IMPROVED ION-PAIR HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC METHOD FOR THE QUANTIFICATION OF A WIDE VARIETY OF NUCLEOTIDES AND SUGAR NUCLEOTIDES IN ANIMAL-CELLS

An improved method including extraction procedures is presented for the analysis of nucleotides in suspension-cultivated animal cells. Quantification was performed by ion-pair high-performance liquid chromatography after perchloric acid extraction. It was found that the amount of perchloric acid taken for extraction influenced the yield and that cell washing procedures caused deterioration of the analysis results for triphosphates. More than thirty nucleotides and sugar-nucleotides were separated within 25 min using a Supelcosil reversed-phase column (3-mu-m) with tetrabutylammonium hydrogensulphate as pairing agent and methanol-pH gradient elution. Cultivated hybridoma cells showed variations in intracellular nucleotide concentrations as well as relative amounts during different growth phases, which could reflect the physiological state of a cell culture.