Abstract
High Performance Liquid Chromatography is the accepted method for the study of nucleotides in cell and tissue extracts. A variety of chromatographic separations have been described but to date the use of microparticulate anion exchange procedures remains the most popular. However the accurate analysis of cell extracts requires careful attention to both sample preparation and chromatographic operation. This study records the results of some 14 years experience and many thousands of analyses. Optimal conditions for the acid extraction of cellular nucleotides to maximise recovery, minimise nucleotide Interconversions, maximal removal of protein and excess acid extractant have been defined. For RBC's these are 1 part cells to 2.5 parts 12% TCA with acid removal with ether or Alamine /Freon. Protein removal was 99.8% efficient Cyclic nucleotides e.g cXMP are excellent internal standards. For the reliable separation of standard, normal and abnormal cell extracts by anion exchange chromatography over time with any one column, adherence to the following conditions was critical. Buffer pH needs to be adjusted to maintain difficult separations e.g. ADP/NADP, GMP/IMP, buffer molarity should be regularly reduced to counter column ageing and maintain resolution and potential interferences such as EDTA, Ficol-hypaque should be avoided.
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