Mar 11, 2026

Fecal or caecal waters sample preparation for untargeted GCMS-based metabolomic V.1

DOI
https://dx.doi.org/10.17504/protocols.io.5qpvodqw9g4o/v1
Fecal or caecal waters sample preparation for untargeted GCMS-based metabolomic
  • 1Université Clermont Auvergne, INRAE, UNH, Plateforme d’Exploration du Métabolisme, MetaboHUB Clermont, Clermont-Ferrand, France;
  • 2INRAE
  • MetaboHUB-Clermont PFEM
Carole MIGNÉ
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DOI: https://dx.doi.org/10.17504/protocols.io.5qpvodqw9g4o/v1
External link: https://eng-pfem.isc.inrae.fr/
Protocol Citation: Carole MIGNÉ, Charlotte JOLY, Stéphanie DURAND, Estelle Pujos-Guillot 2026. Fecal or caecal waters sample preparation for untargeted GCMS-based metabolomic. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvodqw9g4o/v1
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: September 22, 2025
Last Modified: March 11, 2026
Protocol  Integer ID: 227888
Keywords: fecal water sample, caecal water sample, preparation, mass spectrometry, gas chromatography, untargeted metabolomic, derivatization, caecal waters sample preparation for untargeted gcm, caecal waters sample preparation, caecal water samples ready for analysis, based metabolomic, sample preparation, untargeted gcm, pfem, gcm
Funders Acknowledgements:
Agence Nationale de la Recherche au titre de France 2030
Grant ID: ANR- 21-ESRE-0035
Agence Nationale de la Recherche
Grant ID: ANR-11-INBS-0010
Abstract
The following protocol describes the preparation of fecal or caecal water samples for non-targeted analysis by mass spectrometry coupled to gas chromatography as proposed by the PFEM (MetaboHUB-Clermont).
The steps cover the sample preparation and quality control.
The expected outcome of this protocol is a list of fecal or caecal water samples ready for analysis by GCMS.
Materials
Sigma 3-16PK centrifuge (Fischer Bioblock Scientific)

Protocol materials
Methoxyamine hydrochlorideMerckCatalog #89803-1G
PyridineMerck MilliporeSigma (Sigma-Aldrich)Catalog #360570
BSTFA + 1% TMCS Silylation ReagentThermo FisherCatalog #TS-38833
NaN3Merck MilliporeSigma (Sigma-Aldrich)Catalog #S2002-5G
HeptaneMerck MilliporeSigma (Sigma-Aldrich)Catalog #246654-100mL
ULC/MS grade methanolBiosolveCatalog #0013684101BS
L-VALINE (1-13C, 99%)EurisotopCatalog #CLM-470-MPT-PK
Before start
Randomise the order in which samples are prepared and organize them according to this order.

Sample preparation
Fecal (FW) or caecal (CW) water samples were obtained from stools which are more or less dry depending on the species.

Weigh samples as described in the table 1 and stored at -80 °C . Then, freeze dry or steal (or glass) ball agitator each sample.

SpeciesQuantity of stoolsmilliQ water vol
Rat100 mg1 mL
mini Pig300 mg3 mL
Human5 to 8 gnot neccessary
Mouse50 mg0,5 mL
Table 1 : stools quantity and milliQ water volumes depending on the species

FW or CW come from homogenized stool samples (with or without water) ultra-centrifuged at 4 °C 50000 rpm (171500 g, Beckman Ti 70.1 rotor) 02:00:00 .

Remove the supernatant and store it in Eppendorf tubes.
Add2 µL of NaN3Merck MilliporeSigma (Sigma-Aldrich)Catalog #S2002-5G (an anti-microbial agent : 100 mg/mL ) per/g of FW or CW to the supernatants.

FW or CW aliquots depending to obtained volume and store at-80 °C in Eppendorf tubes to derivatization.

Thaw FW or CW sample at 4 °C , vortex 00:00:10 then add a 2-fold volume of ice-cold ULC/MS grade methanolBiosolveCatalog #0013684101BS into FW or CW samples (Table2).

SpeciesFW or CW volumes
Mouse300 µL
Rat300 µL
Mini Pig500 µL
Human500 µL
Table 2 : FW or CW volumes depending on the species

Vortex and keep the samples at -20 °C 00:30:00

Centrifuge the samples (Sigma 3-16PK, Fuscher Bioblock Scientific) 15000 rpm, 4°C, 00:10:00

Transfer 90 µL supernatant to glass vial. Spike10 µL of 100 mg/mL IS L-VALINE (1-13C, 99%)EurisotopCatalog #CLM-470-MPT-PK ) to vial.

At the same time, a derivatization control sample (sample was substitued by milliQ water) was prepared in order to remove the background noise produced during sample preparation, derivatization, and GC/MS analysis.
Dry samples completely using a EZ2.3 genevac system (Biopharma Technologies France).

Equipment
new equipment
NAME
Genevac EZ-2
BRAND
EZ-2
SKU
https://www.spscientific.com/Products/Centrifugal_Evaporators___Sample_Concentrators/Genevac/EZ-2_Series/EZ-2_Series/
SPECIFICATIONS


Derivatisation for GCMS analysis :
Prepare a fresh solution of Methoxyamine hydrochlorideMerckCatalog #89803-1G (15 mg/mL ) in PyridineMerck MilliporeSigma (Sigma-Aldrich)Catalog #360570 . The volume of this solution depends on the number of samples to be analysed (80 µL for each samples).
Dissolve the dry samples in adding 80 µL of methoxylamine solution to each vial.

Vortex vigorously for 00:01:00 and incube at 37 °C for 24:00:00 (in order to inhibit the cyclization of reducing sugars and the decarboxylation of α-keto acids).

Add 80 µL of BSTFA + 1% TMCS Silylation ReagentThermo FisherCatalog #TS-38833 in the mixture and derivatize at70 °C 01:00:00

Wait a few minutes for samples to cool and transfer 50 µL of derivatized mixture in a glass vial containing 100 µL of HeptaneMerck MilliporeSigma (Sigma-Aldrich)Catalog #246654-100mL prior to injection. Store blank and biological samples at 4 °C until further analysis

As well as a sample pool is formed from 10 µL of each extracted and derivatized sample to monitor the drift of the spectrometer during GCMS analysis.
Transfer 50 µL of sample pool in a glass vial containing 100 µL of heptane prior to injection, repeat this step 8 times (for preprocessing analysis). Store pool samples at 4 °C until further analysis.

Protocol references