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Background <p>Short-chain fatty acids (SCFAs), including acetate, propionate, and butyrate, are key microbial metabolites that play crucial roles in modulating host–microbiome interactions, immune function, and metabolic homeostasis. Due to their significant involvement in both physiological and pathological processes, there is increasing demand for reliable, sensitive, and high-throughput analytical methods to quantify SCFAs in biological matrices. Liquid biopsies, such as plasma and serum, offer a minimally invasive means to monitor SCFA levels, making them valuable for both research and clinical applications.</p> Methods <p>In this study, we introduce a workflow for the quantitative profiling of acetate, propionate, and butyrate using direct infusion mass spectrometry (DI-MS). The method employs a Q Exactive HF Hybrid Quadrupole-Orbitrap mass spectrometer coupled with the TriVersa NanoMate™ robotic nanoflow ion for automated sample introduction. To improve ionization efficiency and detection sensitivity, SCFAs were extracted with isopropanol (IPA) and chemically derivatized prior to analysis. Method validation included determination of limits of detection (LOD), limits of quantification (LOQ), linearity, reproducibility, and assessment of matrix effects. The method was further applied to both plasma and serum samples to demonstrate its utility in clinically relevant matrices.</p> Results <p>The developed DI-MS-method demonstrated good analytical performance, including low limits of detection (LOD) for all three SCFAs − 0.1 µM for acetate, 0.01 µM for propionate, and 0.05 µM for butyrate - alongside wide linear dynamic ranges (r² ≥ 0.991). The assay showed high reproducibility, with low within-run (CV &lt; 15%) and between-run (CV &lt; 18%) variability for all three SCFA. The optimized workflow employing DI-MS analysis was successfully applied to plasma and serum samples, demonstrating efficient recovery, high reproducibility, and suitability for clinically relevant matrices.</p> Conclusions <p>This validated analytical workflow is well-suited for the quantification of SCFAs in clinical and research settings. Its applicability was confirmed through successful analysis of human plasma and serum samples, yielding concentration profiles consistent with previously published data. The simplicity, sensitivity, and reproducibility of the method make it a valuable tool for investigating SCFA-related pathways and for exploring their relevance in clinical investigations.</p> Clinical trial number <p>Not applicable.</p> Graphical Abstract <p></p>

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Direct infusion mass spectrometry quantitation of short-chain fatty acids as a tool for liquid biopsy profiling

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年4月20日

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  • Arijan Pasic,
  • Svitlana Rozanova,
  • Anna Lena Fisse,
  • Jeremias Motte,
  • Maximilian Schröder,
  • Philip Lennart Poser,
  • Dirk Winkelhardt,
  • Julian Uszkoreit,
  • Ralf Gold,
  • Katrin Marcus,
  • Katalin Barkovits

摘要

Background

Short-chain fatty acids (SCFAs), including acetate, propionate, and butyrate, are key microbial metabolites that play crucial roles in modulating host–microbiome interactions, immune function, and metabolic homeostasis. Due to their significant involvement in both physiological and pathological processes, there is increasing demand for reliable, sensitive, and high-throughput analytical methods to quantify SCFAs in biological matrices. Liquid biopsies, such as plasma and serum, offer a minimally invasive means to monitor SCFA levels, making them valuable for both research and clinical applications.

Methods

In this study, we introduce a workflow for the quantitative profiling of acetate, propionate, and butyrate using direct infusion mass spectrometry (DI-MS). The method employs a Q Exactive HF Hybrid Quadrupole-Orbitrap mass spectrometer coupled with the TriVersa NanoMate™ robotic nanoflow ion for automated sample introduction. To improve ionization efficiency and detection sensitivity, SCFAs were extracted with isopropanol (IPA) and chemically derivatized prior to analysis. Method validation included determination of limits of detection (LOD), limits of quantification (LOQ), linearity, reproducibility, and assessment of matrix effects. The method was further applied to both plasma and serum samples to demonstrate its utility in clinically relevant matrices.

Results

The developed DI-MS-method demonstrated good analytical performance, including low limits of detection (LOD) for all three SCFAs − 0.1 µM for acetate, 0.01 µM for propionate, and 0.05 µM for butyrate - alongside wide linear dynamic ranges (r² ≥ 0.991). The assay showed high reproducibility, with low within-run (CV < 15%) and between-run (CV < 18%) variability for all three SCFA. The optimized workflow employing DI-MS analysis was successfully applied to plasma and serum samples, demonstrating efficient recovery, high reproducibility, and suitability for clinically relevant matrices.

Conclusions

This validated analytical workflow is well-suited for the quantification of SCFAs in clinical and research settings. Its applicability was confirmed through successful analysis of human plasma and serum samples, yielding concentration profiles consistent with previously published data. The simplicity, sensitivity, and reproducibility of the method make it a valuable tool for investigating SCFA-related pathways and for exploring their relevance in clinical investigations.

Clinical trial number

Not applicable.

Graphical Abstract