Individual stool samples were first lyophilized under vacuum using a freeze dryer (Christ Alpha) and an equal amount of dry mass weight for each sample was combined to prepare the pooled sample for each group. 40 mg of each pooled sample was added to 80 mg of glass homogenisation beads (acid washed, 425-600 μm, Sigma-Aldrich, USA) and processed using a two-phase modified Bligh and Dyer extraction protocol (Fukawa et al., 2016). Briefly, polar and lipid metabolites were extracted by sequential addition of methanol (Optima grade, Fisher Scientific), 3.8 mM tricine solution (Sigma-Aldrich) and chloroform (Gradient grade, Merck) (1:0.5:1 v/v/v, total 2 mL) to the sample. The mixture was vortexed for 2 min following each addition of solvent. The mixture was then centrifuged at 4 °C, 16,000 g for 20 min. This resulted in separation of the sample into two fractions – the top methanol-tricine solution layer contained the polar metabolites while the bottom chloroform layer contained the lipid species. The top polar layer was collected and the remaining mixture was re-extracted using a mixture of methanol and tricine solution (9:10 v/v), followed by centrifugation at 4 °C, 16,000 g for another 10 min. The resulting polar layer was combined together with the first polar extract and stored at -80 °C before subsequent mass spectrometry analysis.

Each polar extract was analysed in triplicate using an ultra-performance liquid chromatography system (AQUITY UPLC, Waters) in tandem with a mass spectrometer (QExactive, Thermo Scientific). A C18 UPLC column (ACQUITY UPLC HSS T3 column, 2.1 × 100 mm, 1.8 μm, Waters) was used for separation and the mobile phase comprised of two solvents. ‘A’ being water with 0.1% formic acid (Merck) and ‘B’ being methanol with 0.1% formic acid. The UPLC program is as follows: the column was first equilibrated for 0.5 min at 0.1% B. The gradient was then increased from 0.1% B to 50% B over 8 min before being held at 98% B for 3 min. The column was washed for a further 3 min with 98% acetonitrile (Merck) with 0.1% formic acid and finally equilibrated with 0.1% B for 1.5 min. The solvent flow rate was set at 0.4 mL/min; a column temperature of 30 °C was used. The eluent from the UPLC system was directed into the MS.

High-resolution mass spectrometry was then performed in both positive and negative electrospray ionization (ESI) modes, with a mass range of 70 to 1050 m/z and a resolution of 70,000. Sheath and auxiliary gas flow was set at 30.0 and 20.0 (arbitrary units) respectively, with a capillary temperature of 400 °C. The spray voltages were 1.25 kV for positive and 1.5 kV negative mode ionization. Mass calibration was performed using standard calibration solution (for QExactive, Thermo) prior to injection of the samples. A quality control (QC) sample comprising of equal aliquots of each sample was run at regular intervals during the batch LC-MS runs.

The raw LC-MS data obtained was processed using a XCMS-based peak finding algorithm [89]. The QC samples were used to adjust for instrumental drift. Detected mass peaks were assigned putative metabolite identities by matching the respective masses (< 10 ppm error) with the KEGG and Human Metabolome Database (HMDB). Where possible, the identities of selected metabolites of interest were confirmed based on mass spectral comparison with available metabolite standards.

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