Simultaneous determination of purine nucleotides, their metabolites and beta-nicotinamide adenine dinucleotide in cerebellar granule cells by ion-pair high performance liquid chromatography.

The method described here allows the quantitative simultaneous determination of adenosine 5'-triphosphate, adenosine 5'-diphosphate, adenosine 5'-monophosphate, adenosine, guanosine 5'-triphosphate, guanosine 5'-diphosphate, guanosine, inosine 5'-monophosphate, inosine, uric acid, xanthine, hypoxanthine and beta-nicotinamide adenine dinucleotide by ion-pair high performance liquid chromatography. The chromatographic analysis requires 26 min per sample and allows the separation of the mentioned metabolites in a time as short as 16 min. Primary cultures of rat cerebellar granule cells were incubated in serum-free medium containing 25 mM KCl for 1.5-48 h and their acid extracts were injected onto column. Uric acid, inosine 5'-monophosphate, inosine, beta-nicotinamide adenine dinucleotide, adenosine, adenosine 5'-monophosphate, guanosine 5'-diphosphate, adenosine 5'-diphosphate, guanosine 5'-triphosphate and adenosine 5'-triphosphate were identified and quantified, while hypoxanthine, xanthine and guanosine were below the detection limit. This method makes use of a single-step sample pre-treatment procedure which allows a greater than 91% recovery of the compounds of interest and provides the assay of the metabolites of interest in little amounts of cell extracts. Therefore, this method is suitable to evaluate the energetic state in a variety of cell types, both under normal and dismetabolic conditions, such as after the induction of apoptosis or necrosis.