Analysis of Short-Chain Fatty Acids/Organic Acids (3-NPH Derivatives) in Fecal Specimens from SPF and Antibiotic-Fed Mice

Significance of the Topic

Quantitative profiling of short-chain fatty acids (SCFAs) and related organic acids is vital to understanding gut microbiota interactions with host physiology and disease mechanisms. These metabolites influence immune responses, metabolic disorders, and overall gut health.

Study Objectives and Overview

This study aimed to enhance the LC/MS analysis of C2–C5 SCFAs and selected organic acids in mouse fecal samples by employing 3-nitrophenylhydrazine (3-NPH) derivatization. By comparing untreated SPF mice to antibiotic-treated mice with depleted microbiota, the work sought to reveal microbial contributions to acid profiles.

Methodology

The analytical workflow involved aqueous derivatization of carboxylic and carbonyl-containing acids using 3-NPH, pyridine as catalyst, and a carbodiimide condensing agent at room temperature for 30 minutes. Samples were diluted in methanolic formic acid prior to LC/MS/MS analysis using MRM transitions optimized for 3-NPH derivatives.

Instrumentation Used

• LC/MS system: Shimadzu LCMS-8060
• HPLC column: Mastro C18, 2.1 mm × 150 mm, 3 μm
• Mobile phases: 0.1 % formic acid in water (A) and acetonitrile (B) with gradient elution
• Ionization: ESI in positive/negative modes; Nebulizer 2.0 L/min; drying gas 10.0 L/min; heat block 400 °C

Main Results and Discussion

• MRM chromatograms achieved clear separation of six SCFAs and key organic acids.
• Antibiotic-fed mice showed a marked reduction in SCFA levels (C2–C5) compared with SPF controls, confirming microbial origin.
• Succinic acid increased significantly in antibiotic-treated mice, indicating altered metabolic flux when gut flora are suppressed.
• Variability in straight-chain versus branched SCFA ratios suggests individual microbiome differences.

Benefits and Practical Applications

The method provides sensitive and specific quantification of volatile and hydrophilic acids without the losses typical of GC/MS. It is directly applicable to gut microbiome research, metabolic disease studies, and pharmacological evaluations.

Future Trends and Potential Uses

Advances may include expanded metabolite panels, streamlined sample preparation, and integration with high-throughput platforms. These developments will enhance understanding of host–microbiome interactions, personalized nutrition strategies, and disease diagnostics.

Conclusion

3-NPH derivatization coupled with LC/MS/MS offers a robust platform for SCFA and organic acid analysis in complex biological matrices. The approach reliably distinguishes microbiota-driven metabolic changes, providing a valuable tool for biomedical and clinical research.

References

• Narushima S, Sugiura Y, Oshima K, Atarashi K, Hattori M, Suematsu M, Honda K. Characterization of the 17 strains of regulatory T cell-inducing human-derived Clostridia. Gut Microbes. 2014;5(3):333-9.