A
deoxycytidine analog,
gemcitabine (dFdC), is effective for treating solid
tumors and
hematological malignancies. After being transported into
cancer cells, dFdC is phosphorylated to dFdC
triphosphate (dFdCTP), which is subsequently incorporated into the
DNA strand, thereby inhibiting
DNA synthesis. Intracellular dFdCTP is the critical determinant for dFdC cytotoxicity, so therapeutic
drug monitoring or in vitro testing of the capability of
cancer cells to accumulate dFdCTP may be informative for optimizing dFdC administration. We have developed a new isocratic-elution high-performance liquid chromatography method for quantifying dFdCTP in
cancer cells. Samples (500 microL) were eluted isocratically using 0.06 M Na(2)HPO(4) (pH 6.9) containing 20%
acetonitrile, at a constant flow rate of 0.7 mL/min and at ambient temperature. Separation was carried out using an
anion-exchange column (TSK gel
DEAE-2SW; 250 mm x 4.6 mm inside diameter, particle size 5 microL) and monitored at 254 nm. The standard curve was linear with low within-day and interday variability. The lower detection limit (20 pmol) was as sensitive as that of the previous gradient-elution method. dFdCTP was well separated from other
nucleoside triphosphates. The method could measure dFdCTP in cultured or primary leukemic cells treated in vitro with dFdC. The method was also applicable to simultaneous determination of dFdCTP and
cytarabine triphosphate, the results of which demonstrated
ara-CTP production augmented by dFdC pretreatment. Thus, our isocratic high-performance liquid chromatography assay method will be of great use because of its sensitivity and simplicity as well as its applicability to
biological materials.