About 1 g of finely chopped seed samples were taken for initial extraction with chloroform and methanol (2:1) followed by agitation at room temperature for 1 h and centrifuged for 5 min at 3500× g. Fatty acid profiling was performed at the School of Agriculture and Food sciences, University of Queensland laboratory. The GC-MS (Shimadzu QP2010, Shimadzu Coporation, Tokyo, Japan) was used at oven temperature of 100 °C, injector temperature 250 °C, total program time was 39 min, and helium used as the carrier gas. The inlet pressure used for gas chromatography was 0.4 kPa, at linear gas velocity of 42.7 cm/s, column (Restek stabilwax capillary column; 30 m × 0.25 mm ID × 0.5 µm film thickness) flow 1.10 mL/min with a split ratio of 1:1 and injection volume of 0.2 µL. For mass spectrometry, the ion source temperature used was 200 °C, the interface temperature was 250 °C and the mass range was 35–500 atomic mass units. Identification of the compounds was done by comparing their retention times and mass spectra with corresponding data from a standard food industry FAME Mix (Restek Corporation, Bellefonte, PA, USA).
Fame mix
Manufactured by Restek
Sourced in United States
FAME Mix is a calibration standard for use in the analysis of fatty acid methyl esters (FAMEs) by gas chromatography. It contains a mixture of common FAME compounds that can be used to identify and quantify FAME components in sample extracts.
Automatically generated - may contain errors
Lab products found in correlation
Qp2010, by Shimadzu (1 mentions)
Stabilwax capillary column, by Restek (1 mentions)
Astaxanthin, by Merck Group (1 mentions)
Tocopherol, by Merck Group (1 mentions)
Apocarotenal, by Merck Group (1 mentions)
Canthaxanthin, by Merck Group (1 mentions)
Zeaxanthin, by Merck Group (1 mentions)
β cryptoxanthin, by Merck Group (1 mentions)
Lycopene, by Merck Group (1 mentions)
β carotene, by Merck Group (1 mentions)
Lutein, by Merck Group (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
Ethanol, by Merck Group (1 mentions)
N hexane, by Merck Group (1 mentions)
Acetone, by Merck Group (1 mentions)
Dichloromethane, by Merck Group (1 mentions)
Agilent 5975, by Agilent Technologies (1 mentions)
Rt 2560 capillary column, by Restek (1 mentions)
5 protocols using fame mix
1
Fatty Acid Profiling of Seed Samples
2
Carotenoids and Tocopherols Quantification
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Lutein, zeaxanthin, β-cryptoxanthin, canthaxanthin, astaxanthin, apocarotenal, physalien, β-carotene, lycopene and tocopherols (α, β, γ and δ) were from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Acetonitrile, methanol, ethanol, dichloromethane, acetone, n-hexane and ascorbic acid were of analytical-grade purity and purchased from Merck (Darmstadt, Germany). Water was prepared using a Millipore Simplicity 185 S.A., 67120, water purification system (Molshem, France). The standard FAME Mixture of 37 components was purchased from Food Industry FAME Mix, RESTEK (Bellefonte, PA, USA) (lot 24676).
3
Exosome Lipid Fatty Acid Profiling
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The fatty acid profile of total exosome lipids was determined by fatty acid methyl ester (FAME) analysis. Total lipid extract was trans-esterified in 2 ml of methanol H2SO4 2% at 70°C for 1 h. FAMEs were analysed by GC-MS on an Agilent 5975 in series with GC (Hewlett Packard 6890 series) (Agilent Technologies). Quantification was achieved by normalisation with an internal standard of nonadecanoic acid methyl ester and the response factors for the various fatty acids were calculated with weighted methyl ester calibrators FAME mix (Restek, Lisses, France) [21 (link)].
4
Quantifying Intramuscular Fat Composition
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For IMF analysis, the tissue samples of the LT muscle (30 g) were freeze-dried and pulverized. Thereafter, an examination of the IMF content of LT muscle tissue samples was conducted using the ether extraction method according to the Association of Official Analytical Chemists regulations [34 ]. The expression of IMF content was reported as g of lipid in 100 g of muscle tissue. After the extraction of lipids, they were converted into FA methyl esters (FAMEs) using the method described in a previously published study [5 (link)]. The FA composition was evaluated with the method established in our previous study [15 (link)] by using a gas chromatography-flame ionization detector (GC-FID, SCION 456-GC, Bruker Daltonics, Fremont, CA, USA). Separation was performed on an RT-2560 capillary column (RESTEK, Bellefonte, PA, USA). The FAMEs were identified and quantified with a 37-component standard FAME Mix (RESTEK). Individually, FA composition values were reported as g per 100 g of total FAs. Subsequently, sums of SFAs (C12:0 + C14:0 + C16:0 + C18:0 + C20:0), MUFAs (C16:1n-7 + C18:1n-9 + C20:1n-9), and n-6 polyunsaturated FAs (PUFAs) (C18:2n-6 + C18:3n-6 + C20:2n-6 + C20:3n-6 + C20:4n-6) were estimated. Lastly, ratios of MUFAs to SFAs, MUFAs to n-6 PUFAs, and n-6 PUFAs to SFAs were then calculated.
5
Margarine Fatty Acid Profiling
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The fat was extracted from the margarine samples at 50 • C, and the resulting fat phase was filtered through filter paper with anhydrous Na 2 SO 4 [18] (link). The fatty acid methyl ester (FAME) preparation assumed the solubilization of margarine's fat in n-hexane. Then, 0.2 mL methanolic potassium hydroxide (2 mol/L) was added as a transesterification agent, following the method described by Jirarattanarangsri [19] . The standard used contained a FAME Mix of 37 components (FAME Mix, Restek, Bellefonte, PA, USA), and the identification of margarine FAME was made by comparing their retention time with those of the FAME Mix standard. Additionally, the resulting mass spectra were confronted with the ones from the GC-MS database (NIST MSSearch 2.0). The GC-MS (GC MS QP 2010 Plus, Shimadzu, Kyoto, Japan) following the method described by Oroian et al. [20] (link), using a SUPELCOWAX10 capillary column (60 m length, 0.25 mm in diameter, with 0.25 µm film thickness, Supelco Inc., Bellefonte, PA, USA) and helium gas with a flow rate of 0.8 mL/min and a split ratio of 1/24; the injection volume was set at 0.001 mL.
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