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

•Baseline separation of ATP and its 9 degradation products with reverse phase UPLC.•Volatile buffers allow simultaneous detection by UV–Vis and MS.•Phosphate acid wash and iron chelator are required to limit peak tailing.•Applicable to complex matrix of contracted skeletal muscle extracts. 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.