Sequant zic hilic column

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.

LC-MS/MS analysis of malonate and succinate was performed using an LCMS-8060 mass spectrometer (Shimadzu, UK) with a Nexera UHPLC system (Shimadzu, UK). Samples were stored in a refrigerated autosampler (4 °C) upon injection of 5 μl into a 15 μl flowthrough needle. Separation was achieved using a SeQuant ZIC-HILIC column (3.5 μm, 100 Å, 150 × 2.1 mm, 30 °C column temperature; Merck Millipore, UK) with a ZIC-HILIC guard column (200 Å, 1 × 5 mm). A flow rate of 200 μl/min was used with mobile phases of: A) 10 mM ammonium bicarbonate (pH unchanged); and B) 100% acetonitrile. A gradient of 00.1 min, 80% MS buffer B; 0.1–4 min, 80–20% B; 4–10 min, 20% B; 10–11 min, 20–80% B; and 11–15 min, 80% B was used. The mass spectrometer was operated in negative ion mode with multiple reaction monitoring (MRM), and spectra were acquired using the LabSolutions software (Shimadzu, UK), with compound quantities calculated from relevant standard curves in MS extraction buffer (50% (v/v) methanol, 30% (v/v) acetonitrile, 20% (v/v) MS-grade water) compared with 1 nmol of relevant internal standard either [¹³C₃]-malonate or [¹³C₄]-succinate for malonate and succinate, respectively (Supplementary Fig. 1).

Chromatographic separation of water-soluble metabolites of UP was achieved by applying 3 μl of dissolved sample on a SeQuant ZIC-HILIC Column (3.5-μm particles, 100 × 2.1 mm; Merck, Darmstadt, Germany), combined with a Javelin particle filter (Thermo Scientific) and a SeQuant ZIC-HILIC Precolumn (5-μm particles, 20 × 2 mm; Merck) using a linear gradient of mobile phase A (5 mM NH₄OAc in CH₃CN/H₂O (5/95, v/v)) and mobile phase B (5 mM NH₄OAc in CH₃CN/H₂O (95/5, v/v)). The LC gradient program was 100% solvent B for 2 min, followed by a linear decrease to 40% solvent B within 16 min, then maintaining 40% B for 6 min, then returning to 100% B in 1 min and 5 min 100% solvent B for column equilibration before each injection. The column temperature was set to 30 °C; the flow rate was maintained at 200 μl/min. The eluent was directed to the HESI source of the QE-MS from 1.85 to 20.0 min after sample injection.

Dried metabolite extracts were resuspended in 50 μL of methanol–water (1:1, v/v) and analyzed using a method as described previously [51 (link)]. Metabolite separation was achieved by a SeQuant ZIC-HILIC column (100 mm × 2.1 mm i.d., 3.5 µm) (Merck, Germany) using a Waters I-Class Acquity UPLC system (Waters, UK). The UPLC system was coupled to a Vion IMS QToF system (Waters, UK) [51 (link)].
Significant differences were analyzed by a two-tailed Student’s t test with Microsoft Excel 2016. The principal component analysis plots were generated using SIMCA 14.1 (Umetrics, Umea, Sweden). PCA was applied to the data after mean centering and UV scaling.

We analyzed plasma and urinary concentrations of DMA, TMA, and TMAO 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 [16 (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 an 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). Diethylamine 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. The urinary concentration of each methylamine was corrected for urine Cr concentration, which was represented in ng/mg Cr.