Liquid chromatography method for simultaneous quantification of ATP and its degradation products compatible with both UV-Vis and mass spectrometry.

ATP and its degradation products are essential metabolic and signaling molecules. Traditionally, they have been quantified via high-performance liquid chromatography (HPLC) with UV-Vis detection while utilizing phosphate buffer mobile phase, but this approach is incompatible with modern mass detection. The goal of this study was to develop an ultra-performance liquid chromatography (UPLC) method free of phosphate buffer, to allow for analysis of adenine nucleotides with UV-Vis and mass spectrometry (MS) simultaneously. The final conditions used an Acquity HSS T3 premier column with a volatile ammonium acetate buffer to successfully separate and quantify ATP-related analytes in a standard mixture and in extracts from non-contracted and contracted mouse hindlimb muscles. Baseline resolution was achieved with all 10 metabolites, and a lower limit of quantification down to 1 pmol per inject was observed for most metabolites using UV-Vis. Therefore, this method allows for the reliable quantification of adenine nucleotides and their degradation products via UV-Vis and their confirmation and/or identification of unknown peaks via MS.