Jun 12, 2026
Version 2
Untargeted Metabolomics : Extraction of stool Metabolites V.2
This protocol is a draft, published without a DOI.
- 1CEA
- MetaboHub-IDF
- MetaboHUB
External link: https://joliot.cea.fr/drf/joliot/Pages/Entites_de_recherche/medicaments_technologies_sante/SPI/lims.aspx
Protocol Citation: Emeline CHU-VAN, florence castelli 2026. Untargeted Metabolomics : Extraction of stool Metabolites. protocols.io https://dx.doi.org/Version created by Emeline CHU-VAN
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 11, 2026
Last Modified: June 12, 2026
Protocol Integer ID: 318924
Keywords: METABOHUB, large cohorts, human health, biomarker discovery, untargeted metabolomic, extraction of stool metabolites, stool metabolome, internal metabolite standards in the extraction process, internal metabolite standard, coverage of stool metabolome, stool metabolite, accurate comparison of relative metabolite abundance, relative metabolite abundance, methanolic protein precipitation from stool, controlled extraction process, extraction process, extraction, methanolic protein precipitation, samples within the large cohort, extraction of stool metabolite, stool metabolome, stool metabolite, coverage of stool metabolome, internal metabolite standards in the extraction process, untargeted metabolomic, metabolomic, methanolic protein precipitation from stool, internal metabolite standard, accurate comparison of relative metabolite abundance, relative metabolite abundance, extraction, controlled extraction process, extraction process, stool, methanolic protein precipitation, samples within the large cohort
Funders Acknowledgements:
MetEx+
Grant ID: 21-ESRE-0035
METABOHUB
Grant ID: 11-INBS-0010
Abstract
The extraction of stool metabolites for untargeted metabolomics involves crushing and methanolic protein precipitation from stool samples, aiming to maximize coverage of stool 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.
Materials
Precellys :
Cryolys :
Tubes precellys :
Freezer -80°C :
Vortex shaker :
Centrifuge :
plate reader (562 nm)
Evaporator :
Lobind microtubes :
Micropipettes :
Protocol materials
Acetonitrile ULC-MSBiosolveCatalog #0001204101BS
ColchicineMerck MilliporeSigma (Sigma-Aldrich)Catalog #C9754
Imipramine hydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #I7379
Alanine-C13Merck MilliporeSigma (Sigma-Aldrich)Catalog #489867
D-Glucose-1-*13*CMerck MilliporeSigma (Sigma-Aldrich)Catalog #297046
Metformine (1,1-Dimethylbiguanide hydrochloride)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D150959
2-aminoanthraceneMerck MilliporeSigma (Sigma-Aldrich)Catalog #A38800
Formic acid 99.0+% qualite LC/MS optimaFisher ScientificCatalog #10780320
L-Aspartic acid-*15*NMerck MilliporeSigma (Sigma-Aldrich)Catalog #332135
Ethylmalonic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #102687
Amiloride hydrochloride hydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #A7410
Water LC-MS ChromasolvFisher ScientificCatalog #15665350
DihydrostreptomycinMerck MilliporeSigma (Sigma-Aldrich)Catalog #D7253
RoxithromycinMerck MilliporeSigma (Sigma-Aldrich)Catalog #R4393
prednisoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #P6254
AtropineMerck MilliporeSigma (Sigma-Aldrich)Catalog #A0132
2-methyl-4-chlorophenoxyacetic acid (MCPA)Merck MilliporeSigma (Sigma-Aldrich)Catalog #45555
DinosebMerck MilliporeSigma (Sigma-Aldrich)Catalog #45453
Ethanol, absolute 99.8%Thermo FisherCatalog #10342652
DimetridazoleMerck MilliporeSigma (Sigma-Aldrich)Catalog #31707
2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid (AMPA)Merck MilliporeSigma (Sigma-Aldrich)Catalog #A0326
Pierce BCA protein assayThermo ScientificCatalog #23227
AMMONIUM CARBONATE, 99.999%Merck MilliporeSigma (Sigma-Aldrich)Catalog #379999
Ammonia solution 25% for HPLMerck MilliporeSigma (Sigma-Aldrich)Catalog #5438300100
ampicillinMerck MilliporeSigma (Sigma-Aldrich)Catalog #59349
Safety warnings
This protocol involves the use of volatile solvents that may pose health risks. Therefore, it is essential to handle these solvents within a designated chemical fume hood and to wear appropriate personal protective equipment (gloves, goggles, lab coat, etc.).
Wear appropriate clothing, closed shoes, lab coat, glasses, gloves, extractor hood
Use of CMR solvents, use of nitrogen
Wear your Personal Protective Equipment (PPE) at least a lab coat, protective eyewear, gloves, long pants and closed toe shoes
Ethics statement
For experiments involving humans, the CPP (commission de protection des personnes physique) agreement.
Randomization
Reorganize stool samples in random order on dry ice (the same randomization is applied for the entire process (sample prep - LC-MS analysis - data treatment)
Store at -80 °C until extraction or proceed to aliquoting step.
Sample extraction
1h 0m 50s
Thaw samples during about 01:00:00 but keep the samples fresh (at +5°C or on ice)
1h
Transfer stool samples in Precellys tubes (about 3 mm3 )
Precellys tubes reference : P000973-LYSK0-A.0 (CK14)
Add 1 mL Ethanol containing internal standards at 37.5 ng/mL
Ethanol, absolute 99.8%Thermo FisherCatalog #10342652
Internal standards :
DimetridazoleMerck MilliporeSigma (Sigma-Aldrich)Catalog #31707
2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid (AMPA)Merck MilliporeSigma (Sigma-Aldrich)Catalog #A0326
2-methyl-4-chlorophenoxyacetic acid (MCPA)Merck MilliporeSigma (Sigma-Aldrich)Catalog #45555
DinosebMerck MilliporeSigma (Sigma-Aldrich)Catalog #45453
vortex 00:00:05
5s
Crush samples with homogenizer (Precellys with Cryolys to keep samples fresh)
Crushing parameters :
speed 6500 rpm
cycle dutation 00:00:30
number of cycles 3
pause between cycles duration 00:00:15
temperature 5 °C
Equipment
Precellys Evolution Touch
NAME
Homogenizer
TYPE
Bertin Technologies
BRAND
P002511-PEVT0-A.0
SKU
LINK
Equipment
Cryolys Evolution
NAME
cooling system compatible with the Precellys Evolution tissue homogenizer
TYPE
Bertin Technologies
BRAND
P000671-CLYS2-A.0 Cryolys Evolution
SKU
LINK
45s
20000 x g, 5°C 15 min
supernatants transfer
300 µL for Hilicneg
300 µL for C18pos
300 µL for QCpool (re-aliquot by 300 µL or a multiple of 300 µL)
Store the pellets at -20 °C for future proteins assay
Dry supernatants under a nitrogen stream at 30 °C using a
Equipment
TurboVap LV
NAME
Evaporator
TYPE
BIOTAGE
BRAND
TurboVap® LV
SKU
Store extracts at -20 °C until analysis.
Protein assay (BCA kit)
20m
Pierce BCA protein assayThermo ScientificCatalog #23227
| A | B | C | D | |
| Dilution schene for standard test tube protocol and microplate procedure (working range = 20 - 2000 µg/mL) | ||||
| vial standard | volume of diluent (µL) | volume and source of BSA (µL) | final BSA concentration (µg/mL) | |
| A | 0 | 300 of stock | 2000 | |
| B | 125 | 375 of stock | 1500 | |
| C | 325 | 325 of stock | 1000 | |
| D | 175 | 175 of vial B dilution | 750 | |
| E | 325 | 325 of vial C dilution | 500 | |
| F | 325 | 325 of vial E dilution | 250 | |
| G | 325 | 325 of vial F dilution | 125 | |
| H | 400 | 100 of vial G dilution | 25 | |
| I | 400 | 0 | 0 = blank | |
Standards (BSA) preparation (in lobind tubes if samples re in lobind tubes)
Diluent = water
| 1 | 2 | 3 | 4 | 5 | |
| A | Blank | std A | std A | sample 1 | sample 1 |
| B | Blank | std B | std B | sample 2 | sample 2 |
| C | Blank | std C | std C | sample 3 | sample 3 |
| D | Blank | std D | std D | sample 4 | sample 4 |
| E | Blank | std E | std E | sample 5 | sample 5 |
| F | Blank | std F | std F | sample 6 | sample 6 |
| G | Blank | std G | std G | sample 7 | sample 7 |
| H | Blank | std H | std H | sample 8 | sample 8 |
| 6 | 7 | 8 | 9 | 10 | |
| A | sample 9 | sample 9 | sample 17 | sample 17 | sample 25 |
| B | sample 10 | sample 10 | sample 18 | sample 18 | sample 26 |
| C | sample 11 | sample 11 | sample 19 | sample 19 | sample 27 |
| D | sample 12 | sample 12 | sample 20 | sample 20 | sample 28 |
| E | sample 13 | sample 13 | sample 21 | sample 21 | sample 29 |
| F | sample 14 | sample 14 | sample 22 | sample 22 | sample 30 |
| G | sample 15 | sample 15 | sample 23 | sample 23 | sample 31 |
| H | sample 16 | sample 16 | sample 24 | sample 24 | sample 32 |
| 11 | 12 | |
| A | sample 25 | sample 33 |
| B | sample 26 | sample 33 |
| C | sample 27 | sample 34 |
| D | sample 28 | sample 34 |
| E | sample 29 | sample 35 |
| F | sample 30 | sample 35 |
| G | sample 31 | H2O |
| H | sample 32 | H2O |
Colored reagent preparation :
Reagent B diluted 50-fold in reagent A
total amount = (standards + samples) x replicates x (volume of colored reagent per sample)
Blank = colored reagent only
Typical dilution of sample according to matrix :
- cell pellets = 5-fold
- stools = 50-fold
- organs = 5-fold or more if samples weigh around 50 - 100 mg
Recontitute pellets with 150 µL H2O (or more if necessary)
Pipette 20 µL of each blank / standard / sample in well plate
Add 200 µL of colored reagent
Incubate the plate 100 rpm, 40°C, 00:20:00
20m
Read the plate at 562 nm (reading between 540 and 590 nm can be used).
| A | B | C | D | E | F | G | H | I | |
| Wells | Concentration (µg/mL) | Value | Mean value | Standard deviation | Coefficient of variation (%) | Back calculated concentration (µg/mL) | Exactitude (%) | ||
| Standard A | A2 | 2000 | DO | DO | SD | < 15% | Conc | < 15% | |
| A3 | DO | ||||||||
| Standard B | B2 | 1500 | DO | DO | SD | < 15% | Conc | < 15% | |
| B3 | DO | ||||||||
| Standard C | C2 | 1000 | DO | DO | SD | < 15% | Conc | < 15% | |
| C3 | DO | ||||||||
| Standard D | D2 | 750 | DO | DO | SD | < 15% | Conc | < 15% | |
| D3 | DO | ||||||||
| Standard E | E2 | 500 | DO | DO | SD | < 15% | Conc | < 15% | |
| E3 | DO | ||||||||
| Standard F | F2 | 250 | DO | DO | SD | < 15% | Conc | < 15% | |
| F3 | DO | ||||||||
| Standard G | G2 | 125 | DO | DO | SD | < 15% | Conc | < 15% | |
| G3 | DO | ||||||||
| Standard H | H2 | 25 | DO | DO | SD | < 15% | Conc | < 15% | |
| H3 | DO |
Expected results
Substract the average absorbance measurement of the blank replicates from themeasurements of all other replicates (standards / samples).
Curve fitting = quadratic
Expected result
Results are validated if :
- CV < 15% for standards and samples
- exactitude < 15% for standards
- Calculated concentrations for samples are std A > conc > std G
Samples reconstitution for Hilic analysis in 2 steps
15m 10s
Resuspend dried extracts :
According to protein assay, samples are reconstituted with a ratio 1 mg proteins / 50 µL.
But for stool sample, a ratio 50 µg / 50 µL can be well, it depend on the sensitivity of the MS detector.
If the final volumes are lower than 75 µL or heigher than 300 µL, the ratio can be adapted.
Reagents for mobile phases :
Water LC-MS ChromasolvFisher ScientificCatalog #15665350
AMMONIUM CARBONATE, 99.999%Merck MilliporeSigma (Sigma-Aldrich)Catalog #379999
Ammonia solution 25% for HPLMerck MilliporeSigma (Sigma-Aldrich)Catalog #5438300100
Acetonitrile ULC-MSBiosolveCatalog #0001204101BS
*ES (external standards : mixture of 14 authentic chemical standards covering the mass range of interest):D-Glucose-1-*13*CMerck MilliporeSigma (Sigma-Aldrich)Catalog #297046
L-Aspartic acid-*15*NMerck MilliporeSigma (Sigma-Aldrich)Catalog #332135
Ethylmalonic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #102687
Amiloride hydrochloride hydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #A7410
prednisoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #P6254
Metformine (1,1-Dimethylbiguanide hydrochloride)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D150959
AtropineMerck MilliporeSigma (Sigma-Aldrich)Catalog #A0132
ColchicineMerck MilliporeSigma (Sigma-Aldrich)Catalog #C9754
Imipramine hydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #I7379
Alanine-C13Merck MilliporeSigma (Sigma-Aldrich)Catalog #489867
2-aminoanthraceneMerck MilliporeSigma (Sigma-Aldrich)Catalog #A38800
ampicillinMerck MilliporeSigma (Sigma-Aldrich)Catalog #59349
DihydrostreptomycinMerck MilliporeSigma (Sigma-Aldrich)Catalog #D7253
RoxithromycinMerck MilliporeSigma (Sigma-Aldrich)Catalog #R4393
Mobile phases preparation for Hilic analysis :
Mobile phase A = Ammonium carbonate 10 mM - pH 10.5
in 1L of water add 0.9606 mg ammonium carbonate
adjust pH at 10.5 with ammonia solution
Mobile phase B = 100% acétonitrile
First step = 40% (of final volume) of mobile phase A containing ES* 2.5x
0 µL Vortex 00:00:05
5s
Second step = 60% (of final volume) of mobile phase B
0 Mass Percent Vortex 00:00:05
5s
20000 x g, 5°C, 00:15:00
15m
Transfer supernatant into 0.2 mL vials
Samples reconstitution for C18 analysis in 1 step
15m 5s
Resuspend dried extracts :
According to protein assay, samples are reconstituted with a ratio 1 mg proteins / 50 µL.
But for stool sample, a ratio 50 µg / 50 µL can be well, it depend on the sensitivity of the MS detector.
if the final volumes are lower than 75 µL or heigher than 300 µL, the ratio can be adapted.
reagents for mobile phases :
Water LC-MS ChromasolvFisher ScientificCatalog #15665350
Acetonitrile ULC-MSBiosolveCatalog #0001204101BS
Formic acid 99.0+% qualite LC/MS optimaFisher ScientificCatalog #10780320
*ES (external standards : mixture of 14 authentic chemical standards covering the mass range of interest):
D-Glucose-1-*13*CMerck MilliporeSigma (Sigma-Aldrich)Catalog #297046
L-Aspartic acid-*15*NMerck MilliporeSigma (Sigma-Aldrich)Catalog #332135
Ethylmalonic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #102687
Amiloride hydrochloride hydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #A7410
prednisoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #P6254
Metformine (1,1-Dimethylbiguanide hydrochloride)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D150959
AtropineMerck MilliporeSigma (Sigma-Aldrich)Catalog #A0132
ColchicineMerck MilliporeSigma (Sigma-Aldrich)Catalog #C9754
Imipramine hydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #I7379
Alanine-C13Merck MilliporeSigma (Sigma-Aldrich)Catalog #489867
2-aminoanthraceneMerck MilliporeSigma (Sigma-Aldrich)Catalog #A38800
DihydrostreptomycinMerck MilliporeSigma (Sigma-Aldrich)Catalog #D7253
RoxithromycinMerck MilliporeSigma (Sigma-Aldrich)Catalog #R4393
Mobile phases preparation for C18 analysis :
mobile phase A = Water 0.1% HCOOH
add 1 mL formic acid in 1L water
mobile phase B = Acétonitrile 0.1% HCOOH
add 1 mL formic acid in 1L acetonitrile
add a mix mobile phase A / mobile phase B / ES* (90/5/5)
Vortex 00:00:05
5s
20000 x g, 5°C, 00:15:00
15m
Transfer supernatant into 0.2 mL vials
QC reconstition
According to protein assay, samples are reconstituted with a ratio 1 mg proteins / 50 µL.
But for stool sample, a ratio 50 µg / 50 µL can be well, it depend on the sensitivity of the MS detector.
For QC reconstitution, final volume = average of all sample volumes
Hilic analysis - reconstitution in 2 steps
C18 analysis - reconstitution in 1 step
Prepare 2-fold, 4-fold and 8-fold diluted QC samples (in mobile phases)
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