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Updated: May 29, 2026
HPLC-based Assay to Monitor Extracellular Nucleotide/Nucleoside Metabolism in Human Chronic Lymphocytic Leukemia Cells
Published on: July 20, 2016
Determination of clofarabine triphosphate concentrations in leukemia cells using sensitive, isocratic
Takahiro Yamauchi1, Rie Nishi, Takanori Ueda
1Department of Hematology and Oncology, University of Fukui, 23 Shimoaizuki, Matsuoka, Eiheiji, Fukui 910-1193, Japan. tyamauch@u-fukui.ac.jp
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 μl) 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.
