MSACL 2019 EU Abstract

Fully-Automated LC-MS Method for Uracil and Dihydrouracil in Human Plasma

Doriane Toinon (1), Tiphaine Robin (1,2), Stephane Moreau (3), Franck-Saint-Marcoux (2)
(1) Shimadzu, Paris, France (2) CHU of Limoges, Limoges, France (3) Shimadzu, Duisburg, Germany

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Doriane Toinon (Presenter) Shimadzu France

Relevant Financial Disclosures (within past 24 months) Salary Shimadzu France Conflict mitigation NOT REQUIRED.

INTRODUCTION: Fluoropyrimidines (5-fluorouracil or capecitabine) are anticancer drugs that can lead to severe or lethal toxicities in case of dihydropyrimidine dehydrogenase (DPD) deficiency. In France, health authorities recommend the determination of uracil concentration to guide dosing of fluoropyrimidines. Numerous LC-MS/MS methods have been proposed but they include complex liquid-liquid or solid-phase extraction procedures.
OBJECTIVES: To answer to the need of high throughput analysis our objective was to develop a method where the extraction was carried out by a programable liquid handler directly coupled to a LC-MS/MS system.
METHODS: The extraction procedure was performed by CLAM-2030 coupled to a LCMS-8060 triple quadrupole mass spectrometer (Shimadzu Corporation, Kyoto) used in positive electrospray ionization mode. Acquisition method targeted MRM transitions for uracil, dihydrouracil, uracil-13C,15N2 and dihydrouracil-13C,15N2. The method was fully validated according to the International Standards Organization (ISO) 15189 standard (repeatability, reproducibility, recovery, matrix effect and selectivity). The whole procedure was applied to 50 real patient samples and its results were compared to a validated LC-MS/MS method used in routine at the Limoges University Hospital.
RESULTS: Once on board, 20 µL of plasma were automatically delivered to a tube containing a PTFE (polytetrafluoroethylene) filter, to which reagents were added, mixed and then filtered. About 24 minutes were needed to obtain the first result and then, extraction (10 min) and separation (14 min) were performed in parallel with the system producing a result every 14 minutes. A lower limit of quantitation of 5 ng/ml was easily reach for both uracil and dihydrouracil (DPD deficiency being diagnosed for uracil >16 ng/ml). The procedure was successfully validated and agreement was obtained with the previously validated method used in Limoges (R²> 0.9; n=50).
CONCLUSION: We propose a LC-MS/MS method for the measurements of uracil and dihydrouracil with no human intervention once primary tube is on board. The simplicity of operation and the minimization of user involvement in the sample preparation process will help obtain high throughput for the monitoring of 5-fluorouracil and capecitabine treatements.