Authors (including presenting author) :
Wong YL (1), WY Yu (1), CY Law (1), Fung F (1), Wong CK (1), Lam CW (1)(2)
Affiliation :
(1) Division of Chemical Pathology, Department of Pathology, Queen Mary Hospital (2) Department of Pathology, Li Ka Shing Faculty of Medicine, University of Hong Kong
Introduction :
Patients with Dihydropyrimidine Dehydrogenase (DPD) deficiency face higher risks of severe toxicity from fluoropyrimidine anti-cancer drugs such as 5-fluorouracil and its prodrugs capecitabine and tegafur. These drugs are not broken down efficiently and build up to toxic levels in the body. The European Medicine Agency (EMA) recommends pre-treatment DPD deficiency testing. And announcement from 國家藥監局 on the revision of the instructions for fluorouracil injection (2025 No. 18) stated that current sales packaging and/or product instructions for locally available fluorouracil injections must include safety information related to dihydropyrimidine dehydrogenase deficiency.
A LC-MS/MS has been developed and validated test to measure plasma uracil, which is elevated in DPD-deficient patients. This is the first HA test for the screening of DPD deficiency before fluoropyrimidine treatment, which helps clinicians to adjust the medication dosage to enhance patient safety.
Objectives :
This study aims to develop and validate a robust Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) method for the simultaneous quantification of plasma uracil, 5,6-dihydrouracil, and 5-fluorouracil. The methodical validation of the assay’s performance characteristics—including sensitivity, specificity, accuracy, and precision—ensured the highest quality and reliability of the results, solidifying the assay as a trusted clinical tool. The developed method has participated in an external quality assurance program based in France (ASQUALAB) for quality assurance management, meeting international standards.
Methodology :
Plasma sample was extracted and the uracil, dihydrouracil peak were separated through the Ultra Performance Liquid Chromatography and detected by the Mass Spectrometer for quantification with high specificity and sensitivity. Chromatographic separation was performed using a UPLC C18 column, ionized analyte molecules were generated by electrospray ionization (ESI). The intact molecular ions were then selected and underwent collision-induced dissociation in a collision cell. Product ions generated were detected in mass spectrometer. Analysis of typical transitions using multiple reaction monitoring (MRM) allow determination of analytes with high specificity.
Result & Outcome :
The development and implementation of a robust LC-MS/MS assay for plasma uracil measurement has contributed to the first HA-wide service for the screening of DPD deficiency before fluoropyrimidine chemotherapy and therapeutic drug monitoring of 5-Fluorouracil by the Division of Chemical Pathology at QMH, as recommended by the Drug Office. Under this service, genotyping is not required, even for patients screened positive for partial or complete DPD deficiency by plasma uracil. While fluoropyrimidines are contraindicated in patients with complete DPD deficiency, patients with partial DPD deficiency may be started on a reduced dose and the option of therapeutic drug monitoring of intravenous infusion of 5‐fluorouracil will be available as a part of the service to improve the drug safety of fluoropyrimidines.
