A robust high-throughput method for quantification of short-chain fatty acids in humans and mice

Introduction  Short-chain fatty acids (SCFAs) have been known for long as pivotal microbiome-derived metabolites [1]. SCFAs exert various metabolic effects and undergo differential regulation in metabolic diseases such as diabetes mellitus [2], [3]. However, due to their volatile nature, SCFAs are challenging analytes in metabolomics and high-throughput methods are lacking. To overcome current technical limitations, we aimed to establish and validate a robust method for the exact quantification of SCFAs from biomaterial for enabling high-throughput measurements in human and murine model studies.

Material and Methods  Briefly, after addition of isotopically labelled internal standards, eight different SCFAs, namely acetic, propionic, butyric, iso-butyric, valeric, iso-valeric, hexanoic and heptanoic acid were extracted from 100 µL plasma or about 10 mg homogenized stool by liquid-liquid extraction with tert-butyl methyl ether as organic phase. Importantly, all steps were performed on-ice or with pre-cooled consumables. Next, SCFAs were derivatized with N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide to ameliorate analytical properties. Derivatized extracts were injected into a gas chromatography-coupled mass spectrometry (GC-MS) system operating in single ion monitoring mode. The method was validated due to bioanalytical method validation guidelines by Europeans Medicines Agency (EMA).

Results  The method revealed excellent linearity with R2≥0.999 for all analytes ranging from 1-5000 µM. Accuracy and intra-day precision was assessed at up to four concentration levels within the range of 87.67-118.6% and 0.710-16.01%, respectively. Of note, accuracy and precision were also assessed for reduced sample volume of only 50 µL mouse plasma. Moreover, there were no interfering matrix effects from biomaterial. Lower limit of quantification (LLOQ) was estimated by signal-to-noise approach and confirmed by repeated measures at LLOQ. Robustness and ruggedness of the method was indicated by different analysts and instrumental settings. As proof-of-principle, SCFAs were quantified from different biomaterial. At last, we set-up the method for an isotope-labelled acetic acid tracer.

Conclusion  Taken together, we established a robust straight-forward method to measure SCFAs in up to 50 samples within 24 h from blood and stool and validated it by EMA guidelines. This method shall serve as promising tool for the analysis of microbiome-host and inter-organ crosstalk in rodent models and to decipher the role of SCFAs in human metabolic diseases.

Publication History

Article published online:
26 May 2023

• References

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