Development and validation of a rapid and sensitive HPLC method for the quantification of 5-fluorocytosine and its metabolites.
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 microg/mL. Limit of quantification for four of the five compounds was 1 microg/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.
Serve
Kinta M
KM
Washington State University, Pullman, 99164, USA.
Yáñez
Jaime A
JA
Remsberg
Connie M
CM
Davies
Neal M
NM
Black
Margaret E
ME
D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000032:analysis* / Q000378:metabolism*; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid / Q000191:economics / Q000379:methods*; D005437:Flucytosine / Q000032:analysis* / Q000378:metabolism*; D051381:Rats; D012680:Sensitivity and Specificity; D013997:Time Factors
D000964:Antimetabolites, Antineoplastic; D005437:Flucytosine