Agilent jet stream esi source

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.

Modern TQ-MS provides the ability to detect and quantify a large number of metabolites. The present quantification of targeted metabolites was performed on Agilent 1290 Infinity II LC and Agilent 6495 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 Scherzo SM-C18 column (2 mm × 100 mm, 3 μm; Imtakt, Kyoto, Japan). The flow rate and injection volume were set at 0.35 mL/min and 1 μL, respectively. Mobile phase A consisted of 0.1% formic acid in water, and phase B consisted of 0.1% formic acid in methanol. The linear gradient used for elution and equilibration of the initial gradient for subsequent runs was as follows: 5% B from 0–3 min, 5–90% B from 3–10 min, 90% B from 10–12 min, 5–95% B from 12–13 min, 5% B from 13–15 min. Quantification was performed in the multiple reaction monitoring (MRM) mode, and the optimal conditions for each metabolite were determined by flow injection of individual standards (100 ng/mL in 20% methanol) into the ESI source in the positive and negative ion modes. ¹³C₆-Phenylalanine was used as an internal standard. The compound retention times and MRM transitions are summarized in Table S1.

MS detection was performed using an Agilent G6460C Triple Quadrupole mass spectrometer equipped with an Agilent Jet Stream ESI source (Agilent Technologies, Waldbronn, Germany) and an IT-TOF-MS system with an electrospray ionization (ESI) interface (Shimadzu, Kyoto, Japan). The nitrogen generator is a Nitrocraft NCLC/MC from Air Liquid (Spain).
Agilent source conditions were optimized to achieve the best sensitivity for all compounds: drying gas temperature of 250 °C and flow of 5 L/min, nebulizer gas pressure of 55 psi (Nitrocraft NCLC/MS from Air Liquid), sheath gas temperature of 400 °C and flow of 12 L/min. The capillary voltage was set to 3000 V in positive mode with a nozzle voltage of 0 V. The fragmentor was 152 and the cell accelerator voltage was 2 for each toxin in this method.
IT-TOF-MS source conditions were nebulizing gas flow, 1.5 L/min, heat block temperature and CDL temperature, 200 °C and detector voltage, 1.65 kV.