SQ was co-determined with FAMEs by GC-FID under the conditions proposed in Regulation (EEC) No. 2568/91 [29 ]. Transesterification took place using a methanolic solution of potassium hydroxide at room temperature. A capillary, TR-FAME column (60 m × 250 μm i.d., 0.25 μm) (ThermoScientific, Bellefonte, PA, USA) was used. The separation conditions were as follows: Carrier gas: Helium (1.1 mL/min), the injector and detector temperature were set at 240 °C, and the injection volume was 2 μL (split ratio 50:1). The temperature was programmed at 100 °C for 5 min, raised from 100 to 240 °C within 15 min, and held constant at 240 °C for 40 min. The identification of SQ was based on the retention time recorded for the SQ standard. Quantification was carried out using appropriate external calibration curves. Samples were analyzed in duplicate (CV% = 5.4, n = 5).
Tr fame column
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The TR-FAME column is designed for the separation and analysis of fatty acid methyl esters (FAMEs) using gas chromatography. It is a capillary column that provides high-resolution separation of a wide range of FAME compounds. The column features a specific stationary phase that enables the efficient and reliable identification and quantification of FAMEs in various sample matrices.
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Lab products found in correlation
3900 gas chromatograph, by Agilent Technologies (2 mentions)
Hpx 87h column, by Aminex (2 mentions)
Agilent chromatograph 7890a, by Agilent Technologies (1 mentions)
Fame mixture, by Merck Group (1 mentions)
Xcalibur 2, by Thermo Fisher Scientific (1 mentions)
Finnigan focus gc, by Thermo Fisher Scientific (1 mentions)
Trace gc ultra system, by Thermo Fisher Scientific (1 mentions)
Trace 1300, by Thermo Fisher Scientific (1 mentions)
Trace 1300 chromatograph, by Thermo Fisher Scientific (1 mentions)
11 protocols using tr fame column
1
GC-FID Analysis of Squalene
2
Fatty Acid Profiling by GC-FID/MS
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Total lipids were extracted from frozen tissue using a modified Folch extraction method. Chloroform:methanol (2:1 v/v) was added to 20 mg of tissues with deuterated tridecanoic acid as an internal standard. Samples were homogenised with ceramic beads (MP biomedicals 6540-434) at 5 Hz for 2 minutes at room temperature. Extracted lipids were dried under nitrogen stream and subsequently saponified and derivatised into fatty acid methyl esters (FAME) using 10% boron trifluoride in methanol. Gas chromatography was performed on a Thermo Finnigan Focus GC coupled to a FID detector or an Agilent 7890B GC connected to a 5977A MSD, both using a Thermo Scientific TR-FAME column (length: 30 m, internal diameter: 0.25 mm, film size: 0.25 μm) with helium as carrier gas (1.5 ml/min). Inlet and FID detector temperature was set at 230 °C and 250 °C respectively, MSD detector temperature was set at 230 °C. Oven programme were as follows: 100 °C 2 min, 25 °C/min to 150 °C, 2.5 °C/min to 162 °C, 162 °C 3.8 min, 4.5 °C/min to 173 C, 173 °C 5 min, 5.0 °C/min to 210 °C, 40 °C/min to 230 °C, 230 °C for 0.5 min. Results were presented and analysed as molar percentage of total FAME.
3
Fatty Acid Profiling by Gas Chromatography
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The fatty acids methyl esters (FAMEs) profile, expressed as % m/m using the peak area, was determined using an Agilent 7890A Chromatograph (Agilent Technologies, Santa Clara, SA, USA) coupled to a flame ionization detector (FID). The separation of fatty acid methyl esters was accomplished with a TR-FAME column, 50 m × 0.22 mm, i.d. 0.25 μm film thickness (Thermo Fisher Scientific, Waltham, MA, USA). The injector was set at 250 °C and the detector at 260 °C. The oven temperature was initially retained at 160 °C for 2 min, then raised with a rate of 1 °C min−1 up to 165 °C and kept for 30 min, then raised again with a rate of 3 °C min−1 up to 200 °C and kept for 8 min, resulting to a total run time of 57 min. Helium was used as carrier gas at a flow rate of 3 mL min−1. Each sample was injected twice, in split mode (50:1) with an injection volume of 1 μL, using an autosampler. FAMEs analysis was performed at Hellenic Food Authority (EFET).
4
Microbial Cultivation and Lipid Analysis
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Cell growth was recorded as optical density at 600 nm. Cell mass was harvested during the cultivations after centrifugation (5000 rpm, 10 min at 4°C). Cell dry weight was determined gravimetrically after drying the harvested cells in an oven at 80°C to a constant weight. Extraction of lipids was performed with a modified method described previously using Folsch solution (Chloroform: Methanol = 2:1 vol.vol-1, [41 (link)]. Non-lipid cell mass was calculated after subtraction of intracellular lipids from the total cell mass. Quantification of glucose, glycerol and organic acids was carried out using high-performance liquid chromatography (HPLC; Kontron Instruments, United Kingdom) with separation on an Aminex HPX-87H column at 60°C with 0.005 M H2SO4 and detection via refractive index or by UV absorption at 210 nm. Ammonia concentration in the supernatant was determined by photometric measurements using a kit from Macherey Nagel, Germany. GC analysis of the fatty acid methyl esters was performed as reported [42 (link)] with a Varian 3900 gas chromatograph equipped with a flame ionization detector (FID) and a TR-FAME column (Thermo Scientific, Germany, 50 m X 0.22 mm X 0.25 μm).
5
Fatty Acid Profiling by GC-FAME Analysis
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The fatty acid composition was determined by GC as fatty acid methyl esters (FAMEs). FAMEs were prepared by transesterification with a methanolic solution of potassium hydroxide at room temperature according to Regulation (EEC) No. 2568/91 and its amendments. A capillary, TR-FAME column (60 m × 250 μm i.d., 0.25 μm) (ThermoScientific, Bellefonte, PA, USA) was used. The separation conditions were as follows: Carrier gas: helium (1.1 mL/min), the injector and detector temperature were set at 240 °C and the injection volume was 2 μL (split ratio 50:1). The temperature was programmed at 100 °C for 5 min, raised from 100 to 240 °C within 15 min, and held constant at 240 °C for 40 min. Identification of FAMEs was based on their retention times to the commercial standard FAME mixture (Sigma Chemical Co., St. Louis, MO, USA) in line with the standardized reference method. The percentages of the individual FAMEs were calculated from the total area of the peaks recorded on the chromatogram. Samples were analyzed in duplicate. Before analysis, the repeatability of the method was checked (CV% = 1.2, 1.5, 4.4, 0.4 and 1.2 for palmitic (C16:0), palmitoleic (C16:1), stearic (C18:0), oleic (C18:1), linoleic (C18:2) acids, respectively, n = 5).
6
Cell Growth and Metabolite Analysis
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Cell concentration was measured optically, at 600 nm and correlated with cell dry weight determined directly. The concentrations of glucose, glycerol, butanol, 1,3 propanediol, ethanol, acetic, butyric, formic and lactic acids in supernatant were determined by HPLC using an Aminex HPX-87H column (300 × 7.8 mm) and the detection was assessed by refractive index and ultraviolet detectors. The operating conditions were as follows: mobile phase, H2SO4 0.005 M; flow rate, 0.6 ml min−1; temperature, 60 °C.
For the determination of the cell wall fatty acid composition, GC analysis of the fatty acid methyl esters was performed as previously reported [31 (link)] with a Varian 3900 gas chromatograph equipped with a flame ionization detector (FID) and a TR-FAME column (Thermo Scientific, Germany, 50 m × 0.22 mm × 0.25 µm).
For the determination of the cell wall fatty acid composition, GC analysis of the fatty acid methyl esters was performed as previously reported [31 (link)] with a Varian 3900 gas chromatograph equipped with a flame ionization detector (FID) and a TR-FAME column (Thermo Scientific, Germany, 50 m × 0.22 mm × 0.25 µm).
7
Volatile Profiling of Macadamia Kernels via SPME-GC-MS
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Volatile compounds of macadamia kernel samples were analysed via solid-phase microextraction (SPME) injection. Macadamia kernel samples (0.5 g) were placed into 20 mL solid phase micro extraction (SPME) autosampler vials and then incubated for 15 min at 60 °C with mixing at 500 rpm. After incubation, samples were exposed to a 2 cm 50/30 μm DVB/CAR/PDMS SPME fibre (Supelco, Inc., Bellefonte, PA) over the headspace for 30 min. The SPME needle was immediately injected into a Shimadzu GC-QP2010 ULTRA™ gas chromatography system (Shimadzu Corporation, Kyoto, Japan) with MS detector. A Thermo Scientific TR-FAME column (60 m Length, 0.25 mm ID, 0.25 μm film) was used to separate compounds for detection. Volatile compounds were identified by comparing the peaks of the mass spectrum against the NIST14 library. A chromatogram for a random kernel sample has been provided in Supplementary Fig. 1 .
8
Quantifying Liver Lipid Composition
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Total lipids were extracted from 15 mg of frozen liver tissue using a modified Folch extraction method. Deuterated tridecanoic acid (C13D25O2H, Cambridge Isotopes; Andover, MA, USA) was used as internal standard. Total lipids were saponified and derivatized into fatty acid methyl esters using boron trifluoride (Morrison and Smith, 1964 (link)). Samples were resuspended in hexane and analyzed on a Thermo Finnigan Focus GC coupled to a FID detector. A Thermo Scientific TR-FAME column (length: 30 m, inter diameter: 0.25 mm, film size: 0.25 μm; Thermo Scientific; Surrey, UK) was used with helium as carrier gas at 1.9 ml/min. Inlet temperature was 230°C and detector temperature was 250°C. Temperature program was as follows: 100°C for 2 min, 25°C/min for 2 min, 2.5°C/min for 4.8 min, hold for 3.8 min, 4.5°C/min for 2.4 min, hold for 5 min, 5°C/min for 7.4 min, 40°C/min for 0.5 min and hold for 0.5 min. Chromatograms were analyzed and quantified using Xcalibur 2.0 (Thermo Scientific; Surrey, UK). Identification of FAME peaks was made by comparing retention times against external standards (Restek 35077 Food industry FAME mix; Bellefonte, PA, USA; and Supelco 46904 Vaccenic Methyl ester; Sigma-Aldrich, St Louis, MO, USA). Data were normalized to internal standard and followed by adjustment for tissue weight. Results are expressed as molar percentages normalized to control animals.
9
Fatty Acid Profiling of Avocado Oil
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Two grams of avocado oil were saponified with 5% methanolic KOH to obtain potassium salt of fatty acids. After acidification with 6N HCl, fatty acids were extracted several times with diethyl ether and later methylated using methanol/H2SO4 under reflux. After cooling, fatty acids methyl esters (FAMEs) were recovered with diethyl ether and subsequently analyzed using Trace GC Ultra system (Thermo Fisher Scientific Co., USA) equipped with Thermo TR-FAME column (70% Cyanopropyl Polysilphenylene Siloxane, 30 m × 0.25 mm i.d) with 0.25 µm film thickness using N2 at 30 mL/min and a flame ionization detector operating at a flow rate of 350 mL/min (H2). The injector temperature was set at 200 °C with an initial temperature of 140 °C, which was increased to 200 °C by five °C/min, then kept isothermal for 3 min. FAMEs were identified by comparing their relative retention times with external standards analyzed under the same conditions.
10
Analyzing Short-Chain Fatty Acids in Feces
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To determine the contents of SCFAs, 100-mg fecal samples were collected in 2-mL centrifuge tubes, 100 μL methanol was added, and the tubes were centrifuged for 10 min at 12,000×g at 4 °C. Supernatant (100 mL) was placed into new 2-mL centrifuge tubes, mixed with 25 % metaphosphoric acid at a 5:1 ratio, and stored overnight at 4 °C. Next, the samples were centrifuged for 10 min at 12,000×g at 4 °C. The SCFA contents were analyzed using GC (TRACE 1300, Thermo Fisher Scientific, USA) with a TR-FAME column (60 m × 0.25 mm, ID × 0.25 μm). The detection temperature was 230 °C, the sample volume was 1 μL, and the carrier gas was nitrogen at a flow rate of 3 mL/min [17 (link)].
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