Determination of 5-Fluorouracil, 5,6-dihydro-5-Fluorouracil, Uracil and Dihydrouracil by UPLC-MS/MS in plasma and its application in hepatocellular carcinoma.

Authors

Zhiqiang Tong, Tingting Ding, Yuexin Li, Dongqing Gao, Zhonghua Sun, Xuemei Chen, Shuai Jiang, Mei Dong

Published in

Analytical biochemistry. Pages 116267. Oct 06, 2026. Epub Oct 06, 2026.

Abstract

Aimed to develop and validate an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous determination of 5-Fluorouracil (5-FU), 5,6-dihydro-5-Fluorouracil (5-FUH₂), Uracil (U), and Dihydrouracil (UH₂) concentrations in human plasma, and to apply 5-FU therapeutic drug monitoring(TDM).
UPLC-MS/MS method was adopted using 5-bromouracil (5-Bru) as internal standard (IS).The determination was performed on Waters Acquity UPLC BEH Amide column (100 mm×2.1 mm,1.7μm) with mobile phase consisted of acetonitrile and water. An electrospray ion source was used to carry out negative ion scanning with multiple reaction monitoring (MRM).The method was applied to 10 patients receiving FOLFOX-HAIC. 5-FU exposure was estimated as AUC = C_ss × TCI, and UH₂/U ratio was explored as a DPD-related metabolic marker.
Accuracy, precision, recovery, matrix effect, and stability were examined to meet ICH M10 guidelines for validation of biological methods.All four analytes were quantified over 20-1000 ng/mL with a lower limit of quantification of 20 ng/mL. Mean extraction recovery was approximately 52%-58%, with matrix effects close to 100%.Among 10 patients with advanced hepatocellular carcinoma, the Css of 5-FU ranged from 202.39 to 583.70 ng/mL, the AUC of 5-FU ranged from 9.31 to 26.85 mg·h/L, and the plasma concentration of 5-FUH₂ ranged from 352.96 to 892.59 ng/mL. The UH₂:U concentration ratio ranged from 0.49 to 7.83.
We developed a rapid 5-minute UPLC-MS/MS method for simultaneously determining 5-FU, 5-FUH₂, U and UH₂ in human plasma and applied it to patients treated with FOLFOX-HAIC. This method can comprehensively evaluate the 5-FU exposure and pyrimidine metabolism of patients with hepatocellular carcinoma.

PMID:
42838441
Bibliographic data and abstract were imported from PubMed on 07 Oct 2026.

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