Jan 20, 2026

Public workspaceOrganic acid derivatization protocol for mass spectrometry analysis

DOI
https://dx.doi.org/10.17504/protocols.io.261ger8y7l47/v1
  • Manon Gilson1,
  • Guillaume Bayon-Vicente1,
  • Simone Krings1,
  • Ruddy Wattiez1,
  • Baptiste Leroy1
  • 1UMons
Manon Gilson
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DOI: https://dx.doi.org/10.17504/protocols.io.261ger8y7l47/v1
Protocol Citation: Manon Gilson, Guillaume Bayon-Vicente, Simone Krings, Ruddy Wattiez, Baptiste Leroy 2026. Organic acid derivatization protocol for mass spectrometry analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.261ger8y7l47/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: February 07, 2025
Last Modified: January 20, 2026
Protocol Integer ID: 119778
Keywords: Mass spectrometry, organic acids, derivatization , derivatisation, organic acid derivatization protocol for mass spectrometry analysis, mass spectrometry analysis, organic acid derivatization protocol, using mass spectrometry analysis, mass spectrometry, using mass spectrometry, detection of organic acid, organic acid, derivatization
Abstract
This protocol outlines the derivatization and the detection of organic acids using mass spectrometry analysis. This technique is highly sensitive and allow the detection of low-concentration samples.
Materials
Prepare the following solutions in 66% MS-grade acetronitrile:
  • 200 mM 3-NPH HCl in ACN 66%
  • 50 mM EDC in ACN 66%
  • 7% pyridine in ACN 66%
  • 5% formic acid (in MS-grade water)
Troubleshooting
Organic acid derivatization protocol for mass spectrometry analysis
Prepare the following solutions in 66% MS-grade acetronitrile (ACN):
  • 200 mM 3-NPH HCl in ACN 66%
  • 50 mM EDC in ACN 66%
  • 7% pyridine in ACN 66%
  • 5% formic acid (in MS-grade water)
Dilute the samples if necessary (This technique is highly sensitive and allows the analysis of samples with low concentration).
  • Caution: MOPS (3-(N-morpholino)propanesulfonic acid) saturates 3-NPH, preventing proper derivatization of organic acids. A dilution is necessary to achieve a maximum MOPS concentration of 1 mM
Prepare the reaction mixture:
  • Pipette 50 µL of the sample.
  • Add 25 µL of 200 mM 3-NPH HCl.
  • Add 25 µL of 50 mM EDC.
  • Add 25 µL of 7% pyridine solution.
Incubate the samples at 30°C for 5 hours at 500 rpm.
Add 125 µL of 5% formic acid to stop the reaction