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6 protocols using luna silica

1

Quantifying Plasma TMAO Levels

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Plasma was stored at −80°C until analysis. The TMAO levels in plasma were determined using stable isotope dilution liquid chromatography tandem mass spectrometry (6460 Series Triple Quadrupole LC/MS; Agilent), as described previously.19Plasma (50 μL) was aliquoted to a 1.5‐mL tube and mixed with 200 μL of a 10 μmol/L internal standard composed of d9‐TMAO in methanol. The samples were vortexed for 1 minute, and then the supernatant was recovered following centrifugation at 15 000g at 4°C for 25 minutes. The supernatant (2 μL) was injected directly into a silica column (4.6×250 mm, 5 μm Luna silica, catalog no. 00G‐4274‐E0; Phenomenex) at a flow rate of 0.5 mL/min−1 with 80% solvent A (0.1% formic acid in water) and 20% solvent B (methanol). TMAO and d9‐TMAO were monitored in the positive multiple reaction monitoring mass spectrometry mode using characteristic precursor–production transitions including m/z 76/58 and m/z 85/66, respectively. To calculate the TMAO concentration, various concentrations of a TMAO standard were added to control plasma to prepare the calibration curves.
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2

Spectroscopic Characterization of Compounds

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Optical rotations were measured on a Rudolph Research Analytical AUTOPOL IV digital polarimeter. UV absorptions were measured by an Agilent Cary 60 UV–vis spectrophotometer in MeOH or MeCN, while IR spectra were recorded with an Agilent Cary 630 FTIR. All NMR spectra were acquired in CDCl3 or CD3CN with residual solvent referenced as an internal standard (7.27 and 1.94 ppm for 1H and 77.0 and 1.29 ppm for 13C, respectively). All 1H NMR spectra were recorded on a Varian 500 or 600 MHz Direct Drive instrument equipped with cold-probe detection, and 13C NMR spectra were recorded at 125 or 150 MHz, respectively. Analytical LC/MS was performed on a Phenomenex Kinetex C18 column (50 × 2.1 mm, 2.6 μm) on either an Agilent 6120 single quadrupole, an Agilent 6230 LC/ToF-MS, or an Agilent 6540 LC/QToF-MS with electrospray ionization detection, the latter two of which were utilized for HRESIMS. All HPLC analysis was performed on a Shimadzu LC20-AT system equipped with a photodiode array detector (M20A) using semipreparative [Phenomenex Luna C18 (250 × 10 mm, 5 μm)] or analytical [Phenomenex Luna C18 (250 × 4.6 mm, 5 μm) and Phenomenex Luna Silica (250 × 4.6 mm, 5 μm)] conditions. All solvents were obtained from Fisher Scientific and were HPLC grade (>99% purity) unless otherwise stated.
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3

Lipidomic Analysis by LC-MS/MS

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Phenomenex Luna silica (3 μm, 1.5 mm × 200 mm) was selected as the chromatographic column. Lipids were extracted under A phase (chloroform/methanol/ammonia = 89.5:10:0.5) and B phase (chloroform/methanol/ammonia/water = 55:39:0.5:5.5). Extraction began with a 95% gradient of A phase from 0 to 5 min, then a linear decrease to 60% (in 7 min) for 4 min, a further decline to 30% for 15 min, and return to 95% for the last 5 min. Mass spectrometry multiple reaction monitoring was established for lipid identification and quantitative analysis (Lam et al., 2017 (link), 2018 (link)).
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4

Quantifying Plasma TMAO Levels

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The collected blood samples were immediately processed and stored at −80°C until analysis. Plasma TMAO levels were quantified by stable isotope dilution liquid chromatography tandem mass spectrometry (6460 Series Triple Quadrupole LC/MS; Agilent) using a d9-(trimethyl)-labeled internal standard, as previously described (23 (link)).
In total, 50 μL of plasma was added to a 1.5 mL Eppendorf tube and mixed with 200 μL of 10 μmol/L internal standard composed of d9-TMAO in methanol. The samples were vortexed for 1 min, and then the supernatant was aliquoted following centrifugation at 15,000 g and 4°C for 25 min. Two microliters of supernatant was injected directly into a silica column (4.69 × 250 mm, 5 μm Luna silica, catalog no. 00G-4274-E0; Phenomenex) at a flow rate of 0.5 mL/min−1 with 80% solvent A (0.1% formic acid in water) and 20% solvent B (methanol). TMAO and d9-TMAO were monitored in the positive multiple reaction monitoring mass spectrometry mode using characteristic precursor–production transitions including m/z 76/58 and m/z 85/66, respectively. To calculate TMAO concentrations, serial concentrations of TMAO standard were added to control plasma to prepare a calibration curve.
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5

HPLC Analysis of Tocopherol Profiles

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Analysis of tocopherols was performed by HPLC according to Brazaityte et al.’s [25 (link)] methodology with some modifications. About 1 mg of oil was weighed in an Eppendorf tube, and then 1 mL of n-hexane with 1% of BHT was added to the tube. Afterward, the samples were filtrated through a 0.45 µm polytetrafluoroethylene (PTFE) membrane syringe filter (VWR International, Radnor, PA, USA) and were analyzed by HPLC/FLD (fluorescence detector) (Agilent Technologies, Santa Clara, CA, USA). The HPLC measurements were performed using a normal phase column (Phenomenex Luna Silica, 5 μm, 250 mm × 4.6 mm). The HPLC 10A system, equipped with an RF-10A fluorescence detector (Shimadzu, Japan), was used for analysis. Peaks were detected at an excitation wavelength of 295 nm and an emission wavelength of 330 nm. The mobile phase (0.5% isopropanol in hexane) was used at a flow rate of 1 mL min−1. The α-tocopherol, γ-tocopherol, and δ-tocopherol were identified according to the analytical standard. The α-tocopherol, γ-tocopherol, and δ-tocopherol content were expressed per 100 g of oil.
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6

Purification and Characterization of Natural Compounds

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1 H, 13 C, and 2D NMR spectra were recorded on a Bruker AVANCE III NMR spectrometer (Bruker, Billerica, Massachusetts, US), acquiring 1 H data at 400 MHz and 13 C data at 100 MHz, using standard experiments from Bruker pulse programs library. High-resolution mass spectrometry (HR-MS) was determined using an LTQ Orbitrap XL (Thermo Fisher Scientific, Waltham, Massachusetts, U.S.). The compositions of the essential oil were analyzed by an ITQ 900 mass spectrometer coupled to a TRACE GC Ultra gas chromatography (Thermo Fisher Scientific, Waltham, Massachusetts, U.S.). Methanol (MeOH) extracts were fractionated on silica gel 60 (230-400 mesh ASTM, Merck) and then purified with semi-preparative normal-phase column (luna silica (2), 250 × 10 mm, 5 μm, Phenomenex) on an Agilent 1100 HPLC (Agilent Technologies, Santa Clara, California, U.S.).
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