The fatty acid (FA) composition of dietary fats [cow's milk cream, rich in saturated FAs (SFAs); refined olive oil, rich in monounsaturated FAs (MUFAs); and refined olive oil plus eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), rich in MUFAs and omega-3 long-chain polyunsaturated FAs (PUFAs)] was determined by the method described in EEC/796/2002, 7 using a gas chromatography system (HP-5890, Hewlett-Packard) equipped with flame ionization detector and a SP-2380 capillary column (Supelco, 30 m x 0.32 mm) packed with cyan propyl siloxane (0.25 µm). The initial column temperature was 165 ºC, which was held for 10 min, then programmed from 165 ºC to 200 ºC at 1.5 ºC/min. Injector and detector temperature were 250 ºC, with the carrier gas H 2 . The FA composition of different dietary fats is detailed in Table 1.
Sp 2380 capillary column
Manufactured by Merck Group
Sourced in United States, Germany
The SP-2380 capillary column is a chromatography column used for the separation and analysis of fatty acid methyl esters (FAME). The column features a highly polar stationary phase that allows for the effective separation of a wide range of FAME compounds. The column dimensions and technical specifications are tailored to provide reliable and consistent separation performance.
Automatically generated - may contain errors
Lab products found in correlation
Hp 5890, by Hewlett-Packard (5 mentions)
Supelco 37 component fame mix, by Merck Group (2 mentions)
Hp 6890 gc system, by Agilent Technologies (2 mentions)
Hp 5973 electron impact mass spectrometer, by Agilent Technologies (2 mentions)
Agilent 7890a gc, by Agilent Technologies (1 mentions)
Agilent 4890d, by Agilent Technologies (1 mentions)
Silica gel 60 plate, by Merck Group (1 mentions)
Boron trifluoride, by Merck Group (1 mentions)
2 7 dichlorofluorescein, by Merck Group (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Gc fid, by Agilent Technologies (1 mentions)
Flash ea 1112, by Thermo Fisher Scientific (1 mentions)
Qp2010, by Shimadzu (1 mentions)
Gc 7820a, by Agilent Technologies (1 mentions)
37 component fame mix, by Merck Group (1 mentions)
Protein assay dye reagent, by Bio-Rad (1 mentions)
21 protocols using sp 2380 capillary column
1
Dietary Fat Composition Analysis
2
Fatty Acid Profiling of Lipid Samples
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The nutritional makeup of the LM samples was established using methods recommended by the AOAC [27 ]. The total lipids were extracted from 3.0 g of LM or 0.1 g of subcutaneous fat samples using chloroform/methanol (2:1 v/v) according to the procedures described by Lee et al. [24 (link)]. The extracted lipids were saponified and esterified according to the AOCS method [28 ] for preparation of fatty acid methyl esters (FAMEs). The prepared FAMEs were analyzed using a Thermo Electronic (Austin, TX, USA) gas chromatography apparatus (Model TRACE GC Ultra) equipped with an automatic sampler Model AS-3000 (Thermo Electronic Co., Waltham, MA, USA). A 0.25 mm i.d. by 60 m long fused silica SP-2380 capillary column (Supelco, Inc., Bellefonte, PA, USA) was used to separate the methyl esters, which were detected using a flame ionization detector (FID). The injection temperature was 240 °C, and the column temperature was programmed from 130 °C to 220 °C at 4 °C/min. Helium was the carrier gas, with a flow rate at 1.6 mL/min and a split ratio of 30:1. Furthermore, the identification and quantitation of individual FAMEs in the sample were also completed according to the AOCS method [28 ].
3
Fatty Acid Profiling of Dairy and Plant Oils
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The fatty acid composition of cow's milk cream, refined olive oil, and refined olive oil plus omega-3 long-chain PUFAs was determined, in triplicate from the same lot, by the method described in EEC/796/2002 [17] using a gas chromatography system (HP-5890, Hewlett-Packard) equipped with flame ionization detector and a SP-2380 capillary column (Supelco, 30 m 0.32 mm) packed with cyanopropyl siloxane (0.25 μm) (Table 1). The initial column temperature was 165 ºC, which was held for 10 min, then programmed from 165 ºC to 200 ºC at 1.5 ºC/min. Injector and detector temperature were 250 ºC, with the carrier gas H2. For fatty acid composition in postprandial TRLs (named TRL-SFAs from cow's milk cream, TRL-MUFAs from refined olive oil, and TRL-MUFAs+3 from refined olive oil plus omega-3 long-chain PUFAs), aliquots of 100 μL were lyophilized. A solution composed of 2.64 mL of methanol:toluene:dimethoxypropane:sulphuric acid (16.5:5:1:1) and heptane was added on the lyophilized residue. After shaking, and incubating the mixture at 80 °C for 1 h, the upper phase was transferred to another vial and dried with a stream of N2 gas. The resulting extract was dissolved in heptane and the fatty acid methyl esters were analyzed into a gas chromatography system as described above (Table 2).
4
Analyzing Fatty Acid Composition in Milk, Oils, and Lipoproteins
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The fatty acid composition of cow’s milk cream, refined olive oil, and refined olive oil plus omega-3 long-chain PUFAs was determined, in triplicate from the same lot, by the method described in EEC/796/2002 [17 ] using a gas chromatography system (HP-5890, Hewlett-Packard, Waldbronn, Germany) equipped with flame ionization detector and a SP-2380 capillary column (Supelco, 30 m × 0.32 mm) packed with cyanopropylsiloxane (0.25 µm) (Supplementary Materials Table S1 ). The initial column temperature was 165 °C, which was held for 10 min, then programmed from 165 °C to 200 °C at 1.5 °C/min. Injector and detector temperature were 250 °C, with the carrier gas H2.
For fatty acid composition in postprandial TRLs (named TRL-SFAs from cow’s milk cream, TRL-MUFAs from refined olive oil, and TRL-PUFAs from refined olive oil plus omega-3 long-chain PUFAs), aliquots of 100 µL were lyophilized [18 (link)]. A solution composed of methanol: toluene: dimethoxypropane: sulphuric acid (16.5:5:1:1) and heptane was added on the lyophilized residue. After shaking and incubating the mixture at 80 °C for 1 h, the upper phase was transferred to another vial and dried with a stream of N2 gas. The resulting extract was dissolved in heptane and the FA methyl esters were analyzed into a gas chromatography system as described above (Table 1 ).
For fatty acid composition in postprandial TRLs (named TRL-SFAs from cow’s milk cream, TRL-MUFAs from refined olive oil, and TRL-PUFAs from refined olive oil plus omega-3 long-chain PUFAs), aliquots of 100 µL were lyophilized [18 (link)]. A solution composed of methanol: toluene: dimethoxypropane: sulphuric acid (16.5:5:1:1) and heptane was added on the lyophilized residue. After shaking and incubating the mixture at 80 °C for 1 h, the upper phase was transferred to another vial and dried with a stream of N2 gas. The resulting extract was dissolved in heptane and the FA methyl esters were analyzed into a gas chromatography system as described above (
5
Fatty Acid Composition Analysis of Dietary Fats
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The fatty acid composition of dietary fats (cow’s milk cream rich in SFAs, refined olive oil rich in MUFAs, and refined olive oil plus eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) rich in MUFAs and omega-3 long-chain PUFAs) was determined by the method described in EEC/796/2002 [24 (link)]. This involved using a gas chromatography system (HP-5890, Hewlett-Packard, Palo-Alto, CA, USA) equipped with a flame ionization detector and a SP-2380 capillary column (Supelco, Bellefonte, PA, USA, 30 m × 0.32 mm) packed with cyanopropyl siloxane (0.25 μm). The initial column temperature was 165 °C, which was held for 10 min and then programmed to rise from 165 °C to 200 °C, at 1.5 °C min−1. The injector and detector temperatures were 250 °C, with H2 as the carrier gas. The fatty acid composition of the dietary fats in each customized diet (HFD-SFA, HFD-OO and HFD-OO-ω3) is shown in Table 2 and was similar to that described in [25 (link)].
6
Serum Fatty Acid Profiling in Mice and Humans
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For in vivo lipolysis assays in mice, serum samples were collected, and free fatty acid levels were then quantified per the manufacturer’s instructions (Biovision Abcam). Serum fatty acids from patients were analyzed by gas chromatography–mass spectrometry performed by the Analytical Facility for Bioactive Molecules platform at the Hospital for Sick Children (Toronto, Ontario, Canada). Briefly, serum samples (20 μL) were spiked with an internal standard mix and acidified with HCl. Nonesterified fatty acids were acidified and double extracted with hexane. The fatty acids were then converted to their pentafluorobenzyl esters using 1% pentafluorobenzyl bromide/diisopropylamine (1:1) and separated by automated gas chromatography (GC Agilent 7890A, Agilent Technologies) on a fused-silica SP2380 capillary column (30 m × 0.25 mm × 0.2 μm film thickness; Supelco Analytical). Fatty acid ions were detected and measured using a MSD Agilent 5975C quadrupole mass detector (Agilent Technologies). Peaks of fatty acid esters were identified by comparisons with individual fatty acid standards (Supelco Analytical).
7
Quantification of Fatty Acids in Plasma
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The quantification of FAs was performed as in previous studies [50 (link)]. Briefly, the lipid fraction of plasma samples of dams and pups was extracted with a mix of CHCl3/MeOH 2:1 (v/v), and then derivatized to obtain the FA methyl esters (FAMEs), which were determined by gas chromatography in an Agilent 4890D chromatograph (Agilent Technologies, Waldbronn, Germany) equipped with an SP-2380 capillary column (60 m, 0.25 mm i.d., 0.2 µm, Supelco, Bellefonte, PA, USA), a split/splitless injector set at 270 °C and a flame ionization detector set at 300 °C. The split ratio was 1:30. For FAME separation the oven program was configured as follows: initial temperature 150 °C (held at this temperature for 1 min) up to 180 °C at 3 °C/min, from 180 °C (0.5 min) up to 220 °C at 14.5 °C/min, and from 220 °C (3 min) up to 250 °C at 9.9 °C/min, maintaining 9.5 min at 250 °C. The carrier gas was hydrogen (1.72 × 105 Pa). Two µL of the samples were injected, and the FA were identified by comparing the retention time with a standard mix (Supelco 37 component FAME Mix, Sigma-Aldrich Co., St Louis, MO, USA). A total of 26 FAs were identified. The quantification was performed by peak area normalization (the quantitative results are obtained by expressing the area of a given peak as a percentage of the sum of the areas of all the identified peaks).
8
Lipid Extraction and Fatty Acid Analysis
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Total lipids were extracted from cell lysates (0.1% Triton in water) by the method of Bligh and Dyer [14 (link)] after addition of butylated hydroxytoluene (BHT, 50 µM final) as an antioxidant. PC17:0, 17:0, TG17:0 (Sigma, St-Quentin Fallavier, France), were added to serve as internal standards. Lipids were separated on thin layer chromatography (silica gel 60 plates, Merck, Fontenay sous Bois, France) using the solvent system hexane/diethylether/acetic acid 80:20:1 (v/v), revealed under UV light after spraying 0.05% 2′7′-dichlorofluorescein (Sigma) in methanol and identified by comparison with an authentic standard spotted on the same plate. Phospholipids, free fatty acids and triglycerides (TG) spots were scrapped from the plate and transmethylated using boron trifluoride (Sigma, St-Quentin Fallavier, France). The resulting fatty acid methyl esters (FAME) were analyzed by GC using a Hewlett-Packard system equipped with a Supelco SP2380 capillary column (60 m × 0.22 mm) with helium as a carrier gas. FAME were identified by comparison with commercial standards [15 (link)]. Results are expressed as molar percentage of each individual fatty acid calculated from the sum of all detected fatty acids taken as 100%. Quantification of PL, TG and free FA pools was made in correspondence with their fatty acid content using the internal standards.
9
Comprehensive Chemical Characterization of Polysaccharides
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The neutral sugar content of the sample was obtained using the phenol-sulfuric acid method (9 (link)) using galactose as a standard. The uronic acid content was obtained using the m-hydroxybiphenyl method (10 (link)), using galacturonic acid as a standard. Protein content was obtained using the Bradford method (11 (link)), using bovine serum albumin as a standard. The thiobarbituric acid (TBA)-positive material content of the sample was obtained using the TBA method (12 (link)), modified to fit our laboratory conditions and using 2-keto-3-deoxy-D-manno-octulosonic acid (Kdo) as a standard. Sugar components were analyzed using gas chromatography (GC) after converting individual sugar components into alditol acetate derivatives (13 (link)) by hydrolyzing the sample for 90 min at 121ºC using 2 M trifluoroacetic acid. GC analysis was conducted using a GC ACME-6100 (Young-Lin Co., Anyang, Korea) equipped with a SP-2380 capillary column (0.2 μm×30 m, Supelco, Inc., Bellefonte, PA, USA) under standard temperature conditions [60ºC (1 min), 60ºC→ 220ºC (30ºC/min), 220ºC (12 min), 220ºC→250ºC (8ºC/min), and 250ºC (15 min)]. The mole % of each monosaccharide was calculated from the area ratio of its respective peak, the reaction coefficient of each monosaccharide to the flame ionization detector, and the molecular weight of the alditol acetate derivative of each monosaccharide.
10
Comprehensive Compositional Analysis of RGP
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This study analyzed the composition of RGP using various analytical methods. The measurement of neutral sugar content was conducted utilizing the phenol-sulfuric acid method [23 (link)], whereas uronic acid content was analyzed using the m-hydroxybiphenyl method [24 (link)]. Total polyphenol content was assessed using the Folin–Ciocalteu spectrophotometric method [25 (link)], and the protein content was analyzed using the Bradford assay (Bio-Rad, Hercules, CA, USA). The KDO content was determined using the thiobarbituric acid method [26 (link)], and the monosaccharide composition using the alditol acetate method [27 (link)] with slight modifications.
The sugar composition of RGP was analyzed using gas chromatography (GC) with a modified alditol acetate method. A Young-Lin Co. ACME-6100 GC instrument, equipped with a SP-2380 capillary column (Supelco, Bellefonte, PA, USA) and a flame ionization detector (FID), was employed for the analysis. The GC temperature program included the following steps: 60 °C (1 min), 60 → 220 °C (30 °C/min), 220 °C (12 min), 220 → 250 °C (8 °C/min), and 250 °C (15 min). Molecular ratios were determined by calculating peak areas and applying appropriate response factors.
The sugar composition of RGP was analyzed using gas chromatography (GC) with a modified alditol acetate method. A Young-Lin Co. ACME-6100 GC instrument, equipped with a SP-2380 capillary column (Supelco, Bellefonte, PA, USA) and a flame ionization detector (FID), was employed for the analysis. The GC temperature program included the following steps: 60 °C (1 min), 60 → 220 °C (30 °C/min), 220 °C (12 min), 220 → 250 °C (8 °C/min), and 250 °C (15 min). Molecular ratios were determined by calculating peak areas and applying appropriate response factors.
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