Plasma aliquots were stored at −40 °C prior to analysis. The plasma TMAO concentrations were quantified using stable isotope dilution liquid chromatography tandem mass spectrometry (6460 Series Triple Quadrupole LC/MS; Agilent) as previously described52 (link).
The molecular weight of TMAO is 75.22 g/mol. The extracted plasma aliquots were spiked with internal standards comprised of d9-TMAO in methanol. The samples were mechanically vortexed for 1 minute and centrifuged at 15,000 × g for 25 minutes at 4 °C. The analytes were separated on a phenomenex Luna Silica column (4.6 mm × 250 mm, 5 μm particle size) at room temperature. The mobile phase consisted of 80% solvent A (0.1% formic acid in water) and 20% solvent B (methanol) at a flow rate of 0.5 mL/min. The MS/MS analyses for TMAO and d9-TMAO were conducted in the positive multiple reaction monitoring mass spectrometry mode at 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 calibration curves. The data were collected and analysed using the chemistry station provided by Agilent Technology.
The molecular weight of TMAO is 75.22 g/mol. The extracted plasma aliquots were spiked with internal standards comprised of d9-TMAO in methanol. The samples were mechanically vortexed for 1 minute and centrifuged at 15,000 × g for 25 minutes at 4 °C. The analytes were separated on a phenomenex Luna Silica column (4.6 mm × 250 mm, 5 μm particle size) at room temperature. The mobile phase consisted of 80% solvent A (0.1% formic acid in water) and 20% solvent B (methanol) at a flow rate of 0.5 mL/min. The MS/MS analyses for TMAO and d9-TMAO were conducted in the positive multiple reaction monitoring mass spectrometry mode at 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 calibration curves. The data were collected and analysed using the chemistry station provided by Agilent Technology.
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