QUANTITATION OF DEOXYRIBONUCLEOSIDE 5'-TRIPHOSPHATES BY A SEQUENTIAL BORONATE AND ANION-EXCHANGE HIGH-PRESSURE LIQUID-CHROMATOGRAPHIC PROCEDURE

A rapid method for the quantitative determination ofcellular deoxyribonucleoside 5′-triphosphates is described. Cell extracts are first separated by boronate chromatography at pH 8.9, which removes 99% of the ribonucleoside triphosphate (rNTPs) from the deoxyribonucleoside triphosphates (dNTPs). The resulting dNTP fraction is analyzed by gradient high-pressure liquid chromatography utilizing a strong anion-exchange column, which can separate minor rNTP peaks from the corresponding dNTPs. This sequential procedure, which requires less than 1 h per sample for both chromatographic steps, results in the quantitative recovery of greater than 98% of the dNTPs from cell extracts. Nucleotide analogs, such as 1-β-d-arabinofur-anosylcytosine-5′-triphosphate and 5-bromo-2′-deoxy-uridine-5′-triphosphate, can also be quantitated efficiently by this method. © 1992 Academic Press, Inc.