A highly sensitive high-performance liquid chromatography-mass spectrometry method for quantification of fludarabine triphosphate in leukemic cells

A high-performance liquid chromatography (HPLC)-mass spectrometry (MS) method has been developed for the analysis of 9-β-d-arabinofuranosyl-2-fluoroadenine 5′-triphosphate (F-ara-ATP) from biological samples. Quantification is carried out by selected ion monitoring of the parent ion. Baseline separation of the monophosphate (F-ara-AMP) and diphosphate (F-ara-ADP) is achieved using the volatile ion-pairing reagent dimethylhexylamine. This method is selective and sensitive with an on-column detection limit of ∼50 fmol. It also permits simultaneous monitoring of endogenous adenosine phosphates. The utility of the assay has been demonstrated by the analysis of F-ara-ATP in human leukemic cells after incubation with 9-β-d-arabinosyl-2-fluoroadenine (F-ara-A) at clinically relevant concentrations.