Fasting blood samples were taken between the 26th and the 28th week of gestation. Plasma was prepared by centrifugation and was stored at −80°C until analysis. Total lipid extraction was carried out with chloroform/methanol (2:1 v/v) and PC, which contributes about 75% of plasma phospholipids, was isolated by solid-phase extraction on aminopropylsilica cartridges and eluted with chloroform/methanol (3:2 v/v). Fatty acid methyl esters were generated by reaction of purified PC with 2% sulfuric acid (v/v) at 50°C for 2 hours, extracted into hexane and separated by gas chromatography. A BPX-70 column (30 m × 220 μm; film thickness 0.25 μm) fitted to a Hewlett-Packard HP6890 gas chromatograph was used for separation with helium as the running gas and detection of fatty acid methyl esters by flame ionization before quantification using the ChemStation software in absolute concentration (μg/mL plasma). Plasma PC fatty acids were expressed as percentages of total plasma PC fatty acids, and the ratio of total n-3 to n-6 PUFAs were calculated accordingly.
Bpx 70 column
Manufactured by Hewlett-Packard
Sourced in United States
The BPX-70 column is a laboratory equipment used for gas chromatographic analysis. It is designed to effectively separate and analyze complex mixtures of volatile organic compounds. The column features a high-quality stationary phase that provides efficient separation, enabling accurate identification and quantification of the sample components.
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8 protocols using bpx 70 column
1
Gestational Plasma Phospholipid Fatty Acid Profile
2
Fatty Acid Profiling by GC-FID and GC-CACI-MS/MS
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Samples (200 mg) were homogenized and lipids were extracted using a standard protocol [36 (link)]. Extracts were methylated overnight at 40°C in 1% methanolic sulfuric acid and subsequently transmethylated [37 (link)], using a modified protocol as described [38 (link)]. The resulting fatty acid methyl esters (FAME) were dissolved in heptane and stored at -20°C until analyses. FAME were quantitatively analyzed using a HP 5890 series II GC-FID with a BPX 70 column (length: 60 m, inner diameter: 0.32 mm, film: 0.25 μm; Hewlett Packard, Palo Alto, CA, USA) with H2 was used as a carrier gas, and a GC-flame ionization detector and structural identification by gas chromatography-covalent adduct chemical ionization tandem mass spectrometry (GC-CACI-MS/MS) as previously described [39 (link)–42 (link)]. An equal weight FAME mixture (68A; Nu-Chek Prep, Inc., Elysian, MN, USA) was used to calculate response factors on a daily basis [41 (link)]. All GC analyses were performed in triplicate.
3
Determination of RBC EPA and DHA
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Determinations of RBC EPA and DHA were performed according to the method by Fisk et al.22 (link). Plasma and RBC lipids were extracted into chloroform: methanol and fatty acid methyl esters were formed by heating the lipid extract with methanolic sulphuric acid. The fatty acid methyl esters were separated by gas chromatography on a Hewlett Packard 6890 gas chromatograph fitted with a BPX-70 column. Fatty acid methyl esters were identified by comparison with runtimes of authentic standards and data were expressed as weight % of total fatty acids.
4
Erythrocyte Omega-3 Fatty Acids Analysis
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Fasting blood samples were collected from participants by a nurse into 4-mL sodium citrate vacutainer tubes (BD Vacutainer®, Franklin Lakes, NJ) and centrifuged at 4°C (4000g for 10 min). After centrifugation, erythrocytes were collected with a disposable pasteur pipette and transferred into eppendorfs, which were stored in a − 80°C freezer until further analysis. Erythrocyte EPA and DHA were assessed using gas chromatography as described elsewhere (19 ). Briefly, erythrocyte lipids were extracted into chloroform–methanol, and fatty acid methyl esters (representing the erythrocyte fatty acids) were formed by heating the lipid extract with methanolic sulfuric acid. The fatty acid methyl esters were separated by gas chromatography on a Hewlett Packard 6890 gas chromatograph fitted with a BPX-70 column using the settings and run conditions described elsewhere (19 ). Fatty acid methyl esters were identified by comparison with run times of authentic standards. Data are expressed as weight % of total fatty acids. O3I was calculated by summing the percentages of EPA and DHA according to Harris and von Schacky (5 (link)).
5
RBC Fatty Acid Profiling by GC
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Concentrations of EPA and DHA in red blood cells (RBCs) were measured using gas chromatography [23 (link)]. Briefly, RBC lipids were extracted into chloroform methanol and fatty acid methyl esters (representing the RBC fatty acids) were formed by heating the lipid extract with methanolic sulphuric acid. The fatty acid methyl esters were separated by gas chromatography on a Hewlett Packard 6890 gas chromatograph fitted with a BPX-70 column. Fatty acid methyl esters were identified by comparison with run times of authentic standards. Fatty acids are expressed as a % of total fatty acids present.
6
Fatty Acid Profiling via GC-FID and GC-MS/MS
7
Fatty Acid Composition Analysis
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Fasting blood samples were collected at each of the 9 clinic visits during the 12-mo intervention, from which plasma phosphatidylcholine, platelets, and MNCs were isolated and analyzed for FA composition. The preparation and analysis of blood samples has been described previously (15 (link)). Briefly, FAs were analyzed by GC, performed on a Hewlett Packard 6890 gas chromatograph fitted with a BPX-70 column (30 m × 0.22 mm × 0.25 μm). The instrument was controlled by and data were collected using HPChemStation (Hewlett Packard). FAMEs were identified by comparison of retention times with those of authentic standards run previously.
8
Erythrocyte Fatty Acid Profiling
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Sample collection and fatty acid determination were outlined elsewhere [10 (link)]. In brief, blood samples were collected into 4 mL sodium citrate vacutainer tubes and centrifuged at 4 °C (4000× g for 10 min). After centrifugation, plasma was collected with a disposable Pasteur pipette, transferred into separate Eppendorf probes and stored in a −80 °C freezer until further analysis. Erythrocyte lipids were extracted into chloroform:methanol and fatty acid methyl esters (representing the erythrocyte fatty acids) were formed by heating the lipid extract with methanolic sulphuric acid. The fatty acid methyl esters were separated by gas chromatography on a Hewlett Packard 6890 gas chromatograph fitted with a BPX-70 column using the settings and run conditions described by Fisk et al. [17 (link)]. Fatty acid methyl esters were identified by comparison with runtimes of authentic standards and data were expressed as weight % of total fatty acids.
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