Masshunter workstation

The analysis of pharmacokinetics was conducted using an LC-MS/MS system consisting of an Agilent 1260–6460 liquid chromatography instrument (Agilent, Agilent Technologies Inc., Palo Alto, CA, USA) equipped with a quaternary pump, a vacuum degasser, a thermo-stated column oven, and an autosampler (set at 4 °C), which were coupled to a triple quadrupole mass spectrometer. Separation was performed on an Agilent C18 column (3.0 × 50 mm, 2.7 μm) at 25 °C. The mobile phase consisted of (A) acetonitrile and (B) water containing 0.1% formic acid with a gradient elution program (20–30% A at 0–1 min; 30–50% A at 1–2 min; 50–60% A at 2–3 min; 60–70% A at 3–6 min). The flow rate was kept constant at 0.4 mL/min and the injection volume was 2 μL. The ion source was ESI- Agilent Jet S; the gas flow rate was set at 5 L/min; nebulizer 45 psi; sheath gas temperature 300 °C; sheath gas flow 11 L/min; capillary 3500 V; and nozzle voltage 500 V.
The MassHunter^TM Workstation (Agilent, Waldbronn, Germany) was used for data collection and acquisition.

To quantify levels of uremic toxins and related metabolites, targeted profiling was performed on Agilent 1290 Infinity LC and Agilent 6490 Triple Quadrupole MS systems equipped with an Agilent Jet Stream ESI source (Agilent Technologies, Palo Alto, CA, USA). MassHunter Workstation (Ver B.06.00, Agilent Technologies) software was used for data acquisition and analysis. Chromatographic separation was performed using a ZIC®-HILIC column (2.1 mm × 100 mm, 3.5 μm; SeQuant). The flow rate and injection volume were set at 0.4 ml/min and 1 μl, respectively. Mobile phase A consisted of 10 mM ammonium acetate and 0.1% formic acid in water:acetonitrile (5:95 v/v), and phase B consisted of 10 mM ammonium acetate and 0.1% formic acid in water:acetonitrile (50:50 v/v). The linear gradient used for elution and to equilibrate the initial gradient for subsequent runs was 1% B from 0–2 min, 1–55% B from 2–6 min, 55–99% B from 6–7 min, 99% B from 7–9 min, 99–1% B from 9–9.1 min, and 99% B from 9.1–13 min. Quantification was performed in the MRM mode and the optimal conditions for each metabolite were determined by flow injection of individual standards (100 ng/mL in 75% acetonitrile) into the ESI source in the negative ion mode. p-Cresyl sulfate-²H₇ was used as an internal standard. Compound retention times and MRM transitions are summarized in Supplementary Table S5.