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<p>Nicotinamide adenine dinucleotide (NAD<sup>+</sup>) is an essential metabolite contributing to cellular energy needs and its decline is associated with age-related disorders. Comprehensive analysis of the NAD<sup>+</sup> landscape following NAD<sup>+</sup> supplementation therapies would provide a broader understanding of impacts on NAD<sup>+</sup> pathway biology. However, the analysis of NAD<sup>+</sup> 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<sup>+</sup> 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<sup>+</sup> pathway profiling in NAD-Glo samples. A strong correlation (R<sup>2</sup> = 0.94) was demonstrated between the two assays for tissue NAD<sup>+</sup> measurements in mice treated with NAM supplementation. The LC-MS/MS and NAD-Glo data confirmed dose-dependent NAD<sup>+</sup> boosting in mice lung and skin tissues after NAM treatment. In addition, LC-MS/MS analysis revealed that the highest dose of NAM (900&#xa0;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<sup>+</sup> metabolome.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年4月23日

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  • Omprakash Nacham,
  • Jordan W. Brown,
  • Mohammad Mehdi Maneshi,
  • Victoria Kurschner,
  • Mike Sheehan,
  • Renee Sadowski,
  • Chris Ling,
  • Nari Talaty,
  • Robert Johnson,
  • Andrew M. Swensen

摘要

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.