Sep 24, 2025

Public workspaceUrine sample preparation for untargeted MS-based metabolomic V.1

DOI
https://dx.doi.org/10.17504/protocols.io.6qpvr8e8blmk/v1
Urine sample preparation for untargeted MS-based metabolomic
  • Charlotte JOLY1,
  • CENTENO Delphine1,
  • Stéphanie DURAND1,
  • Estelle Pujos-Guillot2
  • 1Université Clermont Auvergne, INRAE, UNH, Plateforme d’Exploration du Métabolisme, MetaboHUB Clermont, Clermont-Ferrand, France;
  • 2INRAE
  • MetaboHUB-Clermont PFEM
Estelle Pujos-Guillot
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DOI: https://dx.doi.org/10.17504/protocols.io.6qpvr8e8blmk/v1
Protocol Citation: Charlotte JOLY, CENTENO Delphine, Stéphanie DURAND, Estelle Pujos-Guillot 2025. Urine sample preparation for untargeted MS-based metabolomic. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr8e8blmk/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: In development
We are still developing and optimizing this protocol
Created: March 29, 2024
Last Modified: September 24, 2025
Protocol Integer ID: 97534
Keywords: untargeted analysis, metabolomics, LCQToF, Mass spectrometry, MetaboHUB, urine sample preparation, urine sample preparation for untargeted m, preparation of urine sample, urine sample preparation, based metabolomic, mass spectrometry, urine sample, list of urine sample, liquid chromatography, sample preparation, untargeted m
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 urine samples for non-targeted analysis by mass spectrometry coupled to liquid chromatography as proposed by the PFEM (MetaboHUB-Clermont).
A pre-analytical normalisation step is carried out based on the osmolality measurement determined for each sample.
The steps cover the sample preparation and quality control.
The expected outcome of this protocol is a list of urine samples ready for analysis by LCMS.

Materials
Model 2020 Osmometer (Advanced instrument)
Sigma 3-16PK centrifuge (Fischer Bioblock Scientific)
Protocol materials
ReagentMilliQ water
ReagentMilliQ water
Troubleshooting
Before start
Randomise the order in which samples are prepared and organize them according to this order.
Two aliquots per sample to be analysed are required. The first will be used to determine the osmolality measurement and the second for metabolomic analysis by LCMS.
Osmolality measurement
Thaw an aliquot of each Sampleurine sample atTemperatureRoom temperature

Centrifuge samples Centrifigation15493 x g, 4°C, 00:10:00
Measure the osmolality value of each Sampleurine sample using an osmometer.
This setting determines the dilution factor that will be carried out on each Sampleurine sample analysed by LCMS.

Model 2020 Osmometer

Calibrate the device using the following three solutions (once per month)
- 50 mOsm/kg (Advanced Instruments Reference :3MA005)
- 850 mOsm/kg (Advanced Instruments Reference :3MA085)
- 2000 mOsm/kg (Advanced Instruments Reference :3LA201)
Each day of use, carry out a quality control measurement (clinitrol) with a value of 290 mOsm/kg. If the measured value differs from the expected value, recalibrate the device.
Critical
Measure the osmolality value of each Sampleurine sample . If it exceeds 2000 mOsm/kg, theSampleurine sample must be diluted in ReagentMilliQ water and analysed again.

Analyze
Sample preparation
Thaw the second aliquot of each Sampleurine sample atTemperatureRoom temperature
Centrifuge Sampleurine sample Centrifigation15493 x g, 4°C, 00:10:00

Add Amount100 µL of Sampleurine sample in Eppendorf tubes

Dilute each Sampleurine sample in ReagentMilliQ water so that each diluted sample has an osmolality value of around 100mOsm/kg

A pooled QC sample is prepared by mixing Amount10 µL from each diluted samples

Each diluted Sampleurine sample is divided into three aliquots : one is reserved for LCMS analysis in positive mode, another is dedicated to analysis in negative mode, and the last is a backup sample.

Store samples for LCMS analysis at Temperature-20 °C