Simultaneous detection and quantification of adenine nucleotides in mammalian cultured cells by HPLC

Adenine nucleotides, such as ATP, ADP, ADP-ribose, AMP, NAD ⁺ and NADH, have central roles in cellular homeostasis and participate in several pathways. Because of their diverse roles in cell biology, accurately quantifying these metabolites is paramount for several research areas such as bioenergetics, signaling, cancer and others. There are several methods available to analyze adenine nucleotides, ranging from enzymatic assays to mass spectrometry approaches, however, to our knowledge, no method in the literature quantifies these six adenine nucleotides simultaneously, mostly due to limitations in sample preparation or detection methods. Here, we developed a method that can detect simultaneously ATP, ADP, ADP-ribose, AMP, NAD ⁺ and NADH using RP-HPLC with UV-Vis detection. This methodological approach is simple, sensitive to physiological concentrations of all nucleotides and detects biologically significant variations in ATP, AMP and NAD ⁺ induced by pharmacological treatments, presenting an accessible and accurate alternative for quantification of these nucleotides in biological samples.