Development of an HPLC-DAD Method for the Extraction and Quantification of 5-Fluorouracil, Uracil, and 5-Fluorodeoxyuridin Monophosphate in Cells and Culture Media of Lactococcus lactis

Abstract

The drug 5-fluorouracil (5-FU) is a common cancer chemotherapeutic, presenting toxicity. Mild toxicity is treated with administration of probiotics. The interaction of these probiotics with the drug may have a crucial effect on its therapeutic efficacy. In the present work, a method for the quantification of uracil, 5-FU, and its active metabolite 5-fluorodeoxyuridin monophosphate in cells and culture medium of the probiotic L. lactis is presented. Extraction using H2O containing 0.05% v/v formic acid (1:5 v/v) was followed by ammonium sulphate protein precipitation and SPE. Analysis was conducted in a Nucleosil column using a gradient of water, formic acid, and acetonitrile. Calibration curves were constructed for 5-FU (5–100 μg/mL), uracil (5–20 μg/mL), and 5-fluorodeoxyuridin monophosphate (5–20 μg/mL) using 5-bromouracil as the internal standard (R2 ≥ 0.999). The photodegradation of 5-FU amounted to 36.2% at 96 h. An administration experiment in the dark revealed a decline in 5-FU concentration in the culture media (88.3%) and uptake by the cells, while the uracil and FdUMP levels increased in the cells. The inactive metabolite 5,6 dihydrofluorouracil was detected in the medium. Our results demonstrate that uptake and metabolism of 5-FU in L. lactis cells leads to a decline in the drug levels and in the formation of both the active and the inactive metabolites of the drug.