LC‐MS/MS method for simultaneous analysis of uracil, 5,6‐dihydrouracil, 5‐fluorouracil and 5‐fluoro‐5,6‐dihydrouracil in human plasma for therapeutic drug monitoring and toxicity prediction in cancer patients

Abstract

The chemotherapeutic drug 5‐fluorouracil (5‐FU) is widely used for treating solid tumors. Response to 5‐FU treatment is variable with 10–30% of patients experiencing serious toxicity partly explained by reduced activity of dihydropyrimidine dehydrogenase (DPD). DPD converts endogenous uracil (U) into 5,6‐dihydrouracil (UH2), and analogously, 5‐FU into 5‐fluoro‐5,6‐dihydrouracil (5‐FUH2). Combined quantification of U and UH2 with 5‐FU and 5‐FUH2 may provide a pre‐therapeutic assessment of DPD activity and further guide drug dosing during therapy. Here, we report the development of a liquid chromatography–tandem mass spectrometry assay for simultaneous quantification of U, UH2, 5‐FU and 5‐FUH2 in human plasma. Samples were prepared by liquid–liquid extraction with 10:1 ethyl acetate‐2‐propanol (v/v). The evaporated samples were reconstituted in 0.1% formic acid and 10 μL aliquots were injected into the HPLC system. Analyte separation was achieved on an Atlantis dC18 column with a mobile phase consisting of 1.0 mm ammonium acetate, 0.5 mm formic acid and 3.3% methanol. Positively ionized analytes were detected by multiple reaction monitoring. The analytical response was linear in the range 0.01–10 μm for U, 0.1–10 μm for UH2, 0.1–75 μm for 5‐FU and 0.75–75 μm for 5‐FUH2, covering the expected concentration ranges in plasma. The method was validated following the FDA guidelines and applied to clinical samples obtained from ten 5‐FU‐treated colorectal cancer patients. The present method merges the analysis of 5‐FU pharmacokinetics and DPD activity into a single assay representing a valuable tool to improve the efficacy and safety of 5‐FU‐based chemotherapy. Copyright © 2012 John Wiley & Sons, Ltd.

Dihydropyrimidine Accumulation Is Required for the Epithelial-Mesenchymal Transition

“…To do so, we used liquid chromatography and mass spectrometry (LC-MS) (Büchel et al, 2012) to determine the cellular concentration of DPYD substrates (uracil and thymine) and immediate products (dihydrouracil (DHU) and dihydrothymine (DHT)) (Figure 5G) (Lohkamp et al, 2010). In HMLE-Twist-ER cells, overexpression or knockdown of DPYD resulted in a corresponding ~10-fold increase or decrease, respectively, in the intracellular DHU/uracil molar ratio (Figure 5H).…”

Fluorouracil plasma monitoring: systematic review and economic evaluation of the My5-FU assay for guiding dose adjustment in patients receiving fluorouracil chemotherapy by continuous infusion

“…[105][106][107] Despite LC-MS/MS methods being found to be sensitive and robust, the instrumentation is not in standard use in routine clinical laboratories in the UK. For more details on a LC-MS/MS method please refer to a paper by Kosovec et al…”

A Critical Review on Clinical Application of Separation Techniques for Selective Recognition of Uracil and 5-Fluorouracil

“…A LC-MS/MS method has been described for simultaneous analysis of Ura, 5-FU and UH2 in human plasma. The developed method combined the analysis of 5-FU pharmacokinetics and DPD activity into a single assay [56]. These assays were found to be suitable for monitoring 5-FU plasma levels in routine clinical practice and may contribute to improved efficacy and safety of 5-FU-based chemotherapies for therapeutic drug monitoring and toxicity prediction in cancer patients [57].…”