Simple, high-throughput measurement of gut-derived short-chain fatty acids in clinically relevant biofluids using gas chromatography-mass spectrometry

Introduction: The quantitative measurement of circulating gut bacteria-derived metabolites has increased in recent years due to their associations with health and disease. While much of the previous attention has been placed on metabolites considered as deleterious to health, a shift to the investigation of short-chain fatty acids (SCFAs) as potential health promotors has been observed.

Objectives: To develop a simple, high-throughput and quantitative assay to measure gut-derived SCFAs in clinically relevant biofluids using gas chromatography-mass spectrometry (GC–MS).

Methods: A short (7.5 min) GC–MS assay was optimized for measurement of seven straight- and branched-chain SCFAs and their deuterated isotopes using a wax-based column for analysis without prior derivatization. The assay was validated using routine criteria to assess precision, accuracy, matrix effects, recovery, and extraction reproducibility. Assay applicability was tested in cohorts of healthy individuals and kidney disease patients.

Results: The assay was demonstrated to be precise, accurate and reproducible with acceptable levels of matrix effect and analyte recovery. Lower limits of detection and quantitation were in the low ng/mL range. An investigation into different blood collection tube chemistries demonstrated that lithium heparin plasma and serum clotting activator tubes are recommended for use in future cross-study comparisons. Kidney disease patient analyses demonstrated variable differences across SCFAs when comparing hemodialysis to earlier stages of chronic kidney disease, demonstrating the suitability of the assay for translation to clinical analyses.

Conclusion: The assay has been validated and identified as reliable for use in larger-scale studies for the analysis of SCFAs in human plasma and serum.