Analysis of Short-Chain Fatty Acids in Biological Samples Using GC-MS

Significance of the Topic

Short-chain fatty acids (SCFAs) such as acetic acid play key roles in host health, impacting metabolic and immunological processes. Accurate quantification in biological samples is challenging due to volatility during sample preparation and interference in LC-MS workflows.

Study Objectives and Overview

This study presents a GC-MS approach for the simultaneous quantitation of seven SCFAs in human plasma. It employs 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) to derivatize carboxylic acids directly in aqueous or methanolic media, followed by MRM detection.

Methodology and Instrumentation

Sample Pretreatment:

• Human pooled plasma (EDTA) spiked with 0–100 μmol/L SCFA standards and 10 μmol/L acetic acid-d4 as internal standard
• Deproteinization by methanol addition, high-speed shaking (30 s), centrifugation (16 000 G, 3 min, 25 °C), supernatant collection
• Derivatization with DMT-MM and n-octylamine, reaction at room temperature for 9 h

GC-MS Conditions:

• Column: BPX-5 (30 m × 0.25 mm I.D., 0.25 μm)
• Carrier gas: He, linear velocity 39.0 cm/s; split injection (30:1) at 250 °C
• Oven program: 60 °C (2 min) → 15 °C/min → 330 °C (5 min)
• MS: Ion source 200 °C; interface 280 °C; MRM mode, loop time 0.3 s

Results and Discussion

All seven SCFA derivatives showed excellent linearity (R2 ≥ 0.9990). Concentrations measured in human plasma were:

• Formic acid: 115.64 μmol/L
• Acetic acid: 184.36 μmol/L
• Propionic acid: 8.02 μmol/L
• Isobutyric acid: 3.97 μmol/L
• n-Butyric acid: 5.89 μmol/L
• Isovaleric acid: 0.82 μmol/L
• n-Valeric acid: 3.25 μmol/L

MRM chromatograms confirmed clear peak separation at 1 μmol/L spike.

Practical Benefits and Applications

This method offers:

• Direct aqueous derivatization without sample drying
• High sensitivity and reproducibility for SCFA profiling
• Compatibility with complex biological matrices
• Utility in microbiome research, clinical metabolomics, and QA/QC settings

Future Trends and Applications

Potential developments include:

• Automation and rapid derivatization workflows
• Integration into high-throughput platforms for large cohort studies
• Expansion to broader metabolite panels in lipidomics
• Clinical diagnostics for gut–microbiome interactions

Conclusion

The described GC-MS method using DMT-MM derivatization enables robust quantitation of seven SCFAs in human plasma, providing a valuable tool for research and industrial applications.

Reference

• Shimadzu Application News No. M273A, First Edition Apr. 2017; Revision A Feb. 2023
• Toray Research Center, TRC News No. 115, May 2012