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Development and validation of a rapid and sensitive HPLC method for the quantification of 5‐fluorocytosine and its metabolites
Author(s) -
Serve Kinta M.,
Yáñez Jaime A.,
Remsberg Connie M.,
Davies Neal M.,
Black Margaret E.
Publication year - 2010
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.1326
Subject(s) - chemistry , chromatography , prodrug , formic acid , reproducibility , high performance liquid chromatography , metabolite , detection limit , intracellular , calibration curve , quantitative analysis (chemistry) , biochemistry
To study the intracellular metabolism of the prodrug 5‐fluorocytosine (5FC), we developed a novel reverse‐phase high‐performance liquid chromatography method to simultaneously detect 5FC and its four major anabolic metabolites: 5‐fluorouracil, 5‐fluorouridine, 5‐fluorouridine‐monophosphate and 5‐fluoro‐2′deoxyuridine‐5′‐monophosphate. Separation of each compound was accomplished under isocratic conditions using a C 18 column and mobile phase of formic acid–water (1 : 99 v/v). The method was validated for both accuracy and reproducibility in cell culture media. Additionally, metabolites were assessed for stability at ambient temperatures and following freeze–thaw cycles. Calibration curves were linear over a range of 1–200 μg/mL. Limit of quantification for four of the five compounds was 1 μg/mL in cell culture media (RSD < 11%). This method was successfully used to monitor intracellular conversion of 5FC to its metabolic products over a 24h period. Copyright © 2009 John Wiley & Sons, Ltd.