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Bstfa 1 tmcs

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BSTFA+1% TMCS is a silylating reagent used in gas chromatography-mass spectrometry (GC-MS) analysis. It is a mixture of N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) and 1% trimethylchlorosilane (TMCS). This reagent is used to derivatize polar compounds, such as alcohols, carboxylic acids, and amines, to increase their volatility and thermal stability for GC-MS analysis.

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10 protocols using bstfa 1 tmcs

1

Targeted Metabolomics Analysis Protocol

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HPLC grade acetonitrile, methanol, and formic acid were purchased from Merck Chemicals (Darmstadt, Germany). Chloroform, pyridine, anhydrous sodium sulfate, BSTFA (1% TMCS), heptadecanoic acid, methoxyamine, and L-2-chlorophenylalanine were purchased from Sigma-Aldrich (St. Louis, MO). Absolute IDQ P180 kit was purchased from Biocrates Life Sciences (Innsbruck, AT) for targeted quantification of amino acids and biogenic amines.
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2

Metabolite Derivatization for GC-MS Analysis

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BSTFA+1% TMCS (N, O-bis (trimethylsilyl) trifluoroacetamide with 1% trimethylchlorosilane), methoxyamine hydrochloride and pyridine, internal standard cis-10-nonadecenoic acid (C19:1, >99% purity), and the other 21 chemical standards of metabolites (shown in Table 2) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). High-performance liquid chromatography grade methanol was purchased from Tedia Co., Inc. (Fairfield, USA).
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3

Quantification of Selenium Compounds

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2 g of purified SeADO protein was acidified using 1 mol/L HCl to pH 3.0 and extracted with ethyl acetate. The organic layer was collected and dried by passing through MgSO4. The solvent was evaporated by rotary evaporator and nitrogen (N-EVAP) to 100 μL. The sample was trimethylsilylated for analysis using BSTFA + 1% TMCS (Sigma). All spectra were recorded on an Agilent 7890A GC system connected to an Agilent 7000B triple quadrupole MSD with electron impact ionization mode. A 1-μL portion of the derivatized extract was injected in splitless mode onto the column. The column used was a DB-5ms (30 m × 250 μm × 0.25 μm film thickness, Agilent J&W ScientiWc, USA) fused silica capillary column. Injector temperature was 280°C and the oven program was as follows: oven temperature was held at 60°C for 2 min and then increased to 240°C at 10°C/min and then increased to 300°C at 20°C/min finally maintained at 300°C for 5 min. Helium was used as the carrier gas for GC at a flow rate of 1.0 mL/min, and the inlet temperature was maintained at 280°C with splitless. Chromatographic data were acquired and processed using MassHunter Workstation Quantitative Software.
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4

GC-MS Analysis of Polar Metabolites

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Polar metabolites were derivatized and analysed by GC-MS (Agilent 7890B-5977A) and identification and abundance of individual metabolites was estimated as previously described 113 (link)
. In brief, dried metabolite samples were washed with methanol (twice), and derivatized overnight at RT with methoxyamine (20 mg/mL in pyridine, Sigma) followed by addition of BSTFA + 1% TMCS (Sigma) for > 1 hr at RT. GC-MS was performed using splitless injection (injection temperature 270°C) onto a 30 m + 10 m × 0.25 mm DB-5MS+DG column (Agilent J&W), with helium carrier gas, in electron impact ionization mode. The oven temperature was initially 70°C (2 min), followed by a temperature increase to 295°C at 12.5°C/min and subsequently to 320°C at 25°C/min (held for 3 min). GAVIN software 114 (link)
was used for metabolite identification and quantification by comparison to the retention times, mass spectra, and responses of known amounts of authentic standards.
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5

Cholesterol and Oxysterols Analytical Protocol

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Cholesterol and COP analytical standards, 5α-cholestane (HPLC grade), BSTFA + 1% TMCS (N,O-Bis(trimethylsilyl)trifluoroacetamide with 1% of trimethylchlorosilane, for GC derivatization) and pyridine (HPLC grade) were purchased from Sigma Aldrich Corp., Poznan, Poland. Methanol (HPLC grade), ethanol (96%, analytical grade), hexane (HPLC grade), chloroform (analytical grade), potassium hydroxide (analytical grade) and BHT (butylated hydroxytoluene, analytical grade) were bought in Avantor Performance Materials Poland S.A., Gliwice, Poland. Nitrogen (purity: ≥99.999%) was provided by Multax S.C., Stare Babice, Poland. Helium (purity: ≥99.9999%) was bought from Air Products and Chemicals, Inc., Warsaw, Poland.
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6

Metabolite Profiling by GC-MS

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High-performance liquid chromatography (HPLC) grade methanol was purchased from the Tedia Company (Inc., Fairfield, USA). BSTFA+1% TMCS (N, O-bis (trimethylsilyl) trifluoroacetamide with 1% trimethylchlorosilane for GC) (> 99.0% purity), methoxyamine hydrochloride (> 98.0% purity) and pyridine (> 99.8% purity), internal standard 2-isopropylmalic acid (> 98.0% purity) and the other 25 chemical standards of metabolites (S1 Table) were commercially obtained from Sigma-Aldrich (St. Louis, MO, USA).
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7

Analytical Method for Plasma C5 Keto Acids

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Here, “C5 keto acids” refers to the two species of 5-carbon-unit monocarboxylic acids carrying either 3-keto or 3-hydroxy radicals. Their plasma levels were measured by organic extraction, trimethylsilylation (BSTFA +1% TMCS, Sigma) and gas chromatography-mass spectrometry in SIM mode (Scion TQ mass analyzer, Brüker). Quantification was calibrated on known amounts of unlabeled analytes relative to stable-isotope-labeled internal standards (3,4,5-13C3 3-ketopentanoate from Eurisotop, Saint Aubin, France and 2,2,3,3,4,4,5,5,6,6-d10 6-hydroxyhexanoic acid from Sigma-Aldrich). The concentrations of plasma C3-carnitine, produced only by triheptanoin due to its odd number of carbons, were also measured as described [20 (link)].
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8

Metabolite Extraction and Derivatization for GC-MS

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The 20-mg freeze-dried powder samples were transferred into a 2 mL tube and soaked in an extraction solvent containing 450 μL of 80% methanol and 10 μL of 0.5 mg mL −1 internal standard adonitol. Samples were vortexed for 30 s and homogenized with a tissue grinder for 4 min at 35 Hz to adequately extract the metabolites. The samples were centrifuged at 4 °C for 15 min at 10000 rpm, then 260 μL of the supernatant was transferred to a fresh tube. The QC samples were combined from 50 μL of each sample. After evaporating in a vacuum concentrator for 6 h, the 50 μL of 20 mg mL −1 methoxyamination hydrochloride in pyridine was added and then incubated at 80 °C for 30 min. The Silylation reaction was performed by adding 70 μL of derivatization reagents (99% BSTFA+1% TMCS; Supelco) at 70 °C for 1.5 h. After gradually cooling the samples to room temperature, 3 μL of 500 mg mL −1 alkane in hexane (C7-C40; Anpel) was added to the QC samples. Finally, all samples were analyzed by gas chromatography coupled with a mass spectrometer (GC-MS). The raw data of metabolites were deposited in the MetaboLights database (Accession Number: MTBLS4752).
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9

Sterol Quantification in Oils by GC

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The content of sterols was determined by GC following a previously described procedure [54 ]. Briefly, lipids (0.05 g) were saponified with 1 M KOH in methanol, and the unsaponifiables were extracted using a mixture of hexane and methyl tert-butyl ether (1:1, v/v). The solvent was evaporated under a nitrogen stream, and dry residues were dissolved in anhydrous pyridine, and silylated with BSTFA + 1% TMCS (Supelco, Bellefonte, PA). The sterol derivatives were separated on an Agilent Technologies 6890 Plus GC (Agilent Technologies. Palo Alto. CA, USA) system equipped with a flame-ionization detector and a DB-35MS capillary column (25 m × 0.20 mm, 0.33 μm; Agilent J&W, USA). Samples were injected in splitless mode. The column temperature was initially set at 100 °C and held for 5 min, then increased to 250 °C at 25 °C/min and held for 1 min, and further increased to 290 °C at 3 °C/min and held for 20 min. The detector was set at a temperature of 300 °C. Hydrogen was used as the carrier gas at a flow rate of 1.5 mL/min. 5α-Cholestane was used as an internal standard. Identification was performed by comparing the retention data of compounds with the standards. Samples of each oil were analyzed in triplicate.
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10

Phytosterol Analysis in Hemp Oils

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For analysis of sterols, a total of 0.05 g of each hemp oil was used. To the samples were added 50 µg of internal standard(5α-cholestane-Supelco, Bellefonte, PA, USA). The samples were saponified with 1 M KOH in methanol, and the unsaponifiables were extracted using a mixture of hexane and methyl tert-butyl ether (1:1, v/v). The solvent was evaporated under a nitrogen stream, and dry residues were dissolved in anhydrous pyridine (Supelco, Bellefonte, PA, USA), and silylated with BSTFA + 1% TMCS (Supelco, Bellefonte, PA, USA). The phytosterols were analyzed using a Hewlett-Packard 6890 gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) in splitless mode with an FID detector and a DB-35MS capillary column (25 m × 0.20 mm, 0.33 μm; Agilent J&W, USA). The detector and injector were set at a temperature of 300 °C. The oven temperature was initially 100 °C for 5 min, increasing at 25 °C/min to 250 °C and then at 3 °C/min to 290 °C. The final temperature was held for 20 min. The carrier gas was hydrogen and the flow rate was 1.5 mL/min. Sterols were identified by comparing their retention times with those of standards. The sterols were determined in duplicate [57 (link)].
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