DETERMINATION OF 2-CHLORO-2'-DEOXYADENOSINE NUCLEOTIDES IN LEUKEMIC-CELLS BY ION-PAIR HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

A specific isocratic ion-pair HPLC method for the quantitation of mono -, di- and triphosphates of 2-chloro-2'-deoxyadenosine (CdA) in leukem ic cells from patients is described. The method is based on an extract ion of nucleotides from cells with a solution of perchloric acid conta ining triethylammonium phosphate followed by an isocratic separation o n an Ultrasphere ODS column (250 x 4.6 mm, 5 mu m) with a mixture of 8 9% triethylammonium phosphate buffer (0.08 M, pH 6.1) and 11% methanol as the eluent. UV absorbance at 265 nm was used. The limit of detecti on was 65 nM. Standard curves for the CdA triphosphate (CdATP) were li near within the concentration range of 200 nM to 12 mu M. The mean ove rall recovery of CdATP was 90% within a concentration range of standar d curves. The within-day and day-to-day coefficients of variation at c oncentrations of 1.44 mu M and 6.25 mu M CdATP were < 10%. The applica bility of the method was demonstrated by in vitro studies of the accum ulation of CdA mono-, di- and triphosphates in CCRF-CEM cells and by d etermination of the cellular pharmacokinetics of CdA nucleotides in le ukemic cells from a patient treated with CdA.