Abstract
The size of intracellular 2′-deoxythymidine-5′-triphosphate (dTTP) pools is an important indicator of cytotoxicity in cells exposed to antiviral drugs and mutagens. We describe an ion paired reverse-phase high performance liquid chromatographic (HPLC) assay for the quantification of thymidine triphosphate in peripheral blood mononuclear cells (PBMC). Cell extracts are first purified by solid phase extraction using strong anion exchange (SAX) cartridges. The resulting fraction is analyzed by isocratic HPLC using a C18 column and a mobile phase consisting of triethylamine phosphate buffer with 2% acetonitrile. UV detection at 267 nm is used. Coefficients of variation for the assay are in the range of 6.0–9.8%. The overall recovery is 86.84%. LOQ for this assay is 48 ng/mL (100 nM), which is equivalent to 5 pmol/106 cells and give a signal to noise ratio of 4:1. The method described is used for the determination of dTTP pools in PHA-stimulated PBMC from healthy volunteers.
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