Plasma TMAO, TMA, and DMA Quantification

The plasma levels of TMAO, TMA, and their dimethylamine (DMA) metabolites were analyzed by liquid chromatography-mass spectrometry (LC-MS) using previously described methods [21 (link)]. For the LC-MS analysis, an Agilent 6410 Series Triple Quadrupole mass spectrometer (Agilent Technologies, Wilmington, DE, USA) with an electrospray ionization source was employed. We used diethylamine as an internal standard. Using an Agilent Technologies 1200 HPLC system, chromatographic separation was carried out on a SeQuant ZIC-HILIC column (150 × 2.1 mm, 5 μm; Merck KGaA, Darmstadt, Germany) protected by an Ascentis C18 column (2 cm × 4 mm, 5 μm; Merck KGaA). The eluate was monitored for DMA, TMA, and TMAO in multiple-reaction-monitoring mode using characteristic precursor-product ion transitions: m/z 46.1→30, m/z 60.1→44.1 and m/z 76.1→58.1, respectively.