Determination of clofarabine triphosphate concentrations in leukemia cells using sensitive, isocratic high-performance liquid chromatography.

摘要

BACKGROUND/AIM: An active metabolite of the anti-leukemia agent clofarabine (Cl-F-ara-A) is an intracellular triphosphate form, Cl-F-ara-ATP. Monitoring this active form could provide crucial information for optimizing treatment regimens based on Cl-F-ara-A. A simple, isocratic HPLC method was developed. MATERIALS AND METHODS: Samples (500 mul) from leukemic cells were loaded onto an anion-exchange column and eluted with a phosphate-acetonitrile buffer (flow rate: 0.7 ml/min) at ambient temperature. The Cl-F-ara-ATP concentration was determined by measuring absorbance at 254 nm. RESULTS: The standard curve was linear, with minimal within-day and inter-day variability. Recovery was excellent; low and high quantitation limits were 10 pmol and 5,000 pmol, respectively. Cl-F-ara-ATP eluted independently of ATP, GTP, UTP, and CTP. Production of Cl-F-ara-ATP was successfully measured in cultured leukemia HL-60 cells treated in vitro with Cl-F-ara-A. CONCLUSION: This method is simple, sensitive and applicable for determination of the Cl-F-ara-ATP content of biological materials.