Simplified Analysis of Aqueous Short Chain Fatty Acids by GC/MS

Significance of the Topic

Short chain fatty acids (SCFAs) are key metabolites in environmental and biological systems, serving as energy sources for colonocytes, indicators of gut microbiota activity, and markers of water or food quality. A rapid, derivatization-free method enhances throughput and reduces complexity in analytical workflows.

Objectives and Study Overview

This study introduces a simplified GC–MS protocol for quantifying aqueous C2–C6 fatty acids (acetic through hexanoic acid) without chemical derivatization or internal standards. The total analysis cycle, including column cooling, is approximately 10 minutes.

Methodology and Instrumentation

Sample Preparation:

• Prepare a 10 mM premixed SCFA standard and dilute to 4–100 µM in deionized water.
• Fill 2 mL vials to the brim to minimize headspace loss of volatile acids.

Instrumentation:

• GC–MS System: Shimadzu GCMS-QP2020 NX
• Auto Injector: AOC-20i Plus
• Auto Sampler: AOC-20s Plus
• Column: SH-Rtx™-WAX (60 m × 0.25 mm i.d., 0.5 µm film)

Chromatographic Conditions:

• Injection Mode: Split (5:1), 1 µL at 240 °C
• Carrier Gas: Helium at constant linear velocity (34.0 cm/s)
• Oven Program: 80 °C hold 2 min → ramp 40 °C/min to 200 °C → ramp 25 °C/min to 240 °C (2 min); total run time 8.60 min

Mass Spectrometry:

• Ion Source Temperature: 200 °C; Interface Temperature: 240 °C
• Acquisition Mode: Selected Ion Monitoring (SIM), loop time 0.30 s

Major Results and Discussion

All targeted SCFAs were baseline-separated in under 9 minutes. Quadratic calibration curves spanning 4–100 µM (0.24–6.0 µg/mL for acetic acid; comparable for others) exhibited good linearity. Repeatability at 10 µM (n=7) yielded RSDs of 3.9–9.2%. Increased variability at low concentrations suggests employing a deuterated acetic acid internal standard to correct bias.

Benefits and Practical Applications

• No derivatization or specialized consumables required, reducing setup time and costs.
• Fast analysis supports high-throughput environmental, clinical, and industrial testing.
• Sensitivity sufficient for direct aqueous measurements without complex extraction.

Future Trends and Potential Applications

• Integration of steam distillation or headspace sampling to extend applicability to solid or fecal samples.
• Use of stable isotope-labeled internal standards for enhanced accuracy at trace levels.
• Automation and coupling with online sample preparation for volatile organic acid profiling.

Conclusion

This work demonstrates a rapid, derivatization-free GC–MS method on the GCMS-QP2020 NX platform for precise quantitation of short chain fatty acids in water (4–100 µM). Incorporation of an internal standard further improves low-level precision.

Instrumental Details

• Shimadzu GCMS-QP2020 NX
• AOC-20i Plus Auto Injector
• AOC-20s Plus Auto Sampler
• SH-Rtx™-WAX column (60 m × 0.25 mm i.d., 0.5 µm)

References

1. Zijlstra J.B., Beukema J., Wolthers B.G., Byrne B.M., Groen A., Dankert J. Pretreatment methods prior to gas chromatographic analysis of volatile fatty acids from faecal samples. J. Chromatogr. 1977;78(2):243–250.
2. Dyer D.C. A new method of steam distillation for the determination of the volatile fatty acids, including colorimetric identification reactions. J. Biol. Chem. 1917;28:445–473.