Mar 12, 2026
Version 1
Large Targeted Metabolomics_Sample Preparation_MTH Tours V.1
- 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
External link: https://metabolomique.med.univ-tours.fr/
Protocol Citation: Staff Members of PMAC 2026. Large Targeted Metabolomics_Sample Preparation_MTH Tours. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1rm57lr2/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: 313069
Keywords: metabolomics, LC-MS, extraction, biofluids, feces, tissues, metabolomic coverage, analysis of metabolite, metabolite, sample preparation, methods after sample preparation, present in biological sample, biological sample, cv of qc sample, qc sample, protein precipitation, data treatment data, sample, system suitability cv of qc sample
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 ESI-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 QC injections, and at least 50% of samples, 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
Solvents
- Methanol HiPerSolv CHROMANORM (VWR International (Avantor))
- Acetonitrile HiPerSolv CHROMANORM (VWR International (Avantor))
- Water milliQ (Merck MilliporeSigma (Sigma-Aldrich))
Consumable Items
- Pipettes tips
- 1.5 mL glass vial
- 200 µL glass insert
- Vials caps
- 1.5 mL Eppendorf tube
- 96-wells plate (200 µL, 500 µL, 800 µL)
Materials
- Micropipettes (200 µL, 1000 µL)
- Planar agitator
- Vortex
- Centrifuge
- Lyophilisator
- SpeedVac (or equivalent)
- N2 evaporator
- Precision balance
Protocol materials
Acetonitrile HiPerSolv CHROMANORMVWR International (Avantor)
Water milliQMerck MilliporeSigma (Sigma-Aldrich)
Acetonitrile HiPerSolv CHROMANORMVWR International (Avantor)
Methanol HiPerSolv CHROMANORMVWR International (Avantor)
Water milliQMerck 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.
Ethics statement
All samples were obtained in accordance with French legislation (Loi Jardé). In French hospitals with a research mission, the use of sample leftovers for research purposes is based on the principle of no opposition. According to Loi Jardé, no requirement of Ethical Committee or Institutional Review Board was required because no new sampling was required. This law also stipulates that patients must be fully informed of this information/practice and patients must have the possibility, at any time, of objecting to the use of their samples in research. The analyses were always performed in accordance with confidentiality rules. Data were coded without mention of first and last names, and the results were produced in a way that does not allow patients to be identified. Patients did not invoke their right to have a right of access, rectification, portability and limitation of the processing of data and/or biological samples.
A) Extraction of biofluids
13h 10m
For biofluids extraction, 3 steps are necessary and expleined in detail below
A.1) Aliquoting
Let samples thaw at Room temperature
Make 2 separate 50 µL aliquots (1 for Reversed-Phase(A.1) & 1 for HILIC (A.2))
For biological fluids, prepare a pooled sample by taking the same volume from each sample. After shaking vigorously, make QC aliquots (generally, 1 QC for every 10 samples)
Cover each plate with aluminum foil
Store at -80 °C pending extraction
A.2) Reversed-Phase
1h
Add Methanol HiPerSolv CHROMANORMVWR International (Avantor) to a 15 mL or 50 mL plastic tube
Remove the aluminum foil
Add 400 µL of Methanol HiPerSolv CHROMANORMVWR International (Avantor) to every well
Cover with a new aluminum foil
Planar agitation: strength 6/10, 00:10:00 , Room temperature
Centrifugation: 3000 rpm, 4°C, 00:30:00 , Acc = 6/9, Dec = 6/9
1h
Remove supernatant: transfer 350 µL to a new 96-well plate 500 µL
Nitrogen evaporation: 40 °C , 00:45:00
In a 15 mL or 50 mL new container, prepare a "recovery solution" of Acetonitrile HiPerSolv CHROMANORMVWR International (Avantor) / Water milliQMerck MilliporeSigma (Sigma-Aldrich) (2:8)
Add 100 µL of recovery solvent to every well
Planar agitation: strength 6/10, 00:10:00 , Room temperature
Centrifugation: 3000 rpm, 4°C, 00:30:00 , Acc = 6/9, Dec = 6/9
Transfer 90 µL to a 96-well plate with 200 µL wells
Cover with plastic film for injection
A.3) HILIC
In a 15 mL or 50 mL tube, add Acetonitrile HiPerSolv CHROMANORMVWR International (Avantor)
Remove the aluminum foil
Add 400 µL of Acetonitrile HiPerSolv CHROMANORMVWR International (Avantor)
Put on a new aluminum foil
Planar agitation: strength 6/10, 00:10:00 ,Room temperature
Centrifugation: 3000 rpm, 4°C, 00:30:00 , Acc = 6/9, Dec = 6/9
Remove supernatant: transfer 350 µL to a new 96-well plate 500 µL
Nitrogen evaporation: 40 °C , 00:45:00
In a 15 mL or 50 mL tube, prepare a "recovery solution" of Acetonitrile HiPerSolv CHROMANORMVWR International (Avantor) /Water milliQMerck MilliporeSigma (Sigma-Aldrich) (8:2)
Add 100 µL of recovery solvent to each well
Planar agitation: strength 6/10, 00:10:00 , Room temperature
Centrifugation: 3000 rpm, 4°C, 00:30:00 , Acc = 6/9, Dec = 6/9
Transfer 90 µL to a glass insert for a 2mL glass vial
Close all vials
B) Extraction of solid (tissue & feces)
For solid sample extraction, 3 steps are necessary and expleined in detail below
B.1) Aliquoting
Lyophilized whole tissue 48:00:00 (at least) :
- Insert frozen sample in a suitable container
- Cover with parafilm
- Pierce multiple times the parafilm
- Allow tubes and sample to return to -80 °C
Weight 3 mg +/- 0.2 mg of lyophilized sample in 1.5 mL Eppendorf tube
Add 1000 µL of Methanol HiPerSolv CHROMANORMVWR International (Avantor) /Water milliQMerck MilliporeSigma (Sigma-Aldrich) (1:1)
TIPS: From experience, ACN/H₂O extraction (1:1) is more suitable for brain extraction.
Agitate vigorously (3000 rpm) during 00:00:05
Planar agitation: strength 7/10, 00:30:00 , Room temperature
Centrifugation: 15000 x g, 4°C, 00:15:00 , Acc = 9/9, Dec = 9/9
- Transfert 950 µL of supernatant in a 1.5 mL Eppendorf tube (Reversed-Phase)
- From the Eppendorf, transfert 375 µL of a 1.5 mL glass vial (HILIC)
B.2) Reversed-Phase
Dry sample at 40 °C , 04:00:00 and under vacuum
Add 100 µL of Acetonitrile HiPerSolv CHROMANORMVWR International (Avantor) /Water milliQMerck MilliporeSigma (Sigma-Aldrich) (2:8)
Agitate vigorously (3000 rpm) during 00:00:05
Planar agitation: strength 7/10, 00:15:00 , Room temperature
Centrifugation: 15000 x g, 4°C, 00:15:00 , Acc = 9/9, Dec = 9/9
Transfert 90 µL in a 200 µL 96-wells plates
QC sample :
- Transfert 5 µL from each wells in a 1.5 mL tube
- Agitate vigorously (3000 rpm) during 00:00:30
- Transfert the whole QC in one well
Cover with a transparent lid for injection
B.3) HILIC
Dry sample at 40 °C , 04:00:00 and under vacuum
Add 100 µL of Acetonitrile HiPerSolv CHROMANORMVWR International (Avantor) /Water milliQMerck MilliporeSigma (Sigma-Aldrich) (8:2)
Agitate vigorously (3000 rpm) during 00:00:15
Planar agitation: strength 7/10, 00:15:00 , Room temperature
Centrifugation: 15000 x g, 4°C, 00:15:00 , Acc = 9/9, Dec = 9/9
- Transfert 90 µL in a 200 µL glass insert
- Set the insert inside the glass vial
Close all vials