Improved determination of uracil and dihydrouracil in plasma after a loading oral dose of uracil using high-performance liquid chromatography with photodiode array detection and porous graphitic carbon stationary phase.
OBJECTIVE
The aim of this study was to develop and validate a high-performance liquid chromatographic method for the measurement of plasma concentrations of uracil and dihydrouracil after administration of an oral loading dose of uracil in the context of evaluation of DPD enzyme activity.


METHODS
Analytes were extracted from 500μL plasma sampler with a mixture of ethyl acetate isopropanol (85:15, v/v) after protein precipitation with solid ammonium sulfate. The extract was inject in the porous graphitic carbon stationary phase, eluted with water and acetonitrile in gradient mode, allowing complete separation of uracil, dihydrouracil and the internal standard (5-fluorouracil). Chromatograms were monitored at 210 and 260nm.


RESULTS
Total chromatographic run time, including reequilibration, was 30min. The assay was linear in the concentration range of 0.2 to 20μgmL(-1). Accuracy was 98.4-105.3%, intra-assay precision was 5.1-12.1% and between-assay precision was of 5.3-10.1%. Analytes were stable in plasma at room temperature up to 6h and for three freeze and thaw cycles. Processed samples are stable up to 12h.


CONCLUSIONS
The developed method was fully validated and has significantly reduced running time when compared to previous assay using porous graphitic stationary phase, allowing complete resolution of uracil, dihydrouracil and internal standard. This assay might be suitable to investigate the eventual correlation between concentrations of uracil and dihydrouracil in plasma after an oral loading dose and DPD enzyme activity, with potential contribution to therapeutic drug monitoring.
Toxicological Analysis Laboratory, Instituto de Ciências da Saúde, Universidade Feevale, Novo Hamburgo, RS, Brazil.
Roberta Zilles
RZ
Hahn
Oncology Department, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil.
Andrés Fernando Andrade
AF
Galarza
Toxicological Analysis Laboratory, Instituto de Ciências da Saúde, Universidade Feevale, Novo Hamburgo, RS, Brazil.
Anelise
A
Schneider
Toxicological Analysis Laboratory, Instituto de Ciências da Saúde, Universidade Feevale, Novo Hamburgo, RS, Brazil.
Marina Venzon
MV
Antunes
Oncology Department, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil.
Gilberto
G
Schwartsmann
Toxicological Analysis Laboratory, Instituto de Ciências da Saúde, Universidade Feevale, Novo Hamburgo, RS, Brazil. Electronic address: rafael.linden@feevale.br.
Rafael
R
Linden
D000284:Administration, Oral; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation* / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D006108:Graphite / Q000737:chemistry*; D006801:Humans; D016062:Porosity; D015203:Reproducibility of Results; D014498:Uracil / Q000008:administration & dosage* / Q000031:analogs & derivatives* / Q000097:blood*
C007419:dihydrouracil; D014498:Uracil; D006108:Graphite
Chemotherapy; Dihydropyrimidine dehydrogenase; Fluoropyrimidine drugs; HPLC-PDA; Porous graphitic carbon