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 (R = 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.