Quantification of TMA, TMAO, and DMA by LC-MS

As previously described [8 (link),14 (link)], plasma concentrations of TMA, TMAO, and DMA were determined by Liquid Chromatography–Mass Spectrometry (LC–MS) analysis. LC–MS analyses were carried out on an Agilent 6410 Series Triple Quadrupole MS (Agilent Technologies) coupled with an electrospray ionization source. Briefly, TMA, TMAO, and DMA were monitored in multiple-reaction-monitoring mode using characteristic precursor-product ion transitions: m/z 60.1→44.1, m/z 76.1→58.1, and m/z 46.1→30, respectively. For chromatographic separation, a SeQuant ZIC-HILIC column (150 × 2.1 mm, 5 μm; Merck KGaA, Darmstadt, Germany) was applied and protected by an Ascentis C18 column (2 cm × 4 mm, 5 μm; Merck KGaA, Darmstadt, Germany). Diethylamine was added to plasma samples as an internal standard. The mobile phase was a mixture (20:80, v/v) of methanol with 15 mmol/L ammonium formate (phase A) and acetonitrile (phase B) at a flow rate of 0.3–1 mL/min.