High-performance liquid chromatographic assay with ultraviolet detection for quantification of dihydrofluorouracil in human lymphocytes: application to measurement of dihydropyrimidine dehydrogenase activity

The anticancer drug 5-fluorouracil (5FU) undergoes extensive biotransformation to 5-dihydrofluorouracil (5FUH2) by the enzyme dihydropyrimidine deshydrogenase (DPD). A new HPLC method with direct UV detection for the determination of 5FUH2 in peripheral lymphocytes has been developed to detect DPD deficiency in patients treated with 5FU-based therapy. The method has been shown to be valid over the 5FUH2 concentration range of 1.14–37.88 nmol/ml. Optimal enzymatic conditions for DPD activity measurement were studied: incubation time, protein and 5FU concentrations. The assay was successfully cross-validated with the existing method using HPLC with radiochemical detection.