Ascentis c18 column

Plasma levels of TMAO, TMA, and their metabolites dimethylamine (DMA) were determined using a previously described method [8 (link)]. For the liquid chromatography–mass spectrometry (LC–MS) analysis, an Agilent 6410 Series Triple Quadrupole mass spectrometer (Agilent Technologies, Wilmington, DE, USA) with an electrospray ionization source was applied. 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, TMAO, and TMA in multiple-reaction-monitoring mode using characteristic precursor-product ion transitions: m/z 46.1 → 30, m/z 76.1 → 58.1, and m/z 60.1 → 44.1, respectively.

Plasma H₂S and thiosulfate concentrations were measured by an HPLC-Mass Spectrometry (MS) protocol previously validated in our lab [19 (link)] using an Agilent Technologies 1290 HPLC system together with an Agilent 6470 Triple Quadrupole LC/MS (Agilent Technologies, Wilmington, DE, USA) and an electrospray ionization (ESI) source [18 (link)]. Chromatographic separation was carried out using a Supelco C18 column (3 µm, 50 × 2.1 mm; Sigma–Aldrich) protected by an Ascentis C18 column (3 µm, 20 × 2.1 mm, Merck KGaA, Darmstadt, Germany). Solvents used in the elution step were composed of 0.1% formic acid (v/v) in acetonitrile, at a flow rate of 300 µL/min. We measured thiosulfate derivative pentafluorobenzyl (PFB)-S₂O₃H and H₂S derivative sulfide dibimane (SDB). Selected reaction monitoring mode was utilized to detect target compounds with a targeted 415→223 m/z and 292.99→81 m/z, for SDB and PFB-S₂O₃H, respectively. Phenyl 4-hydroxybenzoate (PHB) was used as an internal standard and detection was at 212.99→93 m/z. The percentage of coefficient of variation for the intra-assay variability was 4% and 6% for H₂S and thiosulfate, respectively.

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.