AN ION-PAIRING HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC METHOD FOR THEDIRECT SIMULTANEOUS DETERMINATION OF NUCLEOTIDES, DEOXYNUCLEOTIDES, NICOTINIC COENZYMES, OXYPURINES, NUCLEOSIDES, AND BASES IN PERCHLORIC-ACID CELL-EXTRACTS

An ion-pairing high performance liquid chromatographic method for the direct and simultaneous determination of nucleotides, deoxynucleotides , cAMP, nicotinic coenzymes, oxypurines, nucleosides, and bases in per chloric acid cell extracts is presented. By using an Alltima C-18, 250 X 4.6-mm, 5-mu m particle size column, a high resolution of 38 acid-s oluble compounds, including ATP, GTP, dTTP, CTP, UTP, ADP, GDP, dTDP, CDP, UDP, dATP, dGTP, dCTP, dUTP, dADP, dGDP, dCDP, dUDP, and cAMP, is obtained. Elution is performed with a step gradient from buffer A (co nsisting of 10 mM tetrabutylammonium hydroxide, 10 mM KH2PO4, 0.25% me thanol, pH 7.00) to buffer B (consisting of 2.8 mM tetrabutylammonium hydroxide, 100 mM KH2PO4, 30% methanol, pH 5.50). Perchloric acid extr acts of resting and phytohemagglutinin-stimulated human lymphocytes we re analyzed. Data indicate that this chromatographic method offers, fo r the first time to the best of our knowledge, the possibility of simu ltaneously determining di- and triphosphate nucleosides and their corr esponding deoxynucleosides without any chemical manipulation of sample s except for perchloric acid deproteinization. Hence, the present HPLC assay minimizes the risks of modification or loss of metabolite conce ntration and allows one to obtain, with a single chromatographic run, the complete pattern of those metabolites which are known to be involv ed in energy metabolism and in DNA and RNA synthesis, resulting theref ore of great advantage in cell biology studies. (C) 1995 Academic Pres s, Inc.