A liquid chromatography-tandem mass spectrometry based method for the quantification of adenosine nucleotides and NAD precursors and products in various biological samples.

Adenine nucleotides (AN) are ubiquitous metabolites that regulate cellular energy metabolism and modulate cell communication and inflammation. To understand how disturbances in AN balance arise and affect cellular function, robust quantification techniques for these metabolites are crucial. However, due to their hydrophilicity, simultaneous quantification of AN across various biological samples has been challenging. Here we present a hydrophilic interaction high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) based method for the quantification of 26 adenosine nucleotides and precursors as well as metabolic products of nicotinamide adenine dinucleotide (NAD) in plasma, liver, and adipose tissue samples as well as cell culture supernatants and cells. Method validation was performed with regard to linearity, accuracy, precision, matrix effects, and carryover. Finally, analysis of cell culture supernatants derived from intestinal organoids and RAW 264.7 cells illustrates that the here described method is a reliable and easy-to-use tool to quantify AN and opens up new avenues to understand the role of AN generation and breakdown for cellular functions.