A mixed-mode LC-MS-based method for comprehensive analysis of NAD and related metabolites from biological sample matrices

Nicotinamide adenine dinucleotide (NAD⁺) is an essential metabolite contributing to cellular energy needs and its decline is associated with age-related disorders. Comprehensive analysis of the NAD⁺ landscape following NAD⁺ supplementation therapies would provide a broader understanding of impacts on NAD⁺ pathway biology. However, the analysis of NAD⁺ and its metabolites is challenging owing to their polar nature and low retention on reverse phase columns. We have developed and optimized a mixed-mode (reverse-phase/anion-exchange) chromatography-tandem mass spectrometry (LC-MS/MS) method for analysis of NAD⁺ precursors and their metabolic products from biological sample matrices. Attributes including mobile phase ionic strength and column temperature effects on LC-MS/MS performance were evaluated. Fit-for purpose method qualification was performed with regard to linearity, accuracy, and precision. The method described was developed to be compatible with NAD-Glo assay (bioluminescence-based plate reader assay) conditions for purposes of further validating NAD-Glo and allow for expanded NAD⁺ pathway profiling in NAD-Glo samples. A strong correlation (R² = 0.94) was demonstrated between the two assays for tissue NAD⁺ measurements in mice treated with NAM supplementation. The LC-MS/MS and NAD-Glo data confirmed dose-dependent NAD⁺ boosting in mice lung and skin tissues after NAM treatment. In addition, LC-MS/MS analysis revealed that the highest dose of NAM (900 mg/kg) significantly increased NR, NMN, ADPR, NAM, and m-NAM levels. Overall, we present an LC-MS/MS based orthogonal platform to confirm NAD-Glo data and show applicability of the method to more broadly evaluate the NAD⁺ metabolome.