Targeted metabolomics analysis of 5-FU after co-incubation with intratumoral bacteria

40 μM 5-FU was incubated with 40 μM Gim (MCE, HY-17469-50mg) and 4×10⁸ CFU/mL of <i>C. freundii</i>, <i>P. putida</i>, <i>P. aeruginosa</i>, <i>P. fluorescens</i>, <i>L. reuteri</i>, <i>E. coli</i>, <i>K. pneumoniae</i>, and <i>E. asburiae</i> in a total volume of 600 μL of NB medium. Three incubation time points were set at 0h, 6h, and 24h (n=3). At the end of each incubation, 600 μL of ice-cold acetonitrile was added to stop the reaction. After vortex mixing, 200 μL of the sample was taken, and an equal volume of acetonitrile along with 10 μL of the internal standard 5-Chlorouracil (Millipore Sigma, 224588) was added. After vortex mixing, the sample was centrifuged at 12,500 rpm for 10 minutes at 4°C. 100 μL of the supernatant was collected, mixed with four volumes of pure water, vortexed and centrifuged, and 100 μL of the supernatant was collected for analysis.The samples were analyzed by liquid-chromatography with tandem quadrupole mass spectrometry (LC-MS/MS QQQ) using the AB SCIEX Triple Quad™4500MD. Chromatographic separation was achieved 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 (MRM) in electrospray negative ionization, and the ions measured for the quantitation were: 5-FU: 129.1-&gt;42.1, 5-chlorouracil: 145.1 -&gt; 42.1 and FBAL: 106.0-&gt;85.9.<b> </b>The retention time for 5-FU and FBAL in the column and gradient was 0.82 and 0.76 min, respectively. Quantitative analysis for 5-FU was performed using an internal standard method with a 7-point calibration curve. Calibration curves for the analytes were obtained using 7 calibration standards (100, 200, 500, 1250, 2500, 5000, and 10000 ng/mL for 5-FU and FBAL).