HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC METHOD FOR THE DETERMINATION OF INTRACELLULAR DTTP POOLS

The size of intracellular 2'-deoxythymidine-5'-triphosphate (dTTP) poo ls is an important indicator of cytotoxicity in cells exposed to antiv iral 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 st rong anion exchange (SAX) cartridges. The resulting fraction is analyz ed by isocratic HPLC using a C-18 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/10(6) cells and gi ve a signal to noise ratio of 4:1. The method described is used for th e determination of dTTP pools in PKA-stimulated PBMC from healthy volu nteers.