Determination of 3H-labelled cytosine arabinoside and eight metabolites in cell extracts by high-performance liquid chromatography and scintillation spectrometry.

We have developed a method for the separation and quantitation of radiolabeled cytosine arabinoside and its eight metabolites in cell extracts by anion-exchange gradient high-performance liquid chromatography. Baseline separation of cytosine arabinoside and uracil arabinoside and their respective 5'-mono-, di- and triphosphates, as well as cytosine arabinoside diphosphocholine was obtained with the shortest interval between peaks being 3 min. This degree of separation was found to be essential for quantitation of 3H-labeled metabolites by scintillation counting of 1-min fractions. Application of this procedure to the quantitation of [3H] cytosine arabinoside and its metabolites from HL60 human leukemia cells is demonstrated.