Mar 05, 2025
Extraction Protocol for untargeted LC-MS/MS - Urine
- Mauricio Caraballo1,
- Chris Leone1,
- Kelly Christine Weldon1,
- Morgan Panitchpakdi1
- 1University of California San Diego
- Mauricio Caraballo: MSCollaboratory
Protocol Citation: Mauricio Caraballo, Chris Leone, Kelly Christine Weldon, Morgan Panitchpakdi 2025. Extraction Protocol for untargeted LC-MS/MS - Urine. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjnxengk5/v1
Manuscript citation:
Weldon, K.C., Panitchpakdi, M., Caraballo-Rodríguez, A.M. et al. Urinary Metabolomic Profile is Minimally Impacted by Common Storage Conditions and Additives. Int Urogynecol J (2025). https://doi.org/10.1007/s00192-025-06069-2
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: April 25, 2024
Last Modified: March 05, 2025
Protocol Integer ID: 98887
Keywords: Urine, Metabolomics, Storage, MSCollaboratory
Funders Acknowledgements:
Gordon and Betty Moore Foundation
Grant ID: GBMF12120
Disclaimer
This is not a peer-review protocol and it has been created and edited based on inputs from several users and collaborators of the Dorrestein lab.
Abstract
This protocol details the preparation of urine samples for untargeted metabolomics analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS). It covers sample collection, homogenization, metabolite extraction, and preparation for LC-MS/MS analysis.
Protocol materials
Methanol, Optima™ LC/MS Grade, Fisher ChemicalFisher ScientificCatalog #A456-4
Sample description
Sample description
Urine (biological sample)
Note
Minimum volume: 25uL. This is an average volume of urine obtained from mice. For human samples, typically we can work with 100-200 uL
Equipment
Equipment
Equipment
Vortex Mixer
NAME
Vortex
TYPE
Fisher Scientific
BRAND
fisher scientific vortex mixer
SKU
Equipment
Centrifuge 5418
NAME
Centrifuge
TYPE
Eppendorf US
BRAND
Catalog No. 5401000137
SKU
LINK
Equipment
CentriVap
NAME
Benchtop Centrifugal Vacuum Concentrator
TYPE
Labconco
BRAND
7810010
SKU
Solvents
Solvents
Methanol, Optima™ LC/MS Grade, Fisher ChemicalFisher ScientificCatalog #A456-4
Note
Extraction solvent (100% methanol) containing 1uM sulfamethazine (CAS 57-68-1) as an internal standard.
Resuspension solvent (50:50 v/v methanol:water) containing 1uM sulfadimethoxine (CAS 122-11-2) as an internal standard.
Consumables
Consumables
2 mL Qiagen sample tubes (catalog no. 990381)
Shallow polypropylene 96-well plate (Cat. No. 951040048)
Deep polypropylene 96-well plate (Cat. No. 951033405)
Seal plates (Cat. No. ZAFPE50) Note: for LC-MS/MS acquisition
AlumaSeal CS (Sealing Foil, Excel Scientific) (Cat. No. FC100) Note: for long time storage
Sample Extraction & Preparation
Sample Extraction & Preparation
25m 2s
25m 2s
Start with urine sample 25-200 µL and transfer to empty sample tube.
Add extraction solvent in a 1:4 sample:solvent ratio. For instance, 200 µL sample, add 800 µL of prechilled extraction solvent to each sample.
Note
For low volume samples, e.g., 25uL, add 100 µL of prechilled extraction solvent to each sample.
For a 200 µL volume sample, add 800 µL of prechilled extraction solvent to each sample.
Final concentration of 80% MeOH is required.
Mix samples using a vortexer for 00:00:02 .
2s
Place samples at -20 °C for 00:20:00 to aid in protein precipitation.
20m
Centrifuge the samples at 2000 rpm, 4°C for 00:05:00 to pellet the protein precipitate.
Note
If possible, perform this step at4 °C .
5m
In case of low volume samples (e.g., 25 uL from mice), transfer 100 uL. Transfer 800 µL of supernatant per sample into wells of the pre-labeled 96-Well DeepWell.
Note
Remainder can be stored at -80 °C .
In case of low volume samples (e.g., 25 uL urine with 100 uL extraction solvent), transfer 100 uL of centrifuged sample to pre-labeled 96-Well DeepWell.
Dry samples using a centrifugal low pressure system (Centrivap).
Note
Drying time may vary but it is recommended to check the progress every 4 hrs.
4h
Cover your plate using a 96-Well DeepWell Plate compatible cover and store at -80 °C once dry.
Sample preparation for LC-MS/MS
Sample preparation for LC-MS/MS
25m 2s
25m 2s
Re-suspend plates with 250 µL of a 50% MeOH to water solution containing 1 micromolar (µM) of sulfadimethoxine as an internal standard.
Acknowledgements
This work was supported by the Gordon and Betty Moore Foundation, GBMF12120
and https://doi.org/10.37807/GBMF12120.