Determination of 2-chloro-2′-deoxyadenosine nucleotides in leukemic cells by ion-pair high-performance liquid chromatography

Abstract

A specific isocratic ion-pair HPLC method for the quantitation of mono-, di- and triphosphates of 2-chloro-2′-deoxyadenosine (CdA) in leukemic cells from patients is described. The method is based on an extraction of nucleotides from cells with a solution of perchloric acid containing triethylammonium phosphate followed by an isocratic separation on an Ultrasphere ODS column (250×4.6 mm, 5 μm) with a mixture of 89% 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 detection was 65 n M. Standard curves for the CdA triphosphate (CdATP) were linear within the concentration range of 200 n M to 12 μ M. The mean overall recovery of CdATP was 90% within a concentration range of standard curves. The within-day and day-to-day coefficients of variation at concentrations of 1.44 μ M and 6.25 μ M CdATP were < 10%. The applicability of the method was demonstrated by in vitro studies of the accumulation of CdA mono-, di- and triphosphates in CCRF-CEM cells and by determination of the cellular pharmacokinetics of CdA nucleotides in leukemic cells from a patient treated with CdA.