5-Fluorouracil (5-FU) is an anticancer drug extensively used for different cancers. Intracellular metabolic activation leads to several nucleoside and nucleotide metabolites essential to exert its cytotoxic activity on multiple cellular targets such as enzymes, DNA and RNA. In this paper, we describe the development of a method based on liquid chromatography coupled with high resolution mass spectrometry suitable for the simultaneous determination of the ten anabolic metabolites (nucleoside, nucleotide and sugar nucleotide) of 5-FU. The chromatographic separation was optimized on a porous graphitic carbon column allowing the analysis of the metabolites of 5-FU as well as endogenous nucleotides. The detection was performed on an Orbitrap® tandem mass spectrometer. Linearity of the method was verified in intracellular content and in RNA extracts. The limit of detection was equal to 12 pg injected on column for nucleoside metabolites of 5-FU and 150 pg injected on column for mono- and tri-phosphate nucleotide metabolites. Matrix effect was evaluated in cellular contents, DNA and RNA extracts for nucleoside and nucleotides metabolites. The method was successfully applied to i) measure the proportion of each anabolic metabolite of 5-FU in cellular contents, ii) follow the consequence of inhibition of enzymes on the endogenous nucleotide pools, iii) study the incorporation of metabolites of 5-FU into RNA and DNA, and iv) to determine the incorporation rate of 5-FUrd into 18 S and 28 S sub-units of rRNA.

5-氟尿嘧啶（5-FU）是一种广泛用于不同癌症的抗癌药物。细胞内代谢激活会导致一些核苷和核苷酸代谢产物，这些代谢产物对多种细胞靶标（例如酶，DNA和RNA）发挥细胞毒活性至关重要。在本文中，我们描述了一种基于液相色谱与高分辨率质谱联用的方法的开发，该方法适用于同时测定5-FU的十种同化代谢产物（核苷，核苷酸和糖核苷酸）。色谱分离在多孔石墨碳柱上进行了优化，可分析5-FU和内源核苷酸的代谢产物。该检测在串联质谱仪上进行。该方法的线性在细胞内含量和RNA提取物中得到验证。对于5-FU的核苷代谢物，检测限等于在色谱柱上注入12 pg，对于单磷酸和三磷酸核苷酸代谢物，检测限等于在色谱柱上注入150 pg。在细胞内容物，DNA和RNA提取物中的核苷和核苷酸代谢产物中评估基质效应。该方法已成功应用于以下方面：i）测量细胞中5-FU的每种同化代谢产物的比例，ii）跟踪酶对内源核苷酸库的抑制作用，iii）研究将5-FU代谢产物掺入细胞中RNA和DNA，以及iv）确定5-FUrd掺入rRNA的18 S和28 S亚基的比率。在细胞内容物，DNA和RNA提取物中的核苷和核苷酸代谢产物中评估基质效应。该方法已成功应用于以下方面：i）测量细胞中5-FU的每种合成代谢产物的比例，ii）观察到酶对内源核苷酸库的抑制作用，iii）研究将5-FU代谢产物掺入细胞中RNA和DNA，以及iv）确定5-FUrd掺入rRNA的18 S和28 S亚基的比率。在细胞内容物，DNA和RNA提取物中的核苷和核苷酸代谢产物中评估基质效应。该方法已成功应用于以下方面：i）测量细胞中5-FU的每种同化代谢产物的比例，ii）跟踪酶对内源核苷酸库的抑制作用，iii）研究将5-FU代谢产物掺入细胞中RNA和DNA，以及iv）确定5-FUrd掺入rRNA的18 S和28 S亚基的比率。