Quantitative analysis of 5-FU and its bacterial-derived metabolites by untargeted metabolomics

We incubated 5-FU (40 μM) with C. freundii strain (4×108 CFU/mL) in 600 μL of NB medium for 24 h (n=3). A blank control group was included. Post-incubation, reactions were stopped with 600 µL ice-cold acetonitrile. After vortexing, 200 µL aliquots were mixed with an equal volume of acetonitrile and precipitated at -20°C for 1 h. Samples were then revortexed, centrifuged (12,500 rpm, 15 min, 4°C), and 100 µL of supernatant was collected for quantitative analysis using an ultraperformance liquid chromatography (UPLC)-Orbitrap-Exploris-120-MS/MS apparatus with a Kinetex C18 column (2.1 mm × 100 mm, 2.6 μm). The mobile phase consisted of NH4HCO3 (6.5 mM) (A) and acetonitrile (B) at a flow rate of 0.3 mL/min. The gradient elution program was established: 0–1 min, 2% B; 1–3 min, 2–60% B; 3–5 min, 60% B; 5–7 min, 60–100% B; 7–8 min, 100% B; 8–8.1 min, 100–2% B; and 10 min, 2% B. Samples (10 μL of injection volume) were separated and analyzed with the column temperature at 40°C. Data acquisition was performed using Xcalibur software.
Mass spectrometry analysis employed full-scan (60,000 resolution, 60–900 m/z range) and data-dependent MS2 (dd-MS2; 15,000 resolution) modes. Ion source parameters included: 2500 V spray voltage (negative mode), sheath/aux gases at 50/10 arb, and 300°C transfer tube temperature. Full-MS used 100 ms maximum injection time with standard AGC target; dd-MS2 used auto injection time and stepped HCD collision energy (20, 40, 60%). Retention times for 5-FU and FBAL were 0.666 and 0.687 min, respectively. Data processing utilized MS-DIAL (ppm calculation), Sirius (validation), and MZmine (MS2 export).