Bstfa 1 tmcs

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.

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).

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)].

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).

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.

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)].