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Abstract
A simple, fast, and sensitive, isocratic high-performance liquid chromatography technique was developed for the determination of adenine nucleotides (ATP, ADP, AMP) in biological tissues. In conjunction with this technique, a new method of dissolving biological samples and extraction of nucelotides was introduced, using a tissue solubilizer, TMAH (tetramethylammonium hydroxide). The adenine nucleotides were separated on a C-18 column, eluted isocratically with a solvent system consisting of 0.15 M potassium dihydrogen phosphate and disodium hydrogen phosphate, and water at a 60:40 ratio, using an ultra-violet detector at 254 nm. The HPLC technique has sensitivity in the picomol level and is suitable for nucleotide pool and adenylate energy charge calculations. There was no interference from other nucleotides such as the cyclic AMP's, adenine and adenosine. Guanosine diphosphate and guanosine triphosphate gave peaks in the chromatograms but did not interfere.