Large Targeted Metabolomics_LC-MS Parameters_MTH Tours

Staff Members of PMAC

Mar 12, 2026

Version 1

Large Targeted Metabolomics_LC-MS Parameters_MTH Tours V.1

DOI

https://dx.doi.org/10.17504/protocols.io.eq2lyoq9qgx9/v1

Large Targeted Metabolomics_LC-MS Parameters_MTH Tours

Staff Members of PMAC^1,2

¹Plateforme de Métabolomique et d'Analyses Chimiques, US61 ASB, Université de Tours, CHRU Tours, Inserm, Tours, France;
²MetaboHUB-Tours, Tours, France

Jérémy Monteiro

DOI: https://dx.doi.org/10.17504/protocols.io.eq2lyoq9qgx9/v1

External link: https://metabolomique.med.univ-tours.fr/

Protocol Citation: Staff Members of PMAC 2026. Large Targeted Metabolomics_LC-MS Parameters_MTH Tours. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyoq9qgx9/v1Version 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: March 11, 2026

Last Modified: March 12, 2026

Protocol Integer ID: 313071

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

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

I. PURPOSE
Identification and relative quantification (based on intensity) analysis of metabolites present in biological sample for metabolomic coverage.

II. MATERIAL & METHODS
After sample preparation which consisted in a protein precipitation, samples are analyzed by ES-LC-HRMS (Q-Orbitrap) by 3 modalities: C18 in both positive and negative mode & HILIC in positive mode.

III. DATA TREATMENT
Data are analyzed using Workflow4Metabolomics open-source software that allows to detect all features. features are identified with the use of IROA-database, based on the retention time and the m/z

IV. QUALITY CONTROL
-Checking the system suitability
- CV of QC samples (injected/10 injections) < 30 %

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.

For exploratory analyses, features were considered for further analysis only if they were detected in all injections and their CV were less than 30 % after signal normalization. The identification of metabolites was performed based on MS² spectra and retention time compared with the in-house injected database of > 600 compounds from IROA (MSMLS, FAMLS, BACSMLS & OAMLS from Merck).

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

ReagentAluminium oxide powerMerck MilliporeSigma (Sigma-Aldrich) 
ReagentWater milliQMerck MilliporeSigma (Sigma-Aldrich) 
ReagentMethanol HiPerSolv CHROMANORMVWR International (Avantor) 
ReagentIROA MSMLS, OAMLS, BACSMLS, FAMLSMerck MilliporeSigma (Sigma-Aldrich) 
ReagentAcetonitrile HiPerSolv CHROMANORMVWR International (Avantor) 
ReagentFormic acid >99% for LC-MS VWR International (Avantor)Catalog #84865.260 
Reagentformiate ammoniumVWR International (Avantor) 
ReagentCalibration Solution Pierce LTQ Velos ESI Positive IonThermo Fisher 
ReagentCalibration Solution Pierce ESI Negative IonThermo Fisher 
ReagentL-tryptophanMerck MilliporeSigma (Sigma-Aldrich)Catalog #T0254 
ReagentIndoxyl-3-sulfateMerck MilliporeSigma (Sigma-Aldrich) 
ReagentKynurenic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #K3375 
Reagentindole-3-Lactic AcidMerck MilliporeSigma (Sigma-Aldrich) 

Troubleshooting

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]
ReagentWater milliQMerck MilliporeSigma (Sigma-Aldrich)   + 0.1% ReagentFormic acid >99% for LC-MS VWR International (Avantor)Catalog #84865.260  
Stir vigorously during Duration00:01:00   
Ultrasound during Duration00:10:00    to remove air bubble

10m

Phase B:
200 mL + 3.8 mL * [Number of injections]
ReagentAcetonitrile HiPerSolv CHROMANORMVWR International (Avantor)   + 0.1% ReagentFormic acid >99% for LC-MS VWR International (Avantor)Catalog #84865.260  
Stir vigorously during Duration00:01:00  
Ultrasound during Duration00:10:00    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

Adjust the temperature (Temperature55 °C   ) and let it heat the core of the column

Prepare the cleaning solvents (same solvent as the recovery solvent) & ultrasound it during Duration00:05:00    to remove air bubbles

10m

Prepare the piston “lubrication” solution (90% ReagentMethanol HiPerSolv CHROMANORMVWR International (Avantor)   and 10%ReagentWater milliQMerck MilliporeSigma (Sigma-Aldrich)   ) & ultrasound it during Duration00:10:00    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 Duration00:10:00   
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

Gradient:

Retention (min)Flow (mL/min)%B
52
52
52
530
575
50.5100
80.5100
52
70.52

Chromatography HILIC (+)

1d 4h

Phase A:
200 mL + 2.6 mL * [Number of injections]
ReagentWater milliQMerck MilliporeSigma (Sigma-Aldrich)   + Concentration10 millimolar (mM)  Reagentformiate ammoniumVWR International (Avantor)  + 0.1% ReagentFormic acid >99% for LC-MS VWR International (Avantor)Catalog #84865.260  
Stir vigorously during Duration00:01:00  
Ultrasound during Duration00:10:00    to remove air bubble

15m

Phase B:
200 mL + 3.8 mL * [Number of injections]
[95% of
ReagentAcetonitrile HiPerSolv CHROMANORMVWR International (Avantor)   & 5% of ReagentWater milliQMerck MilliporeSigma (Sigma-Aldrich)  ] + Concentration10 millimolar (mM)   Reagentformiate ammoniumVWR International (Avantor)  + 0.1% ReagentFormic acid >99% for LC-MS VWR International (Avantor)Catalog #84865.260  
Stir vigorously during Duration00:01:00  
Ultrasound during Duration00:10:00    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

Adjust the temperature (Temperature40 °C   ) and let it heat the core of the column

Prepare the cleaning solvents (same solvent as the recovery solvent) & ultrasound it during Duration00:05:00    to remove air bubbles

10m

Prepare the piston “lubrication” solution (90% ReagentMethanol HiPerSolv CHROMANORMVWR International (Avantor)   and 10%ReagentWater milliQMerck MilliporeSigma (Sigma-Aldrich)   ) & ultrasound it during Duration00:10:00    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 Duration00:10:00   
Check that the pression is stable; otherwise : purge again

Balancing the system: Gradually reach the flow rate of 0.5 mL/min at 95%B

Gradient:

Retention (min)Flow (mL/min)%B
595
595
.90.595
80.582
510
20.510
40.595
70.595

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 Temperature50 °C   
Let it "cool" Duration01:00:00   
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 ReagentAluminium oxide powerMerck MilliporeSigma (Sigma-Aldrich)  
Rinse thoroughly with ReagentWater milliQMerck MilliporeSigma (Sigma-Aldrich)  
In a glass beaker, cover completely the Sweep Cone and the capillary with ReagentMethanol HiPerSolv CHROMANORMVWR International (Avantor)  
Ultrasound during Duration00:10:00   
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: ReagentCalibration Solution Pierce LTQ Velos ESI Positive IonThermo Fisher  
NEG: ReagentCalibration 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 : ReagentAcetonitrile HiPerSolv CHROMANORMVWR International (Avantor)  /ReagentWater milliQMerck MilliporeSigma (Sigma-Aldrich)   (1:1)
ReagentL-tryptophanMerck MilliporeSigma (Sigma-Aldrich)Catalog #T0254  at Concentration10000 nanomolar (nM)  
ReagentIndoxyl-3-sulfateMerck MilliporeSigma (Sigma-Aldrich)  at Concentration3750 nanomolar (nM)  
ReagentKynurenic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #K3375  at Concentration25 nanomolar (nM)  
Reagentindole-3-Lactic AcidMerck MilliporeSigma (Sigma-Aldrich)  at Concentration500 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

Data Analysis

2d 4h

Metabolite Identification :
m/z and RT of a chemical library (ReagentIROA MSMLS, OAMLS, BACSMLS, FAMLSMerck MilliporeSigma (Sigma-Aldrich)  ) checked with 
Software
Thermo Scientific Xcalibur
NAME

MS² acquired and checked on QC samples
File exported : list of m/z and RT

Features detection:
Raw files are converted 

Software
MSconvert
NAME
Chambers, M., Maclean, B., Burke, R. et al. A cross-platform toolkit for mass spectrometry and proteomics. Nat Biotechnol 30, 91
DEVELOPER
https://proteowizard.sourceforge.io/download.html
SOURCE LINK

Using Workflow4Metabolomics :
         - The data were processed using the XCMS package in R.
         - Molecules were then identified according to the following criteria:
                   -> Retention time delta with reference: 20 seconds
                   -> Experimental vs. theoretical mass delta: 10 ppm
         - Once the Excel data table with the detectedmetabolites was obtained, the data were filtered (following steps)
Software
Workflow4Metabolomics
NAME
doi: 10.1093/bioinformatics/btu813
DEVELOPER
Galaxy
REPOSITORY
https://workflow4metabolomics.usegalaxy.fr/
SOURCE LINK
Data Curation = Signal Normalization & Metabolites Exclusion :
         - Normalization of metabolite areas to the total area of detected metabolites
         - Calculation of the coefficient of variation (CV) for each metabolite in the QCs (analytical variability) and in the samples (biological variability)
         - Removal of metabolites with analytical variability > biological variability
         - Removal of metabolites with analytical variability > 30%

Signal Normalization and Metabolites Exclusion are repetead until there is no further exclusion

Analysis Validation: the analyses were validated by observing the distribution of QCs among the samples using Principal Component Analysis (PCA). For all PCAs, the data were log-transformed and underwent UV scaling normalization.

Software
SIMCA
NAME
Sartorius
DEVELOPER
https://www.sartorius.com/en/products/process-analytical-technology/data-analytics-software/mvda-software/simca
SOURCE LINK

Fusion of modalites = sorting redundancies:
Only the best modality is kept for 1 metabolite, based on RT > Dead Volume (0-1 min) and/or lower CV(metabolite) on QCs
"Analysis Validation" is proceed
Assignment of CHEBI identifiers des for those referenced in the CHEBI database

Software
CHEBI
NAME
Malik, A., Arsalan, M., Moreno, C., Mosquera, J., Félix, E., Kizilören, T., Muthukrishnan, V., Zdrazil, B., Leach, A. R., and O'
DEVELOPER
https://www.ebi.ac.uk/chebi/about
SOURCE LINK
Specific identifiers for the “Homo sapiens - Recon 2_2” metabolic network are also available, allowing them to be viewed via MetExplore3

OpenScience

"Open science has the potential of making the scientific process more transparent, inclusive and democratic.  Open science:
increases scientific collaborations and sharing of information for the benefits of science and society;
makes multilingual scientific knowledge openly available, accessible and reusable for everyone; and
opens the processes of scientific knowledge creation, evaluation and communication to societal actors beyond the traditional scientific community.
Our interconnected world needs open science to help solve complex social, environmental, and economic challenges and achieve the Sustainable Development Goals."
https://www.unesco.org/en/open-science/about

 For Materials & Methods ->

Software
protocols.io
NAME
https://www.protocols.io/welcome
SOURCE LINK

Files -> 

Software
MetaboLights
NAME
Ozgur Yurekten, Thomas Payne, Noemi Tejera, Felix Xavier Amaladoss, Callum Martin, Mark Williams, Claire O’Donovan. MetaboLights
DEVELOPER
https://www.ebi.ac.uk/metabolights/login
SOURCE LINK

Retention (min)	Flow (mL/min)	%B
0	0.5	2
0	0.5	2
2	0.5	2
6	0.5	30
9	0.5	75
9.5	0.5	100
11.8	0.5	100
12	0.5	2
13.7	0.5	2

Retention (min)	Flow (mL/min)	%B
0	0.5	95
0	0.5	95
.9	0.5	95
4.8	0.5	82
9	0.5	10
11.2	0.5	10
11.4	0.5	95
13.7	0.5	95

	Sample Type	Injection Volume (µL)
	Equilibrium(=sample) * 10	5
	Blank	5
	Performance Evaluation * 3	5
	Blank	5
	Blank Solvent #1	5
	Blank Solvent #2	5
	Blank Extraction #1	5
	Blank Extraction #2	5
	Sample QC	5
	Sample *10	5
	Sample QC	5
	Continue the sequence, alternating between samples and a QC	5
	Last Sample QC	5
	QC in MS²	5
	Blank Solvent * 3	5

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Large Targeted Metabolomics_LC-MS Parameters_MTH Tours V.1