High-performance liquid chromatography method for the determination and quantitation of arabinosylguanine triphosphate and fludarabine triphosphate in human cells

A gradient anion-exchange high-performance liquid chromatographic assay was developed for the simultaneous determination and quantitation of the cytot oxic triphosphates of arabinosylguanine (ara-GTP) and fludarabine (F-ara-AT P). The method was validated with respect to selectivity, recovery, lineari ty, precision, and accuracy using authentic standards. To test this assay i n a more complex biological matrix, perchloric acid extracts of circulating human leukemia cells spiked with known concentrations of ara-GTP and F-ara -ATP were examined. Finally, to assess the clinical utility of our method, perchloric acid extracts of circulating human leukemia cells isolated from patients treated with fludarabine and nelarabine were analyzed. The range o f quantitation was 0.0125-10 nmol for the ara- and native NTPs in cellular extracts. This assay should be helpful in establishing the mechanistic rati onales for drug scheduling and combinations of nelarabine and fludarabine, and for correlating the therapeutic efficacy and levels of the cytotoxic tr iphosphates in target cells. (C) 2000 Elsevier Science B.V. All rights rese rved.