Quantitative analysis of 5-FU and its bacterial-derived metabolites (preTA inhibitors testing)

For inhibitor testing, C. freundii strains (4 × 10⁸ CFU/mL) were incubated with 40 μM 5-FU in 600 μL of the NB medium. Each incubation was performed in the presence of one of the previously reported DPD inhibitors—Gim, Eniluracil, Thymidine, Uridine, and Uracil (each at 40 μM). The incubation time points were set at 0 h, 6 h, and 24 h (n = 3) and a blank control group was included. At the end of each incubation, 600 μL of ice-cold acetonitrile was added to stop the reaction. After vortexing, 200 μL of each sample was taken, and an equal volume of acetonitrile along with 10 μL of the internal standard 5-Chlorouracil was added. After vortexing, samples were centrifuged at 12,500 rpm for 10 min at 4°C. The supernatant (100 μL) was collected, diluted with four volumes of pure water, vortexed, and centrifuged again. The resulting supernatant was collected for subsequent analysis.The samples were analyzed using liquid chromatography with tandem quadrupole mass spectrometry (LC-MS/MS QQQ) using the AB SCIEX Triple Quad™4500MD. Chromatographic separation was performed using a Kinetex C18 column (2.1 mm × 100 mm, 2.6 μm). The mobile phase consisted of solution A (26.5 mM HCOOH and 1.0 mM NH4OAC in H2O) and solution B (methanol). A flow rate of 0.3 mL/min was used with the following gradient elution profile: 0–0.50 min, 60% A 40% B; 0.5–2 min, 40–85% B; 2–3 min, 85% B; 3–3.5 min, 85–40% B; 3.5–4 min, 40% B. The injection volume injected was consistently set at 10 μL. This assay used multiple reaction monitoring in electrospray negative ionization, and the ions measured for quantitation were as follows: 5-FU, 129.1->42.1; 5-chlorouracil, 145.1->42.1; and FBAL, 106.0->85.9. The retention time for 5-FU and FBAL in the column and gradient was 0.82 and 0.76 min, respectively. The quantitative analysis of 5-FU was performed using an internal standard method with a seven-point calibration curve. Calibration curves for the analytes were obtained using seven calibration standards (100, 200, 500, 1250, 2500, 5000, and 10000 ng/mL for 5-FU and FBAL). Calibration standards were analyzed in duplicate, and curves were constructed using 1/x-weighted linear regression (R² ≥ 0.995). Limit of detection (LOD): 5-FU: 0.1 ng/mL, FBAL: 0.4 ng/mL, both defined at a signal-to-noise ratio (S/N) ≥ 3.