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MB Sample ID: SA323811

Local Sample ID:6
Subject ID:SU003097
Subject Type:Human
Subject Species:Homo sapiens
Taxonomy ID:9606
Gender:Male and female

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Combined analysis:

Analysis ID AN004903 AN004904
Analysis type MS MS
Chromatography type Ion exchange Reversed phase
Chromatography system Thermo Dionex ICS-5000+ Shimadzu Nexera X2
Column Dionex IonPac AS11-HC (250 x 2mm, 4um) Sigma-Aldrich Discovery HS F5-3 (150 x 2.1 mm, 3 um)
MS Type ESI ESI
MS instrument type Orbitrap Triple quadrupole
MS instrument name Thermo Q Exactive Focus LCMS-8060, Shimadzu Corporation
Ion Mode UNSPECIFIED UNSPECIFIED
Units Peak area/Internal Standard Peak area/Internal Standard

Chromatography:

Chromatography ID:CH003698
Chromatography Summary:Metabolites were detected using an orbitrap-type MS instrument (Q-Exactive focus; Thermo Fisher Scientific) connected to a high-performance IC system (ICS-5000+, Thermo Fisher Scientific) that enabled highly selective and sensitive metabolite quantification owing to the IC separation and Fourier transfer MS principle. The IC instrument was equipped with an anion electrolytic suppressor (Dionex AERS 500; Thermo Fisher Scientific) to convert the potassium hydroxide gradient into pure water before the sample entered the MS instrument. Separation was performed using a Dionex IonPac AS11-HC 4 μm particle size column (Thermo Fisher Scientific). The IC flow rate was 0.25 mL/min, supplemented post-column with 0.18 mL/min makeup flow of MeOH. The potassium hydroxide gradient conditions for IC separation were as follows: from 1 mM to 100 mM (0–40 min) to 100 mM (40–50 min) and to 1 mM (50.1–60 min) at a column temperature of 30 °C. The mass spectrometer was operated in the ESI-positive and negative mode with polarity switching, for all detections. A full mass scan (m/z 70–900) was performed at a resolution of 70,000. The automatic gain control target was set at 3 × 106 ions, and the maximum ion injection time was 100 ms. The source ionization parameters were optimized with a spray voltage of 3 kV, and other parameters were as follows: transfer temperature, 320 °C; S-Lens level, 50; heater temperature, 300 °C; sheath gas, 36; and aux gas, 10.
Instrument Name:Thermo Dionex ICS-5000+
Column Name:Dionex IonPac AS11-HC (250 x 2mm, 4um)
Column Temperature:30 °C
Flow Gradient:From 1 mM to 100 mM (0–40 min) to 100 mM (40–50 min) and to 1 mM (50.1–60 min)
Flow Rate:0.25 mL/min, supplemented post-column with 0.18 mL/min makeup flow of MeOH
Solvent A:The potassium hydroxide gradient conditions
Solvent B:N/A
Chromatography Type:Ion exchange
  
Chromatography ID:CH003699
Chromatography Summary:Cationic metabolite concentrations were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In essence, we employed a triple-quadrupole mass spectrometer equipped with an electrospray ionization (ESI) ion source (LCMS-8060, Shimadzu Corporation, Kyoto, Japan) operated in both positive and negative-ESI and in multiple reaction monitoring (MRM) modes. Analyte separation was achieved on a Discovery HS F5-3 column (2.1 mm I.D. × 150 mm L, 3 μm particle size; Sigma-Aldrich, St. Louis, MO, USA) through a gradient elution with mobile phase A (0.1% formate) and mobile phase B (0.1% acetonitrile). The elution profile was as follows: 100:0 (0–5 min), 75:25 (5–11 min), 65:35 (11–15 min), 5:95 (15–20 min), and 100:0 (20–25 min), with a constant flow rate of 0.25 mL/min and a column oven set at 40 °C.
Instrument Name:Shimadzu Nexera X2
Column Name:Sigma-Aldrich Discovery HS F5-3 (150 x 2.1 mm, 3 um)
Column Temperature:40 °C
Flow Gradient:100:0 (0–5 min), 75:25 (5–11 min), 65:35 (11–15 min), 5:95 (15–20 min), and 100:0 (20–25 min)
Flow Rate:0.25 mL/min
Solvent A:0.1% formate
Solvent B:acetonitrile with 0.1% formate
Chromatography Type:Reversed phase
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