Mar 25, 2025

Public workspaceUntargeted Metabolomics: Extraction of Plasma Metabolites V.2

DOI
dx.doi.org/10.17504/protocols.io.3byl4qo18vo5/v2
  • 1CEA
  • MetaboHub-IDF
emeline.chu-van CHU-VAN
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DOI: dx.doi.org/10.17504/protocols.io.3byl4qo18vo5/v2
External link: https://joliot.cea.fr/drf/joliot/Pages/Entites_de_recherche/medicaments_technologies_sante/SPI/lims.aspx
Protocol Citation: florence castelli, emeline.chu-van CHU-VAN 2025. Untargeted Metabolomics: Extraction of Plasma Metabolites . protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4qo18vo5/v2Version created by emeline.chu-van CHU-VAN
Manuscript citation:
N/A
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: February 19, 2024
Last Modified: March 25, 2025
Protocol Integer ID: 124951
Keywords: METABOHUB, large cohorts, Human health, Biomarker discovery
Funders Acknowledgements:
METABOHUB
Grant ID: 11-INBS-0010
MetEx+
Grant ID: 21-ESRE-0035
Disclaimer
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Abstract
The extraction of plasma metabolites for untargeted metabolomics involves methanolic protein precipitation from a fixed volume of fasting plasma, aiming to maximize coverage of the plasma metabolome. The proposed protocol includes both external and internal metabolite standards in the extraction process to answert to our quality control (QC) procedures. The expected outcome is a normalized and controlled extraction process, allowing for the accurate comparison of relative metabolite abundances across all samples within the large cohorts.
Image Attribution
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Guidelines
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Protocol materials
Reagentwater LC-MS ChromasolvFisher ScientificCatalog #15665350
ReagentAlanine-C13Merck MilliporeSigma (Sigma-Aldrich)Catalog #489867
ReagentampicillinMerck MilliporeSigma (Sigma-Aldrich)Catalog #59349
ReagentAcetonitrile ULC-MSBiosolveCatalog #0001204101BS
ReagentFormic acid 99.0+% qualite LC/MS optimaFisher ScientificCatalog #10780320
ReagentD-Glucose-1-*13*CMerck MilliporeSigma (Sigma-Aldrich)Catalog #297046
ReagentAmiloride hydrochloride hydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #A7410
ReagentAmmonia solution 25% for HPLMerck MilliporeSigma (Sigma-Aldrich)Catalog #5438300100
ReagentEthylmalonic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #102687
ReagentMetformine (1,1-Dimethylbiguanide hydrochloride)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D150959
ReagentAtropineMerck MilliporeSigma (Sigma-Aldrich)Catalog #A0132
ReagentImipramine hydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #I7379
ReagentAMMONIUM CARBONATE, 99.999%Merck MilliporeSigma (Sigma-Aldrich)Catalog #379999
ReagentColchicineMerck MilliporeSigma (Sigma-Aldrich)Catalog #C9754
Reagent2-aminoanthraceneMerck MilliporeSigma (Sigma-Aldrich)Catalog #A38800
ReagentDihydrostreptomycinMerck MilliporeSigma (Sigma-Aldrich)Catalog #D7253
ReagentRoxithromycinMerck MilliporeSigma (Sigma-Aldrich)Catalog #R4393
ReagentL-Aspartic acid-*15*NMerck MilliporeSigma (Sigma-Aldrich)Catalog #332135
ReagentprednisoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #P6254
ReagentMethanol LC-MS CHROMASOLVFisher ScientificCatalog #15614740
ReagentDimetridazoleMerck MilliporeSigma (Sigma-Aldrich)Catalog #31707
Reagent2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid (AMPA)Merck MilliporeSigma (Sigma-Aldrich)Catalog #A0326
Reagent2-methyl-4-chlorophenoxyacetic acid (MCPA)Merck MilliporeSigma (Sigma-Aldrich)Catalog #45555
ReagentDinosebMerck MilliporeSigma (Sigma-Aldrich)Catalog #45453
ReagentAtropineMerck MilliporeSigma (Sigma-Aldrich)Catalog #A0132
ReagentAmmonia solution 25% for HPLMerck MilliporeSigma (Sigma-Aldrich)Catalog #5438300100
ReagentAmiloride hydrochloride hydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #A7410
ReagentprednisoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #P6254
ReagentColchicineMerck MilliporeSigma (Sigma-Aldrich)Catalog #C9754
ReagentImipramine hydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #I7379
ReagentampicillinMerck MilliporeSigma (Sigma-Aldrich)Catalog #59349
ReagentRoxithromycinMerck MilliporeSigma (Sigma-Aldrich)Catalog #R4393
ReagentFormic acid 99.0+% qualite LC/MS optimaFisher ScientificCatalog #10780320
ReagentMetformine (1,1-Dimethylbiguanide hydrochloride)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D150959
ReagentDihydrostreptomycinMerck MilliporeSigma (Sigma-Aldrich)Catalog #D7253
ReagentAcetonitrile ULC-MSBiosolveCatalog #0001204101BS
ReagentD-Glucose-1-*13*CMerck MilliporeSigma (Sigma-Aldrich)Catalog #297046
ReagentL-Aspartic acid-*15*NMerck MilliporeSigma (Sigma-Aldrich)Catalog #332135
Reagentwater LC-MS ChromasolvFisher ScientificCatalog #15665350
ReagentAMMONIUM CARBONATE, 99.999%Merck MilliporeSigma (Sigma-Aldrich)Catalog #379999
ReagentAlanine-C13Merck MilliporeSigma (Sigma-Aldrich)Catalog #489867
Reagent2-aminoanthraceneMerck MilliporeSigma (Sigma-Aldrich)Catalog #A38800
ReagentEthylmalonic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #102687
Before start
To analyze plasma samples, it is essential to collect all samples using the same anticoagulant (preferably EDTA for LC-HRMS analyses)
For human samples analyses in France, a CODECOH ("Déclaration ou autorisation des activités de conservation et de préparation à des fins scientifiques d'éléments issus du corps humain") must be submitted at least 2-3 months in advance to ensure approval before sample reception.
Randomisation
Randomisation
Reorganize plasma samples in random order on dry ice
Store at Temperature-80 °C until aliquoting
Sample aliquoting and quality control sample constitution
Sample aliquoting and quality control sample constitution
Thaw plasma samples (keep them fresh on ice)

Aliquoting : 50µL (or 25µL) depending on available volume
Create a quality control (QC) : pool of a same volume (~20µL) of all samples
Aliquoting QC : 50µL (or more) depending on available volume
Store at Temperature-80 °C until sample extraction
Sample extraction
Sample extraction
Thaw plasma samples (keep them fresh on ice)
Performe metabolite extraction twice (HILIC and C18) from 25µL or 50µL of plasma
Add 4 volumes (100µL or 200 µL) of cold MeOH containing internal standards (IS) at 37.5 ng/mL



extraction solvent :
ReagentMethanol LC-MS CHROMASOLVFisher ScientificCatalog #15614740

Internal standards :
ReagentDimetridazoleMerck MilliporeSigma (Sigma-Aldrich)Catalog #31707 , Reagent2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid (AMPA)Merck MilliporeSigma (Sigma-Aldrich)Catalog #A0326 ,Reagent2-methyl-4-chlorophenoxyacetic acid (MCPA)Merck MilliporeSigma (Sigma-Aldrich)Catalog #45555 , ReagentDinosebMerck MilliporeSigma (Sigma-Aldrich)Catalog #45453



Vortex Duration00:00:05

5s
Incubate on ice Duration01:30:00 until complete protein precipitation

1h 30m
Centrifigation20000 x g, 4°C, 00:15:00

15m
Collect supernatants
Dry supernatants under a nitrogen stream using a
Equipment
TurboVap LV
NAME
Evaporator
TYPE
BIOTAGE
BRAND
TurboVap® LV
SKU
Temperature30 °C

Store at Temperature-80 °C until analysis

sample reconstitution
sample reconstitution
15m 5s
15m 5s
Resuspend dried extracts in 3 volumes of Ammonium carbonate 10mM pH10.5 (with Ammonia solution 25%)+ ES* / Acetonitrile (40:60, v/v) for ZIC-pHILIC analysis
or ultrapure water / acetonitrile (95:5, v/v), containing 0.1% formic acid and ES* for C18 analysis.

reagents for mobile phases:
Reagentwater LC-MS ChromasolvFisher ScientificCatalog #15665350
ReagentAMMONIUM CARBONATE, 99.999%Merck MilliporeSigma (Sigma-Aldrich)Catalog #379999
ReagentAmmonia solution 25% for HPLMerck MilliporeSigma (Sigma-Aldrich)Catalog #5438300100
ReagentAcetonitrile ULC-MSBiosolveCatalog #0001204101BS
ReagentFormic acid 99.0+% qualite LC/MS optimaFisher ScientificCatalog #10780320

*ES (external standards : mixture of 14 authentic chemical standards covering the mass range of interest : ReagentD-Glucose-1-*13*CMerck MilliporeSigma (Sigma-Aldrich)Catalog #297046 , ReagentL-Aspartic acid-*15*NMerck MilliporeSigma (Sigma-Aldrich)Catalog #332135 , ReagentEthylmalonic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #102687 , ReagentAmiloride hydrochloride hydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #A7410 , ReagentprednisoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #P6254 , ReagentMetformine (1,1-Dimethylbiguanide hydrochloride)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D150959 , ReagentAtropineMerck MilliporeSigma (Sigma-Aldrich)Catalog #A0132 , ReagentColchicineMerck MilliporeSigma (Sigma-Aldrich)Catalog #C9754 , ReagentImipramine hydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #I7379 ,
ReagentAlanine-C13Merck MilliporeSigma (Sigma-Aldrich)Catalog #489867
Reagent2-aminoanthraceneMerck MilliporeSigma (Sigma-Aldrich)Catalog #A38800
ReagentampicillinMerck MilliporeSigma (Sigma-Aldrich)Catalog #59349
ReagentDihydrostreptomycinMerck MilliporeSigma (Sigma-Aldrich)Catalog #D7253
ReagentRoxithromycinMerck MilliporeSigma (Sigma-Aldrich)Catalog #R4393

Vortex Duration00:00:05
5s
Centrifigation20000 x g, 4°C, 00:15:00
15m
Transfer supernatant into 0.2 mL vials
QC recontitution
QC recontitution
15m 5s
15m 5s
Resuspend dried extracts in 3 volumes of Ammonium carbonate 10mM pH10.5 (with Ammonia solution 25%)+ ES* / Acetonitrile (40:60, v/v) for ZIC-pHILIC analysis
or ultrapure water / acetonitrile (95:5, v/v), containing 0.1% formic acid and ES* for C18 analysis.

reagents for mobile phases:
Reagentwater LC-MS ChromasolvFisher ScientificCatalog #15665350
ReagentAMMONIUM CARBONATE, 99.999%Merck MilliporeSigma (Sigma-Aldrich)Catalog #379999
ReagentAmmonia solution 25% for HPLMerck MilliporeSigma (Sigma-Aldrich)Catalog #5438300100
ReagentAcetonitrile ULC-MSBiosolveCatalog #0001204101BS
ReagentFormic acid 99.0+% qualite LC/MS optimaFisher ScientificCatalog #10780320

*ES (external standards : mixture of 14 authentic chemical standards covering the mass range of interest : ReagentD-Glucose-1-*13*CMerck MilliporeSigma (Sigma-Aldrich)Catalog #297046 , ReagentL-Aspartic acid-*15*NMerck MilliporeSigma (Sigma-Aldrich)Catalog #332135 , ReagentEthylmalonic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #102687 , ReagentAmiloride hydrochloride hydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #A7410 , ReagentprednisoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #P6254 , ReagentMetformine (1,1-Dimethylbiguanide hydrochloride)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D150959 , ReagentAtropineMerck MilliporeSigma (Sigma-Aldrich)Catalog #A0132 , ReagentColchicineMerck MilliporeSigma (Sigma-Aldrich)Catalog #C9754 , ReagentImipramine hydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #I7379 ,
ReagentAlanine-C13Merck MilliporeSigma (Sigma-Aldrich)Catalog #489867
Reagent2-aminoanthraceneMerck MilliporeSigma (Sigma-Aldrich)Catalog #A38800
ReagentampicillinMerck MilliporeSigma (Sigma-Aldrich)Catalog #59349
ReagentDihydrostreptomycinMerck MilliporeSigma (Sigma-Aldrich)Catalog #D7253
ReagentRoxithromycinMerck MilliporeSigma (Sigma-Aldrich)Catalog #R4393

Vortex Duration00:00:05
5s
Centrifigation20000 x g, 4°C, 00:15:00
15m
Transfer supernatant into 0.2 mL vials
Prepare 2-fold, 4-fold and 8-fold diluted QC samples
Protocol references
Boudah et al. Annotation of the human serum metabolome by coupling three liquid chromatography methods to high-resolution mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 2014 Sep 1:966:34-47. DOI: 10.1016/j.jchromb.2014.04.025