A mixed-mode LC-MS-based method for comprehensive analysis of NAD and related metabolites from biological sample matrices
Abstract 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...
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| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Nature Portfolio
2025-04-01
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| Series: | Scientific Reports |
| Subjects: | |
| Online Access: | https://doi.org/10.1038/s41598-025-97834-2 |
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| Summary: | Abstract 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. |
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| ISSN: | 2045-2322 |