Mixed-Mode LC-MS Method Quantifies NAD+ and Its Metabolites: Preclinical Findings

Synopsis

Measuring NAD+ and its metabolites is technically difficult because these molecules are highly polar and poorly retained by standard chromatography. This study developed a robust mixed-mode LC-MS/MS method combining reverse-phase and anion-exchange separation to accurately quantify NAD+ precursors and metabolic products in biological samples. The method was validated for linearity, accuracy, and precision, and designed to be fully compatible with the NAD-Glo bioluminescence assay, allowing cross-platform confirmation. Strong correlation between assays (R² = 0.94) was demonstrated in mouse tissues after nicotinamide (NAM) supplementation. Both methods showed dose-dependent increases in tissue NAD+, while LC-MS/MS additionally revealed significant rises in NR, NMN, ADPR, NAM, and m-NAM at the highest NAM dose (900 mg/kg). NR and NMN supplementation also significantly increased NAD+ levels, further confirming their effectiveness as NAD+ precursors. This platform enables expanded and reliable profiling of the NAD+ metabolome, supporting research on NAD+-boosting therapies.

Journal

Scientific Reports