Stability of microbiome-derived fatty acids in self-collected stool and blood matrices

Short-chain fatty acids (SCFAs) are widely used as functional readouts of gut microbial activity in vivo. The growing adoption of decentralised study designs and self-collection protocols has amplified the need for reliable room-temperature storage and shipment strategies. However, SCFAs volatility and the persistence of post-collection microbial metabolism raise concerns regarding pre-analytical stability and the interpretability of measured concentrations. We assessed the temporal stability of fatty acids (FAs) across intestinal and systemic matrices under room-temperature storage. Untreated stool was compared with two nucleic acid stabilisation devices (SMART-eNAT and OMNIgene-GUT), while whole blood, plasma, and dried blood spots (DBS) were evaluated as minimally invasive systemic sampling strategies. Profiles were quantified using complementary GC-MS and LC-MS/MS workflows. Untreated stool (n = 32) showed fermentation-driven increases in major SCFAs, whereas immediate freezing preserved baseline profiles. SMART-eNAT maintained faecal FA stability for up to 21 days, while OMNIgene-GUT exhibited baseline and time-dependent alterations. In systemic matrices (n = 20), plasma and whole blood showed an upward drift, whereas DBS declined initially before stabilising after approximately 14 days. FA measurements are highly matrix- and device-dependent. Our findings provide practical guidance for the selection of sampling strategies in microbiome-associated FA studies and emphasise the need for controlled pre-analytical conditions in decentralised microbiome studies.