Rethinking Solid-Phase Microextraction (SPME) for Bioanalysis: Workflow Optimisation in Automated Vacuum-Assisted Cooling SPME for Fatty Acid Monitoring in Faecal Waters

Short-chain fatty acids (SCFAs) are key biomarkers of gut microbiota and intestinal health, yet their analysis is challenging due to high volatility, hydrophilicity, and the complexity of the faecal matrix. This study introduces a novel, green, and fully automated workflow for monitoring SCFAs in faecal water samples. It combines microwave-assisted derivatisation, vacuum-assisted cooling multiple solid-phase microextraction (Vac-HS-cooling-MSPME), and fast gas chromatography–mass spectrometry (GC-MS). On-sample derivatisation with 2,3,4,5,6-pentafluorobenzyl bromide was optimised via design of experiments, reducing reaction time from 3 h to just 2 min at 100 °C while preserving high yields and selectivity. Integration of Vac-HS-cooling-MSPME with a fast GC column (5 m × 0.1 mm ID × 0.4 µm) increased sample throughput from two to approximately six samples per hour, with total chromatographic times of 5.24 min. MSPME specifically improved sensitivity for low-response analytes such as pentafluorobenzyl formate. The method demonstrated excellent linearity (R2 > 0.998), low limits of detection (0.002–0.09 mg/L), good intra- and inter-day precision, accuracies exceeding 85%, and consistent recovery for each analyte (86–90%). The AGREE greenness score improved from 0.65 to 0.68, confirming alignment with green analytical chemistry principles. This system offers a high-throughput, sensitive, and eco-friendly tool for SCFA quantification, confirmed by the application on real paediatric faecal water samples.