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Updated: May 1, 2026

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Design and Analysis of Untargeted Metabolomics Experiments.

Zane G Long1, Rachel W Martin1,2

  • 1Department of Chemistry, University of California, Irvine, California.

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|October 13, 2025
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Summary

Untargeted metabolomics, using liquid chromatography-tandem mass spectrometry (LC-MS/MS), offers a fast and cost-effective method for identifying unknown small molecules in complex biological samples like plants. This guide provides a comprehensive protocol for researchers new to the technique.

Keywords:
LC‐MS/MSelectrospray ionizationmass spectrometryplant metabolitesuntargeted metabolomics

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Area of Science:

  • Analytical Chemistry
  • Biochemistry
  • Plant Science

Background:

  • Untargeted metabolomics identifies unknown small molecules in complex mixtures.
  • It is crucial for biomedical, environmental, and plant metabolite discovery.
  • Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a key detection method.

Purpose of the Study:

  • To provide practical and technical knowledge on untargeted metabolomics using LC-MS/MS.
  • To offer a consolidated protocol for experimental design, data collection, and analysis.
  • To explain terminology and technical details within the context of real-world experiments.

Main Methods:

  • Focus on liquid chromatography-tandem mass spectrometry (LC-MS/MS).
  • Detailed protocols for sample preparation, LC-MS/MS data collection, and data analysis.
  • Utilizes widely available software (MSConvert, MZMine, SIRIUS).

Main Results:

  • Presents a step-by-step protocol applicable to various complex biological samples.
  • Demonstrates the approach using a plant metabolite data set.
  • Includes protocols for preparing samples and assessing LC-MS/MS sensitivity.

Conclusions:

  • Untargeted metabolomics with LC-MS/MS is a powerful tool for metabolite discovery.
  • The provided protocol serves as a starting point for new users in diverse research areas.
  • The methodology is adaptable for analyzing various complex biological samples.