Determination of 5-fluorouracil and dihydrofluorouracil levels by using a liquid chromatography-tandem mass spectrometry method for evaluation of dihydropyrimidine dehydrogenase enzyme activity

Purpose 5-Fluorouracil (5-FU), acting as a pyrimidine antagonist, is a major chemotherapy drug used for the treatment of tumors such as gastrointestinal, breast, ovary, and head and neck cancers. The key and rate-limiting enzyme in 5-FU catabolism is dihydropyrimidine dehydrogenase (DHPDH), whose partial or complete deficiency exposes to a severe 5-FU toxicity in patients. The determination of DHPDH activity in patients before the treatment and setting up a personalized therapy for each patient receiving the drug can help us to prevent the possible risk of toxicity. Methods To isolate peripheral blood mononuclear cells (PBMCs), EDTA-anticoagulated blood samples were collected from randomly selected 47 patients and examined for 5-FU and its metabolite dihydrofluorouracil (FUH2) by using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) to observe DHPDH activity at different intervals (0 and 4th hour) indirectly. Results Intra-assay and interassay CV % values of samples from the measurements of the modified methods are found 1.3-11.9, 2.3-9.4 for 5-FU and 3.1-14.4, 3.3-12.6 for FUH2, respectively. The reference values derived from 45 patients treated with 5-FU are 1.84 +/- A 0.34 ug/gr protein for 5-FU, 40.15 +/- A 11.43 ng/gr protein for FUH2, respectively. FUH2/5-FU ratio is 21.9 +/- A 3.72. In addition, the results determined from two patients, in which the lack of DHPDH is considered, were 3.24 and 4.16 ug/gr protein for 5-FU, 4.1 and 6.7 ng/gr protein for FUH2. FUH2/5-FU ratio is 1.26 and 1.61. Conclusion The measurements of 5-FU, FUH2, and especially their ratio (FUH2/5-FU) by the modified LC-MS/MS method could be used to determine DHPDH enzyme activity.