Simultaneous Detection and Quantification of Adenine Nucleotides in Mammalian Cultured Cells by HPLC

Abstract Adenine nucleotides, including ATP, ADP, ADP-ribose, AMP, NAD+, and NADH, play central roles in cellular homeostasis and are involved in multiple metabolic and signaling pathways. Owing to their broad functional relevance in cell biology, the accurate quantification of these metabolites is essential for diverse research areas such as bioenergetics, cell signaling, and cancer biology. Several analytical methods have been described for the measurement of adenine nucleotides, ranging from enzymatic assays to mass spectrometry-based approaches. In this study, we developed a reverse-phase high-performance liquid chromatography (RP-HPLC) method with UV–vis detection that enables the simultaneous quantification of ATP, ADP, ADP-ribose, AMP, NAD+, and NADH. This method is simple, sensitive within the physiological concentration range of all analytes, and capable of detecting biologically relevant changes in ATP, AMP, and NAD+ levels induced by pharmacological treatments. Therefore, it presents an accessible and reliable alternative for the quantification of these nucleotides in biological samples.