5-FU analysis through mass spectrometry

5-FU from bacterially modified media samples was analyzed by liquid-chromatography with tandem quadrupole mass spectrometry (LC-MS/MS QQQ) using a Waters Xevo-XS with a Waters Acquity I-Class UPLC. Samples were mixed 1:1 with 0.4 μM of a heavy, stable isotope of 5-FU in diH2O (98 atom % 15N, 99 atom % 13C; empirical formula: 13CC3H3F15N2O2; Millipore Sigma) that acted as an internal standard for 5-FU quantitation. Chromatographic separation was achieved using a Thermo Hypercarb column (2.1 × 50 mm, 3u) at room temperature. The mobile phase consisted of solution A: 100 mM NH4OAC in H2O, pH 9.5 using NH4OH, and solution B: 0.1% NH4OH in acetonitrile. A flow rate of 0.3 mL/min was used with the following gradient elution profile: 100% A from 0-6 min, 50% A 50% B from 6-7.1 min, 100% A from 7.1-10 min 50 μL of sample was injected for LC-MS/MS QQQ analysis. This assay used multiple reaction monitoring (MRM) in electrospray negative ionization, and the ions measured for the quantitation were: 5-Fluorouracil: 129.03->42.16 & 129.03->129.0 and C13-5-Fluorouracil: 132.1->60.1 & 132.1->44.1. The retention time for 5-fluorouracil in the column and gradient was 2.65 min. Quantitative analysis for 5-FU was performed using an isotope dilution method with a 7 pt calibration curve (4-fold dilutions starting at 20 μM) run in duplicate with a linear regression coefficient of R2 = 0.9998. The level of quantitation achieved was 0.078 μM and spanned to 20 μM.