The fatty acid profile was obtained by gas chromatography in a Shimadzu GC-2010 Plus Gas Chromatograph (Shimadzu, Tokyo, Japan) using a CPSil 88 fused silica capillary column (50 m × 0.25 mm i.d.), 0.20 m film thickness (Varian, Middelburg, The Netherlands), and helium as the carrier gas (120 kPa). Each fatty acid methyl ester (FAME) was identified by direct comparison with a standard mixture (FAME 37, Supelco, Bellefonte, PA, USA) [27 (link)]. Two samples of each batch were analyzed after the extraction of the fat, and the results are expressed as the percentage of each FAME. On the other hand, Atherogenicity (AI) was calculated from the percentage of lauric (C12:0), myristic (C14:0), and pamitic (C16:0) acids, and the sum of the polyunsaturated (PUFA) and monounsaturated (MUFA) fatty acids. On the other hand, the Thrombogenicity index (TI) was calculated by using the percentage of myristic, palmitic, and stearic (C18:0) acids and the sums of MUFA, omega 6 (n = 6) and the ratio between omega-6 and omega-3 (n = 3).
Cpsil 88 fused silica capillary column
Manufactured by Agilent Technologies
Sourced in Japan
The CPSil 88 fused silica capillary column is a versatile analytical tool designed for gas chromatography applications. It features a cyanopropyl-phenyl stationary phase that provides excellent separation performance for a wide range of analytes, including fatty acid methyl esters, trans fatty acids, and other polar compounds. The column is constructed with high-quality fused silica material, ensuring durability and reproducible results.
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8 protocols using cpsil 88 fused silica capillary column
1
Fatty Acid Profiling and Cardiovascular Risk Indices
2
Fatty Acid Profile Analysis in Burgers
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With regards to fatty acid profile, first fat was extracted from burgers with a chloroform-methanol mixture (2:1) according to [36 (link)]. Then, fatty acid methyl esters (FAME) were obtained by a transmethylation according to ISO 12988-2:2017 [37 ]. Then, FAMEs were injected in a Shimadzu GC-2010 Plus Gas Chromatograph (Shimadzu, Tokyo, Japan), equipped with a CPSil 88-fused silica capillary column (50 m × 0.25 mm i.d.), 0.20 m film thickness (Varian, Middelburg, Netherlands), using helium as the carrier gas (120 kPa). Each fatty acid methyl ester (FAME) was identified by direct comparison with a standard mixture (FAME 37, Supelco, Bellefonte, PA, USA). Two samples of each batch were analyzed, and the results were expressed as the percentage of each FAME.
3
Fatty Acid Profiling of Plasma and Lipoproteins
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Lipids from plasma and lipoprotein fractions were extracted according to Hara and Radin [29 (link)] by using hexane:isopropanol (3:2) and sodium sulfate; then transmethylated with 0.5 M sodium methoxide/methanol following chemical derivatization with 50% BF3-MeOH. All chemicals and solvents used for this method were of analytical grade. For analysis of FA in plasma and lipoprotein fractions, a GC system (Agilent, GC 6890 series, Stockport, UK) equipped with a CP-Sil 88 fused-silica capillary column (100 m × 0.25 mm i.d., with 0.2 μm film thickness; Varian Inc., Oxford, UK) was used. The GC conditions were as follows: the oven temperature was initially set at 110 °C for 4 min after injection and then increased to 240 °C with equilibration time of 2 min. The inlet and flame-ionization detector temperatures were 260 °C, the split ratio was 15:1 and a 2-μL injection volume was used. The hydrogen carrier gas flow to the detector was 25 mL/min, airflow was 400 mL/min, and the flow of nitrogen makeup gas was 40 mL/min. Fatty acid peaks were identified by using a fatty acid methyl ester standard (FAME; Supelco 37 Component FAME mix, Bellefonte, PA, USA) and a TFA reference standard (C18:1 trans-11, methyl ester, Supelco, Bellefonte, PA, USA).
4
Milk Fatty Acid Composition Analysis
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Representative individual milk samples from the two daily milking were monthly analysed for protein, fat and lactose using a MilkoScan FT+ (Foss) standardized for goat milk. In addition, as described by Hara and Radin (1978 (link)), total fat of milk was separated using a mixture of hexane‐isopropane (3/2 v/v). FA transmethylation and methyl esters quantification were effected as above described for the feeds using a gas chromatograph (ThermoQuest Focus, equipped with flame ionization detector; Thermo Electron Corporation) equipped with a CP‐SIL 88 fused silica capillary column (100 m × 0.25 mm (i.d.) with 0.2‐μm film thickness; Varian). Gas chromatograph conditions were those described by Cavaliere et al. (2018 (link)). Fatty acid peaks were identified using pure methyl ester standards and CLA isomers (Larodan Fine Chemicals, AB, Limhamnsgårdens Malmö).
5
Fatty Acid Profiling by GC-FID
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Fatty acid profile was measured according to Santos et al. [23 (link)]. Briefly, 2 mL of n-hexane was added to 0.02 g of oil to obtain fatty acid methyl esters (FAME) by cold transmethylation with methanolic potassium hydroxide. Then, 200 µL of methanolic potassium hydroxide solution (2 N) was added and vigorously mixed. Then, the supernatant was carefully transferred to a glass vial and analyzed by gas chromatography in a Shimadzu GC-2010 Plus Gas Chromatograph (Shimadzu, Tokyo, Japan). This was performed using a CPSil 88 fused silica capillary column (50 m × 0.25 mm i.d.), 0.20 µm film thickness (Varian, Middelburg, The Netherlands), and helium was used as the carrier gas (120 kPa). The used temperature program was a first step of 5 min at 140 °C, followed by an increase of 5 °C/min from 140 to 220 °C, and then maintaining at 220 °C for 15 min. The temperature of the injector and detector was 250 and 270 °C, respectively, and the split ratio was 1:50 with an injection volume of 1 µL. Lastly, each FAME was identified by direct comparison with a standard mixture (FAME 37, Supelco, Bellefonte, PA, USA). All analyses were performed in duplicate, and results are expressed as the relative percentage of each FA on the basis of relative peak areas.
6
Fatty Acid Extraction and Analysis
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Tissue samples were kept at −70°C until fatty acid analysis, to limit the fatty acid degradation. The samples were thawed in an ice water bath for <1 h. As per a protocol modified from Folch, Lees, and Sloane-Stanley [12 (link)], the total lipids were extracted from the muscle with chloroform/methanol (2:1, v/v) by homogenization (Ultra Turrax, 3×15 s, 12,000 revolutions per minute) at room temperature. All solvents contained 0.005% (w/v) of t-butylhy droxytoluene in order to avoid the oxidation of polyunsaturated fatty acids (PUFA). The extraction mixture was stored at 5°C for 18 h in the dark and subsequently washed with 0.02% aqueous CaCl2. The organic phase was dried with Na2SO4 and K2CO3 (10:1, wt/wt) and the solvent was subsequently removed under nitrogen at room temperature. The lipid extract was re-dissolved in toluene and a 25-mg was used for methyl ester preparation. After 20 min an aliquot of chloroform was added to yield a solvent ratio of 2:1 v/v. Fatty acid methyl esters (FAME) were prepared. FAME was separated by capillary gas chromatography on a Cp-Sil 88 fused-silica capillary column (100 m×0.25 mm×0.25 μm), Varian-Chrompack GmbH (Beijing, China).
7
Fatty Acid Profile Analysis in Serum and Muscle
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Total lipids in the serum and gastrocnemius muscle (n = 6 per group) were extracted, using the method described by Bligh and Dyer [30] . The FA in the serum, muscle, and dietary lipids were analyzed by gas chromatography. The FA methyl esters (FAME) were obtained from the basecatalyzed methanolysis of the glycerides (KOH in methanol), after dissolving the lipid extract in high-performance liquid chromatography quality hexane [31] . The total FA profile was recorded by analyzing the FAME on a gas-liquid chromatograph (Shimadzu 2014, Kyoto, Japan), with an autoinjector (AOC-20i) Split/Splitless, equipped with a flame ionization detector and a CP Sil 88 fused silica capillary column (100 m × 0.25 mm × 0.2 μm, film thickness; Varian, Lake Forest, CA, USA), according to the American Oil Chemists' Society Official Method [32] . The FAME were identified by comparing their retention times to those of commercial standards. The values were expressed as a percentage of the total FAME. The detection limit for the main FAME identified ranged from 0.01 to 0.03%. Similar procedure was used in our previous studies [9, 14, 33] .
8
GC-FID and GC-MS Analysis of Fatty Acids
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An Agilent model 6890 N Network System (Palo Alto, CA, USA) equipped with auto injector, fitted with a FID and a CP-Sil 88 fused silica capillary column (100 m x 0.25 mm i.d., Varian, Middelburg, The Netherlands) was used. Injector and detector temperature was 250 °C. Helium was the carrier gas and inlet pressure was set at 194 kPa.
The sample volume injected was 1 µL at a split ratio of 1:100. Total chromatographic time was 90 min, including two ramps. Initial oven temperature was 45 °C. After 4 min, it was raised at 13 °C min -1 to 165 °C and held for 35 min, then increased to 215 °C at 4 °C min -1 and maintained for 30 min. Quantification was carried out using 11:0 as internal standard.
The identification of DMA and FAME was accomplished on an Agilent chromatograph (model 7890A) fitted with a mass spectrometry (MS) electronic impact detector (5975C inert MSD). The filament trap current was 400 µA at 70 eV. Injections were carried out under data system control with an injection volume of 1 µL and split ratio of 1:20. The remaining chromatographic conditions were similar to those described above for GC-FID analysis. The AOCS Lipid Library (http://lipidlibrary.aocs.org/), Wiley 275 and NIST 05 libraries were used to identify the mass spectra.
The sample volume injected was 1 µL at a split ratio of 1:100. Total chromatographic time was 90 min, including two ramps. Initial oven temperature was 45 °C. After 4 min, it was raised at 13 °C min -1 to 165 °C and held for 35 min, then increased to 215 °C at 4 °C min -1 and maintained for 30 min. Quantification was carried out using 11:0 as internal standard.
The identification of DMA and FAME was accomplished on an Agilent chromatograph (model 7890A) fitted with a mass spectrometry (MS) electronic impact detector (5975C inert MSD). The filament trap current was 400 µA at 70 eV. Injections were carried out under data system control with an injection volume of 1 µL and split ratio of 1:20. The remaining chromatographic conditions were similar to those described above for GC-FID analysis. The AOCS Lipid Library (http://lipidlibrary.aocs.org/), Wiley 275 and NIST 05 libraries were used to identify the mass spectra.
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