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Determination of Endogenous Concentrations of Uracil and Dihydrouracil in Dried Saliva Spots by LC-MS/MS

Method Development, Validation, and Clinical Application

Antunes, Marina Venzon, PhD*; Raymundo, Suziane, MSc*; Cezimbra da Silva, Anne Caroline, BSc*; Muller, Victoria Vendramini, BSc*; Vicente Neto, Olavo José, MSc*; Schwartsmann, Gilberto, PhD; Linden, Rafael, PhD*

doi: 10.1097/FTD.0000000000000615
Short Communication

Background: The aim of this study was to develop and validate a liquid chromatography–tandem mass spectrometry (LC-MS/MS) assay for the measurement of uracil (U) and dihydrouracil (UH2) concentrations in dried saliva spots (DSSs), for the evaluation of dihydropyrimidine dehydrogenase (DPD) enzyme activity.

Results: Nine 18-mm diameter DSS discs were extracted with acetate:isopropyl alcohol (85:15, vol/vol) and analyzed by LC-MS/MS. The assay was linear in the range of 10–1000 ng·mL−1, with accuracy between 89% and 112% and precision between 5.7% and 13%. The metabolic ratio [UH2]/[U] was stable in DSS for up to 9 days at 45°C. Concentrations of U and UH2, as well as the metabolic ratio, were highly concordant between matrices. Using a metabolic ratio classification cutoff of 1.16 for the identification of slow DPD metabolizers, 98.7% concordance was achieved between SS and saliva.

Conclusions: DSS samples could be a useful alternative for DPD activity screening, particularly in locations with limited access to highly equipped laboratories.

*Graduate Program on Toxicology and Analytical Toxicology, Universidade Feevale, Novo Hamburgo; and

Oncology Service, Hospital de Clínicas de Porto Alegre, Porto Alegre, Rio Grande do Sul, Brazil.

Correspondence: Marina Venzon Antunes, PhD, Universidade Feevale, ERS 239, no 2755, 93525-075 Novo Hamburgo, Rio Grande do Sul, Brazil (e-mail:

Supported by Universidade Feevale and Fundo de Incentivo à Pesquisa e Eventos do Hospital de Clínicas de Porto Alegre (FIPE-HCPA).

The authors declare no conflict of interest.

Received August 24, 2018

Accepted December 10, 2018

Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.