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.
Quantifying Plasma TMAO Levels
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.
Corresponding Organization : Zhujiang Hospital
Variable analysis
- None explicitly mentioned
- Plasma TMAO levels
- Collected blood samples were immediately processed and stored at −80°C until analysis
- Plasma samples were mixed with 200 μL of 10 μmol/L internal standard composed of d9-TMAO in 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
- Serial concentrations of TMAO standard were added to control plasma to prepare a calibration curve
- Positive control: Serial concentrations of TMAO standard added to control plasma to prepare a calibration curve
- Negative control: Not explicitly mentioned
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