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.

ATP 及其降解产物是重要的代谢和信号分子。传统上，它们是通过高效液相色谱 (HPLC) 和紫外-可见光检测并利用磷酸盐缓冲液流动相进行定量，但这种方法与现代质量检测不兼容。本研究的目标是开发一种无需磷酸盐缓冲液的超高效液相色谱 (UPLC) 方法，以便同时使用紫外-可见光和质谱 (MS) 分析腺嘌呤核苷酸。最终条件使用带有挥发性醋酸铵缓冲液的 Acquity HSS T3 Premier 色谱柱，成功分离和定量标准混合物以及非收缩和收缩小鼠后肢肌肉提取物中的 ATP 相关分析物。所有 10 种代谢物均实现了基线分辨率，并且使用 UV-Vis 观察到大多数代谢物每次注射的定量下限低至 1 pmol。因此，该方法可以通过 UV-Vis 可靠地定量腺嘌呤核苷酸及其降解产物，并通过 MS 确认和/或识别未知峰。