Quantification of TMAO, Choline, and Betaine

Plasma heparin samples were obtained after a 12 h fast and stored at -80 °C. Quantification of TMAO, choline and betaine was performed by LC–MS/MS as done by Missailidis^{29 (link)}, utilizing a protocol designed specifically for this purpose and prepared in a 96-well format. Extracted plasma aliquots were spiked with internal standards, comprised of TMAO-D9 in methanol and water with Proline-13C5 as a recovery standard, and injected on an Agilent 1290 Infinity chromatographic system (Agilent Technologies, Waldbronn, Germany) fitted with an Acquity UPLC Amide column in combination with a VanGuard precolumn (Waters Corporation, Milford, MA, USA). The compounds were detected with an Agilent 6490 Triple Quadrupole mass spectrometer (Agilent Techologies, Santa Clara, CA, USA). Data processing was performed with MassHunter Quantitative Analysis QQQ (Agilent Technologies Inc. Santa Clara, CA, USA). The MS/MS analyses for TMAO, choline, betaine, TMAO- D9 and Proline-13C5, were conducted in multiple-reaction-monitoring (MRM) mode at m/z 76 → 58, m/z 104 → 45, m/z 118 → 58, m/z 85 → 66 and m/z 121 → 74 respectively.