Analysis of 5-fluoro-2′-deoxycytidine and 5-trifluoromethyl-2′-deoxycytidine and their related antimetabolites by high-performance liquid chromatography

An isocratic, ion-paired, reversed-phase high-performance liquid chromatography technique for the quantitative determination of 5-fluoro-2′-deoxycytidine (FdCyd) and 5-trifluoromethyl-2′-deoxycytidine (F₃methyldCyd) and their related antimetabolites is described. Extraction and purification of these compounds from DNA, RNA, and free pools is reviewed diagrammatically. Total analysis time including quantitation of DNA and RNA primary constituents is 45 min. Average combined recoveries for prodrugs and anti-metabolites is above 90% with standard deviations of 0.07 and 0.58 and average precisions of 5.51 and 8.30% for FdCyd and F₃methyldCyd, respectively. Average coefficients of variation were 3.8 ± 0.5% for FdCyd and 7.7 ± 1.0% for F₃methyldCyd. Limits of detection were approximately 1 pmol for unlabelled prodrugs and antimetabolites. FdCyd, when generally tritiated with a specific activity of 18 Ci/mmol, was detected in the 5 · 10^-15-20 · 10^-15 mol (fmol) range depending on sample condition.