title: Development and validation of a rapid and sensitive UPLC-MS/MS method for determination of uracil and dihydrouracil in human plasma.
abstract: Quantification of the endogenous dihydropyrimidine dehydrogenase (DPD) substrate uracil (U) and the reaction product dihydrouracil (UH2) in plasma might be suitable for identification of patients at risk of fluoropyrimidine-induced toxicity as a result of DPD deficiency. In this paper, we describe the development and validation of a rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay for quantification of U and UH2 in human plasma. Analytes were extracted by protein precipitation, chromatographically separated on an Acquity UPLC(®) HSS T3 column with gradient elution and analyzed with a tandem mass spectrometer equipped with an electrospray ionization source. U was quantified in the negative ion mode and UH2 in the positive ion mode. Stable isotopes for U and UH2 were used as internal standards. Total chromatographic run time was 5min. Validated concentration ranges for U and UH2 were from 1 to 100ng/mL and 10 to 1000ng/mL, respectively. Inter-assay bias and inter-assay precision for U were within ±2.8% and ≤12.4%. For UH2, inter-assay bias and inter-assay precision were within ±2.9% and ≤7.2%. Adequate stability of U and UH2 in dry extract, final extract, stock solution and plasma was demonstrated. Stability of U and UH2 in whole blood was only satisfactory when stored up to 4hours at 2-8°C, but not at ambient temperatures. An accurate, precise and sensitive UPLC-MS/MS assay for quantification of U and UH2 in plasma was developed. This assay is now applied to support clinical studies with fluoropyrimidine drugs.
authors.affiliation: The Netherlands Cancer Institute, Department of Clinical Pharmacology, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands; The Netherlands Cancer Institute, Department of Pharmacy and Pharmacology, Louwesweg 6, 1066 EC, Amsterdam, The Netherlands. Electronic address: b.jacobs@nki.nl.
authors.forename: Bart A W
authors.initials: BA
authors.lastname: Jacobs
authors.affiliation: The Netherlands Cancer Institute, Department of Pharmacy and Pharmacology, Louwesweg 6, 1066 EC, Amsterdam, The Netherlands.
authors.forename: Hilde
authors.initials: H
authors.lastname: Rosing
authors.affiliation: The Netherlands Cancer Institute, Department of Pharmacy and Pharmacology, Louwesweg 6, 1066 EC, Amsterdam, The Netherlands.
authors.forename: Niels
authors.initials: N
authors.lastname: de Vries
authors.affiliation: The Netherlands Cancer Institute, Department of Clinical Pharmacology, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands.
authors.forename: Didier
authors.initials: D
authors.lastname: Meulendijks
authors.affiliation: The Netherlands Cancer Institute, Department of Clinical Pharmacology, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands.
authors.forename: Linda M
authors.initials: LM
authors.lastname: Henricks
authors.affiliation: The Netherlands Cancer Institute, Department of Clinical Pharmacology, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands; Utrecht University, Department of Pharmaceutical Sciences, Universiteitsweg 99, 3584CG, Utrecht, The Netherlands.
authors.forename: Jan H M
authors.initials: JH
authors.lastname: Schellens
authors.affiliation: The Netherlands Cancer Institute, Department of Clinical Pharmacology, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands; The Netherlands Cancer Institute, Department of Pharmacy and Pharmacology, Louwesweg 6, 1066 EC, Amsterdam, The Netherlands; Utrecht University, Department of Pharmaceutical Sciences, Universiteitsweg 99, 3584CG, Utrecht, The Netherlands.
authors.forename: Jos H
authors.initials: JH
authors.lastname: Beijnen
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D054067:Dihydropyrimidine Dehydrogenase Deficiency / Q000150:complications; D042943:Dihydrouracil Dehydrogenase (NADP) / Q000378:metabolism; D006801:Humans; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013696:Temperature; D013997:Time Factors; D014498:Uracil / Q000031:analogs & derivatives* / Q000032:analysis / Q000097:blood
chemical_list: C007419:dihydrouracil; D014498:Uracil; D042943:Dihydrouracil Dehydrogenase (NADP)
keywords: 5-Fluorouracil; Capecitabine; Dihydropyrimidine dehydrogenase; Dihydrouracil; UPLC–MS/MS; Uracil