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

Significance of the Topic

Short-chain fatty acids (SCFAs) such as formic, acetic, propionic, and butyric acid play vital roles in human metabolism and gut health. Accurate quantification of SCFAs in biological matrices supports research in microbiome function, metabolic disorders, and nutritional science. However, analytical challenges arise from the high volatility and polarity of these acids, making reliable low-level detection difficult without robust derivatization and sensitive instrumentation.

Objectives and Study Overview

This study aimed to develop and validate a gas chromatography–mass spectrometry (GC-MS) method for quantifying seven SCFAs in human plasma. Key goals included overcoming sample volatility losses during derivatization, ensuring linear calibration over a broad concentration range, and applying a standard addition approach to determine endogenous SCFA levels in pooled human plasma.

Methodology and Instrumentation

Derivatization employed the water-compatible condensing agent DMT-MM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride) with n-octylamine to form stable amide derivatives. Sample preparation steps included:

• Protein precipitation: plasma mixed with methanol and centrifuged.
• Derivatization: addition of DMT-MM and n-octylamine in methanol, incubated 9 h at room temperature.
• Standard addition: spiking with SCFA mixtures (0–100 μmol/L) and using acetic acid-d4 (10 μmol/L) as an internal standard.

GC-MS analysis conditions:

• Column: BPX-5 (30 m × 0.25 mm, 0.25 μm).
• Carrier gas: helium at 39 cm/s linear velocity; split injection (30:1).
• Oven program: 60 °C hold, 15 °C/min to 330 °C.
• MS detection: MRM mode, source 200 °C, interface 280 °C.

Main Results and Discussion

All seven SCFA derivatives exhibited excellent calibration linearity (R2 ≥ 0.9990). Concentrations measured in human pooled plasma were: formic acid 115.6 μmol/L, acetic acid 184.4 μmol/L, propionic acid 8.0 μmol/L, isobutyric acid 3.97 μmol/L, n-butyric acid 5.89 μmol/L, isovaleric acid 0.82 μmol/L, and n-valeric acid 3.25 μmol/L. The results demonstrate reliable quantitation across low micromolar ranges, with low background interference and reproducible peak responses.

Benefits and Practical Applications

• Enables sensitive and accurate measurement of SCFAs in plasma and other biological fluids.
• Compatibility with aqueous samples reduces loss of volatile acids during derivatization.
• Standard addition and isotopic internal standardization improve quantitation in complex matrices.

Future Trends and Potential Applications

• Extension to faecal and intestinal samples for comprehensive microbiome studies.
• Automation of the derivatization workflow to increase throughput.
• Integration with high-resolution MS for untargeted SCFA profiling and discovery of novel metabolites.

Conclusion

A robust GC-MS method using DMT-MM-mediated derivatization and MRM detection offers precise quantitation of seven key SCFAs in human plasma. The approach overcomes volatility challenges and delivers high linearity and reproducibility, supporting diverse applications in biochemical research and clinical investigations.

References

1. Sakai T., Shimadzu application note M273, Apr. 2017.
2. Toray Research Center, Inc., The TRC News No. 115, May 2012.