2.3. Sample preparation

All samples were prepared for analysis via liquid–liquid extraction by mixing 100 µL of an internal standard (IS) mixture containing 6 µg/mL of each deuterium-labelled SCFA in MTBE with 100 µL of the biofluid sample and 100 µL of 1 M hydrochloric acid. The mixture was vortexed for 30 s to ensure complete mixing. The resultant mixture was centrifuged at 2500×g, 4 °C for 15 min to separate the organic (MTBE) and aqueous layers. The organic layer (∼100 µL) was then transferred to a low volume crimp top autosampler vial for analysis. Test samples used for recovery, matrix interference and reproducibility experiments were produced by mixing eight randomly chosen samples of each sample matrix from different healthy individual participants.