard quantification method was applied using FA chromatographic standards (Sigma-Aldrich Inc., St. Louis, MO, USA) and methyl tricosanoate as the internal standard.
The tocopherols content (mg/g) was determined according to the IUPAC 2.432 official method (IUPAC, 1992), following the procedure reported by Rebolleda et al., 2012 [32 (link)]. An Agilent HPLC series 1100 (Agilent Technologies, Santa Clara, CA, USA) with ACE 5 silica column (250 mm × 4.6 mm) was used for the analysis. The eluent consisted of n-hexane/2-propanol, flowing at a rate of 0.8 mL/min. Individual compounds of α-, β-, γ- and δ-tocopherols were identified and quantified using an external calibration curve of the corresponding standard compounds. The determination of fatty acids and tocopherols content was made at least in duplicate.

Fatty acid composition was determined by gas chromatography as reported by Cui et al. [24 (link)]. Fatty acids were separated on a 7890 A series Agilent system GC-FID with a CP-Sil 88 column (100 m × 0.25 mm 0.2 µm, Agilent, Santa Clara, CA, USA). Fatty acid methyl esters (FAMEs) were identified and quantified by comparing the retention times of FAMEs standards and calculating the relative peak areas, respectively. The standard mixture of fatty acid methyl esters (FAMEs, GLC-463) was purchased from NuChek−Prep (Elysian, SP, MN, USA).

Extracted oil (Section 2.2) was dissolved in chloroform and fatty acids were derivatized to methyl esters (FAME) using transesterification procedure with 2% (v/v) sulfuric acid in methanol [16 (link)]. A gas chromatograph (model 6890 N, Agilent Technologies, Santa Clara, CA, USA) fitted with a CP-Sil 88 column (100 m × 0.25 mm i.d., 0.2 μm, Agilent Technologies, Santa Clara, CA, USA) and flame ionization detector were used in quantifying FAME. A temperature gradient program was used in the oven, and hydrogen was used as the carrier gas (constant pressure 206.8 kPa; nominal initial flow rate 2.1 mL min⁻¹ [16 (link)]). The fatty acid composition was calculated as weight percentages using theoretical response factors [17 (link)]. Analyses were performed in duplicate.