TMAO is formed from TMA, which is generated by the metabolism of gut microbiota from dietary precursors (e.g., choline and L-carnitine) [16 (link)]. TMAO and TMA can be metabolized to dimethylamine (DMA). Thus, simultaneous measuring of TMA, TMAO, and DMA and their combined ratios may understand the whole picture of TMA–TMAO metabolic pathway in the pathogenesis of hypertension. We analyzed plasma DMA, TMA, and TMAO levels by LC–MS/MS analysis using an Agilent 6410 Series Triple Quadrupole mass spectrometer (Agilent Technologies, Wilmington, DE, USA) equipped with an electrospray ionization source [27 (link)]. The multiple-reaction-monitoring mode was set up using characteristic precursor-product ion transitions to detect m/z 46.1→30, m/z 60.1→44.1, and m/z 76.1→58.1, for DMA, TMA, and TMAO, respectively. Separation was performed in the Agilent Technologies 1200 HPLC system consisting of autosampler and a binary pump. Chromatographic separation was performed 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, Darmstadt, Germany). Diethyl amine was added to samples as an internal standard. The mobile phase containing methanol with 15mmol/L ammonium formate (phase A) and acetonitrile (phase B) was used at a ratio of 20:80 (phase A: phase B); with the flow rate set as 0.3–1 mL/min.
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