Jun 11, 2026
Version 2
Untargeted Lipidomics - Sample Preparation - MetaboHUB Tours V.2
- 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
External link: https://metabolomique.med.univ-tours.fr/
Protocol Citation: Staff Members of PMAC 2026. Untargeted Lipidomics - Sample Preparation - MetaboHUB Tours. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn5yeqg5d/v2Version 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 10, 2026
Last Modified: June 11, 2026
Protocol Integer ID: 318841
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, lipidomic coverage, analysis of lipid, lipidsearch, lipid, standardized sample preparation workflow for untargeted lipidomics analysis, untargeted lipidomics analysis, untargeted lipidomic, metabohub tours this protocol, standardized sample preparation workflow, metabohub tour, using isopropanol, fecal sample, biological fluid
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
This protocol describes a standardized sample preparation workflow for untargeted lipidomics analysis of biological fluids, tissues, and fecal samples. Lipids are extracted using isopropanol-based precipitation and recovery procedures. The samples are now ready to be injected ( for more information, please refer to our protocol "Untargeted Lipidomics - LC-MS Parameters of Q-Exactive - MetaboHUB Tours" )
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 )
Untargeted lipidomics 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 lipids was performed based on MS² spectra and retention time compared with a well-characterized mix sample, used a reference sample.
Materials
Solvents
- Methanol HiPerSolv CHROMANORM (VWR International (Avantor))
- Acetonitrile HiPerSolv CHROMANORM (VWR International (Avantor))
- Isopropanol HyperSolv 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
Water milliQMerck MilliporeSigma (Sigma-Aldrich)
Acetonitrile HiPerSolv CHROMANORMVWR International (Avantor)
Isopropanol HyperSolv CHROMANORMMerck MilliporeSigma (Sigma-Aldrich)
Water milliQMerck MilliporeSigma (Sigma-Aldrich)
Safety warnings
All sample handling must be carried out under a fume hood, equipped with traditional PPE: lab coat, goggles, and gloves.
Ethics statement
This protocol is used by a service provider platform in collaboration with a hospital and a research laboratory that works with animal models.
1) In the case of research involving human biological samples, the following rules apply:
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.
2) If your research involves animal biological samples, please clearly identify the permits required for your experiments.
A) Extraction of biofluids
13h 10m
For biofluids extraction, 2 steps are necessary and expleined in detail below
A.1) Aliquoting
Let samples thaw at Room temperature
Make 1 50 µL aliquot
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 Isopropanol HyperSolv CHROMANORMMerck MilliporeSigma (Sigma-Aldrich) to a 15 mL or 50 mL plastic tube
Remove the aluminum foil
Add 300 µL of Isopropanol HyperSolv CHROMANORMMerck MilliporeSigma (Sigma-Aldrich) 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 250 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) / Isopropanol HyperSolv CHROMANORMMerck MilliporeSigma (Sigma-Aldrich) /Water milliQMerck MilliporeSigma (Sigma-Aldrich) (6:3:1)
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
B) Extraction of solid (tissue & feces)
For solid sample extraction, 2 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 600 µL of Isopropanol HyperSolv CHROMANORMMerck MilliporeSigma (Sigma-Aldrich) /Water milliQMerck MilliporeSigma (Sigma-Aldrich) (9:1)
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 500 µL of supernatant in a 96-wells plate
- Pool all supernatants by taking 50 µL . After shaking vigorously, make QC aliquots (approximately 1 QC for every 10 injections).
Nitrogen evaporation: 40 °C , 04:00:00
Cover with plastic film for injection
B.2) Reversed-Phase
In a 15 mL or 50 mL new container, prepare a "recovery solution" of Acetonitrile HiPerSolv CHROMANORMVWR International (Avantor) / Isopropanol HyperSolv CHROMANORMMerck MilliporeSigma (Sigma-Aldrich) /Water milliQMerck MilliporeSigma (Sigma-Aldrich) (6:3:1)
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
Transfert 90 µL in a 200 µL 96-wells plates
Cover with a transparent lid for injection