Jun 29, 2026

Large Targeted Metabolomics - LC-MS Parameters of Q-Exactive - MetaboHUB-Tours V.3

DOI
https://dx.doi.org/10.17504/protocols.io.eq2lyoq9qgx9/v3
Large Targeted Metabolomics - LC-MS Parameters of Q-Exactive - MetaboHUB-Tours
  • Staff Members of PMAC1,2
  • 1Plateforme de Métabolomique et d'Analyses Chimiques, US61 ASB, Université de Tours, CHRU Tours, Inserm, Tours, France;
  • 2MetaboHUB-Tours, Tours, France
  • MetaboHUB-Tours
Jérémy Monteiro
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DOI: https://dx.doi.org/10.17504/protocols.io.eq2lyoq9qgx9/v3
External link: https://metabolomique.med.univ-tours.fr/
Protocol Citation: Staff Members of PMAC 2026. Large Targeted Metabolomics - LC-MS Parameters of Q-Exactive - MetaboHUB-Tours. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyoq9qgx9/v3Version created by Jérémy Monteiro
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 29, 2026
Last Modified: June 29, 2026
Protocol  Integer ID: 319991
Keywords: metabolomics, LC-MS, targeted, IROA, Q-Exactive, metabolomic coverage, large targeted metabolomics-lc, analysis of metabolite, metabolite, sample preparation, present in biological sample, cv of qc sample, methods after sample preparation, biological sample, qc sample, protein precipitation, data treatment data, large targeted metabolomic, metabohub, ms parameter
Funders Acknowledgements:
Agence Nationale de la Recherche
Grant ID: ANR-11-INBS-0010 MetaboHUB
Agence Nationale de la recherche au titre de France 2030
Grant ID: ANR-21-ESRE-0035
Disclaimer
N/A
Abstract
After sample preparation which consisted in a protein precipitation, samples are analyzed by HESI-LC-HRMS (Q-Orbitrap) by 3 modalities: C18 in both positive and negative mode & HILIC in positive mode.

This protocol details the calibration of the device, performance monitoring, and the sequences used.
Image Attribution
Laurent Galineau (Plateforme Imagerie Préclinique, US-61 ASB, Université de Tours, CHRU Tours, Inserm, Tours, France ; Université de Tours, INSERM, Imaging Brain & Neuropsychiatry iBraiN U1253, 37032, Tours, France)
Camille Dupuy (Plateforme de Métabolomique et d'Analyses Chimiques, US61 ASB, Université de Tours, CHRU Tours, Inserm, Tours, France; MetaboHUB-Tours, Tours, France; Université de Tours, INSERM, Imaging Brain & Neuropsychiatry iBraiN U1253, 37032, Tours, France)
Guidelines
Metabolomics is the “omics” science studying metabolites and can meet the need for a deeper insight into biological data. It offers a real-time snapshot of a metabolic state and so reflect immediate physiological changes. By revealing metabolic profiles, metabolomics allows for precise diagnosis, tailored treatments, and proactive health management, ensuring highly personalized and effective interventions.
(Nordström, A., Lewensohn, R. Metabolomics: Moving to the Clinic. J Neuroimmune Pharmacol 5, 4–17 (2010). https://doi.org/10.1007/s11481-009-9156-4 )
Large targeted metabolomics analyses were performed on an UPLC Ultimate WPS-3000 system (Dionex, Germany) coupled to a Q-Exactive mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with a HESI source (head electrospray ionisation). Xcalibur 2.2 software (Thermo Fisher Scientific, Bremen, Germany) controlled the system.
Materials
LC-MS Sytem
  • A LC-MS system, designed for high-resolution analysis
  • Columns : reversed phase and HILIC

Reagents
  • Formic acid >99% for LC-MS VWR International (Avantor)
  • Water milliQMerck MilliporeSigma (SigmaAldrich)
  • Acetonitrile HiPerSolv CHROMANORM VWR International (Avantor)
  • Methanol HiPerSolv CHROMANORM VWR International (Avantor)
  • Calibration Solution Pierce ESI Negative Ion (Thermo Fisher)
  • Calibration Solution Pierce LTQ Velos ESI Positive Ion (Thermo Fisher)
  • IROA MSMLS, OAMLS, BACSMLS, FAMLS Merck MilliporeSigma (Sigma-Aldrich)
  • Formiate ammonium VWR International (Avantor)
  • Indoxyl-3-sulfate Merck Millipore Sigma (Sigma-Aldrich)
  • Kynurenic acid Merck Millipore Sigma (Sigma-Aldrich)
  • L-tryptophan Merck Millipore Sigma (Sigma-Aldrich )
  • Indole-3-Lactic Acid Merck Millipore Sigma (Sigma-Aldrich)
Protocol materials
Water milliQMerck MilliporeSigma (Sigma-Aldrich)
Methanol HiPerSolv CHROMANORMVWR International (Avantor)
Aluminium oxide powerMerck MilliporeSigma (Sigma-Aldrich)
Acetonitrile HiPerSolv CHROMANORMVWR International (Avantor)
Formic acid >99% for LC-MS VWR International (Avantor)Catalog #84865.260
formiate ammoniumVWR International (Avantor)
Calibration Solution Pierce LTQ Velos ESI Positive IonThermo Fisher
Calibration Solution Pierce ESI Negative IonThermo Fisher
L-tryptophanMerck MilliporeSigma (Sigma-Aldrich)Catalog #T0254
indole-3-Lactic AcidMerck MilliporeSigma (Sigma-Aldrich)
Indoxyl-3-sulfateMerck MilliporeSigma (Sigma-Aldrich)
Kynurenic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #K3375
Safety warnings
All sample handling must be carried out under a fume hood, equipped with traditional PPE: lab coat, goggles, and gloves.
Chromatography Reversed-Phase (+/-)
3h 20m
Phase A:
  • 200 mL + 5.5 mL * [Number of injections]
  • Water milliQMerck MilliporeSigma (Sigma-Aldrich) + 0.1% Formic acid >99% for LC-MS VWR International (Avantor)Catalog #84865.260
  • Stir vigorously during 00:01:00
  • Ultrasound during 00:10:00 to remove air bubble
10m
Phase B:
  • 200 mL + 3.8 mL * [Number of injections]
  • Acetonitrile HiPerSolv CHROMANORMVWR International (Avantor) + 0.1% Formic acid >99% for LC-MS VWR International (Avantor)Catalog #84865.260
  • Stir vigorously during 00:01:00
  • Ultrasound during 00:10:00 to remove air bubble
10m
Chromatographic System

Equipment
Ultimate 3000
NAME
UHPLC
TYPE
Dionex Softron
BRAND
SRD-3400
SKU
Pump, Column Compartment, AutoSampler
SPECIFICATIONS

Install the column in the oven

Equipment
SecurityGuard ULTRA Cartridges
NAME
UPLC C18 2.1mm ID Columns
TYPE
Phenomenex
BRAND
AJ0-8782
SKU

Equipment
Kinetex 1.7µm XB-C18 100A
NAME
Column
TYPE
Phenomenex
BRAND
00F-4498-AN
SKU
LC Column 150 x 2.1mm
SPECIFICATIONS

5m
Adjust the temperature (55 °C ) and let it heat the core of the column

1h
Prepare the cleaning solvents (same solvent as the recovery solvent) & ultrasound it during 00:05:00 to remove air bubbles
10m
Prepare the piston “lubrication” solution (90% Methanol HiPerSolv CHROMANORMVWR International (Avantor) and 10%Water milliQMerck MilliporeSigma (Sigma-Aldrich) ) & ultrasound it during 00:10:00 to remove air bubbles
15m
Purge the system:
  • 2 times at 100%A and 2 times at 100%B with open valve
  • 2 times with close valve at 50%A and 50%B
  • Set the flow at 2 mL/min at 50%A and 50%B during 00:10:00
  • Check that the pression is stable; otherwise : purge again

Note
In order to purge A and B with the valve closed, check that your LC settings do not send the purge solvents into the column but directly into the waste container.


30m
Balancing the system: Gradually reach the flow rate of 0.5 mL/min at 2%B

1h
Gradient:

Retention (min)Flow (mL/min)%B
00.52
00.52
20.52
60.530
90.575
9.50.5100
11.80.5100
120.52
13.70.52

Chromatography HILIC (+)
1d 4h
Phase A:
  • 200 mL + 2.6 mL * [Number of injections]
  • Water milliQMerck MilliporeSigma (Sigma-Aldrich) + 10 millimolar (mM) formiate ammoniumVWR International (Avantor) + 0.1% Formic acid >99% for LC-MS VWR International (Avantor)Catalog #84865.260
  • Stir vigorously during 00:01:00
  • Ultrasound during 00:10:00 to remove air bubble
15m
Phase B:
  • 200 mL + 3.8 mL * [Number of injections]
  • [95% of
  • Acetonitrile HiPerSolv CHROMANORMVWR International (Avantor) & 5% of Water milliQMerck MilliporeSigma (Sigma-Aldrich) ] + 10 millimolar (mM) formiate ammoniumVWR International (Avantor) + 0.1% Formic acid >99% for LC-MS VWR International (Avantor)Catalog #84865.260
  • Stir vigorously during 00:01:00
  • Ultrasound during 00:10:00 to remove air bubble
15m
Chromatographic System

Equipment
Ultimate 3000
NAME
UHPLC
TYPE
Dionex Softron
BRAND
SRD-3400
SKU
Pump, Column Compartment, AutoSampler
SPECIFICATIONS

Install the column in the oven


Equipment
CORTECS UPLC HILIC 1.6µM
NAME
Column
TYPE
Waters
BRAND


5m
Adjust the temperature (40 °C ) and let it heat the core of the column

1h
Prepare the cleaning solvents (same solvent as the recovery solvent) & ultrasound it during 00:05:00 to remove air bubbles
10m
Prepare the piston “lubrication” solution (90% Methanol HiPerSolv CHROMANORMVWR International (Avantor) and 10%Water milliQMerck MilliporeSigma (Sigma-Aldrich) ) & ultrasound it during 00:10:00 to remove air bubbles
15m
Purge the system:
  • 1 time at 100%A and 1 time at 100%B with open valve
  • 2 times with open valve, 2 times with close valve at 50%A and 50%B
  • Set the flow at 2 mL/min at 50%A and 50%B during 00:10:00
  • Check that the pression is stable; otherwise : purge again
1h
Balancing the system: Gradually reach the flow rate of 0.5 mL/min at 95%B

1h
Gradient:

Retention (min)Flow (mL/min)%B
00.595
00.595
.90.595
4.80.582
90.510
11.20.510
11.40.595
13.70.595

1d
Mass Spectrometry
Mass Spectrometer:
Equipment
Q-Exactive
NAME
Mass Spectrometer
TYPE
ThermoScientific
BRAND
HESI Source
SPECIFICATIONS
MS Parameters :
  • Scan Range: 58-870 m/z
  • Resolution MS1: 70,000
  • Resolution MS2: 15,000
  • Polarity: POS or NEG
  • Microscans: 1
  • Lock mass: None
  • AGC Target: 1E6
  • Maximum Inject Time: 250
  • Sheath Gas Flow Rate: 50
  • Aux Gas Flow rate: 15
  • Sweep Gas flow Rate: 1
  • Spray Voltage (kV): +/-2.5
  • Capillary Temperature (°C): 325
  • S-lens RF level: 60
  • Aux gas Heater temperature (°C): 400
Cleaning the source:
  • Set source temperature to 50 °C
  • Let it "cool" 01:00:00
  • Unplug the Ion MAx API source with HESI probe attached
  • Unscrewed the Sweep Cone and the capillary
  • Immediately plug another cleaned capillary
  • Clean the sweep cone with wet Aluminium oxide powerMerck MilliporeSigma (Sigma-Aldrich)
  • Rinse thoroughly with Water milliQMerck MilliporeSigma (Sigma-Aldrich)
  • In a glass beaker, cover completely the Sweep Cone and the capillary with Methanol HiPerSolv CHROMANORMVWR International (Avantor)
  • Ultrasound during 00:10:00
  • Let it dry
  • Replace the cone and the Ion Max API source with HESI probe
  • Set source temperature back to normal
Reinstall the source & allow it to return to analysis conditions (described above)
Calibration (direct infusion):
  • POS: Calibration Solution Pierce LTQ Velos ESI Positive IonThermo Fisher
  • NEG: Calibration Solution Pierce ESI Negative IonThermo Fisher
  • RMS < 0.4/0.4 in both ionisation mode
Prepared a solution for Performance Evaluation (during sequence injection):
  • Solvent : Acetonitrile HiPerSolv CHROMANORMVWR International (Avantor) /Water milliQMerck MilliporeSigma (Sigma-Aldrich) (1:1)
  • L-tryptophanMerck MilliporeSigma (Sigma-Aldrich)Catalog #T0254 at 10000 nanomolar (nM)
  • Indoxyl-3-sulfateMerck MilliporeSigma (Sigma-Aldrich) at 3750 nanomolar (nM)
  • Kynurenic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #K3375 at 25 nanomolar (nM)
  • indole-3-Lactic AcidMerck MilliporeSigma (Sigma-Aldrich) at 500 nanomolar (nM)
Once calibrated, sequences can be run
Typical Sequences:

Sample TypeInjection Volume (µL)
Equilibrium(=sample) * 105
Blank5
Performance Evaluation * 35
Blank5
Blank Solvent #15
Blank Solvent #25
Blank Extraction #15
Blank Extraction #25
Sample QC5
Sample *105
Sample QC5
Continue the sequence, alternating between samples and a QC5
Last Sample QC5
QC in MS²5
Blank Solvent * 35