2.4.5. Detection of TMAO, TMA, Creatinine, d9-TMAO and d9-TMA by HPLC-MS/MS

Quantification of TMAO, TMA, creatinine, d9-TMAO and d9-TMA was performed using an Q Exactive Focus LCMS (Thermo Fisher Scientific, San Jose, CA, USA) [27]. Chromatographic separation was achieved using an Acquity UPLC BEH Amide (2.1 × 100 mm, 1.7 µm column) analytical column with gradient elution of solvent A (10 mM ammonium formate, pH 3.5) and solvent B (acetonitrile). The column was heated to 40 °C, autosampler and sample pan were maintained at 4 °C. The gradient was 5% A for 0.15 min, to 15% A in 1.2 min, to 20% A in 3 min, to 30% A in 6 min, to 45% A in 7 min, at 45% A for 4 min, to 5% A in 12 min, at 5% A for 3 min. The flow rate was 0.3 mL/min and the total run time was 15 min.

The MS/MS detection was performed on a Q Exactive-Orbitrap mass spectrometer (Thermo Scientific, San Jose, CA, USA) equipped with a heated electrospray ionization (ESI) source. Analytes were detected in positive ionization mode using a parallel reaction monitor (PRM). The spray voltage was set to 3.5 kV and the capillary temperature was set to 320 °C. The ion transitions were m/z 76.07569 → 58.06588 for TMAO, (N) CE = 80, m/z 85.13218 → 66.11606 for d9-TMAO, (N) CE = 100, m/z 60.08078 for TMA, (N) CE = 10, m/z 69.13727 for d9-TMA, (N) CE = 10, and m/z 114.06619 → 72.04531 for creatinine, (N) CE = 60. Signal output was captured and processed with Xcaliber 2.2 SP1.48 (Thermo Fisher Scientific, San Jose, CA, USA).

TMA is an extremely volatile small molecule. It should be operated quickly in the process of sample preparation and detection, and the whole process was kept at low temperature. The samples were detected in random order to minimize errors due to processing time.