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...
Saved in:
| Main Authors: | , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Nature Portfolio
2025-04-01
|
| Series: | Scientific Reports |
| Subjects: | |
| Online Access: | https://doi.org/10.1038/s41598-025-97834-2 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1850172858481770496 |
|---|---|
| author | Omprakash Nacham Jordan W. Brown Mohammad Mehdi Maneshi Victoria Kurschner Mike Sheehan Renee Sadowski Chris Ling Nari Talaty Robert Johnson Andrew M. Swensen |
| author_facet | Omprakash Nacham Jordan W. Brown Mohammad Mehdi Maneshi Victoria Kurschner Mike Sheehan Renee Sadowski Chris Ling Nari Talaty Robert Johnson Andrew M. Swensen |
| author_sort | Omprakash Nacham |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-d6c57052f7b6435480cbb517e1f9a594 |
| institution | OA Journals |
| issn | 2045-2322 |
| language | English |
| publishDate | 2025-04-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj-art-d6c57052f7b6435480cbb517e1f9a5942025-08-20T02:19:58ZengNature PortfolioScientific Reports2045-23222025-04-0115111110.1038/s41598-025-97834-2A mixed-mode LC-MS-based method for comprehensive analysis of NAD and related metabolites from biological sample matricesOmprakash Nacham0Jordan W. Brown1Mohammad Mehdi Maneshi2Victoria Kurschner3Mike Sheehan4Renee Sadowski5Chris Ling6Nari Talaty7Robert Johnson8Andrew M. Swensen9AbbVieAbbVieAbbVieAbbVieAbbVieAbbVieAbbVieAbbVieAbbVieAbbVieAbstract 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.https://doi.org/10.1038/s41598-025-97834-2Nicotinamide adenine dinucleotideNicotinamideNAD-GloTargeted mass spectrometryMixed mode chromatographyNADomics |
| spellingShingle | Omprakash Nacham Jordan W. Brown Mohammad Mehdi Maneshi Victoria Kurschner Mike Sheehan Renee Sadowski Chris Ling Nari Talaty Robert Johnson Andrew M. Swensen A mixed-mode LC-MS-based method for comprehensive analysis of NAD and related metabolites from biological sample matrices Scientific Reports Nicotinamide adenine dinucleotide Nicotinamide NAD-Glo Targeted mass spectrometry Mixed mode chromatography NADomics |
| title | A mixed-mode LC-MS-based method for comprehensive analysis of NAD and related metabolites from biological sample matrices |
| title_full | A mixed-mode LC-MS-based method for comprehensive analysis of NAD and related metabolites from biological sample matrices |
| title_fullStr | A mixed-mode LC-MS-based method for comprehensive analysis of NAD and related metabolites from biological sample matrices |
| title_full_unstemmed | A mixed-mode LC-MS-based method for comprehensive analysis of NAD and related metabolites from biological sample matrices |
| title_short | A mixed-mode LC-MS-based method for comprehensive analysis of NAD and related metabolites from biological sample matrices |
| title_sort | mixed mode lc ms based method for comprehensive analysis of nad and related metabolites from biological sample matrices |
| topic | Nicotinamide adenine dinucleotide Nicotinamide NAD-Glo Targeted mass spectrometry Mixed mode chromatography NADomics |
| url | https://doi.org/10.1038/s41598-025-97834-2 |
| work_keys_str_mv | AT omprakashnacham amixedmodelcmsbasedmethodforcomprehensiveanalysisofnadandrelatedmetabolitesfrombiologicalsamplematrices AT jordanwbrown amixedmodelcmsbasedmethodforcomprehensiveanalysisofnadandrelatedmetabolitesfrombiologicalsamplematrices AT mohammadmehdimaneshi amixedmodelcmsbasedmethodforcomprehensiveanalysisofnadandrelatedmetabolitesfrombiologicalsamplematrices AT victoriakurschner amixedmodelcmsbasedmethodforcomprehensiveanalysisofnadandrelatedmetabolitesfrombiologicalsamplematrices AT mikesheehan amixedmodelcmsbasedmethodforcomprehensiveanalysisofnadandrelatedmetabolitesfrombiologicalsamplematrices AT reneesadowski amixedmodelcmsbasedmethodforcomprehensiveanalysisofnadandrelatedmetabolitesfrombiologicalsamplematrices AT chrisling amixedmodelcmsbasedmethodforcomprehensiveanalysisofnadandrelatedmetabolitesfrombiologicalsamplematrices AT naritalaty amixedmodelcmsbasedmethodforcomprehensiveanalysisofnadandrelatedmetabolitesfrombiologicalsamplematrices AT robertjohnson amixedmodelcmsbasedmethodforcomprehensiveanalysisofnadandrelatedmetabolitesfrombiologicalsamplematrices AT andrewmswensen amixedmodelcmsbasedmethodforcomprehensiveanalysisofnadandrelatedmetabolitesfrombiologicalsamplematrices AT omprakashnacham mixedmodelcmsbasedmethodforcomprehensiveanalysisofnadandrelatedmetabolitesfrombiologicalsamplematrices AT jordanwbrown mixedmodelcmsbasedmethodforcomprehensiveanalysisofnadandrelatedmetabolitesfrombiologicalsamplematrices AT mohammadmehdimaneshi mixedmodelcmsbasedmethodforcomprehensiveanalysisofnadandrelatedmetabolitesfrombiologicalsamplematrices AT victoriakurschner mixedmodelcmsbasedmethodforcomprehensiveanalysisofnadandrelatedmetabolitesfrombiologicalsamplematrices AT mikesheehan mixedmodelcmsbasedmethodforcomprehensiveanalysisofnadandrelatedmetabolitesfrombiologicalsamplematrices AT reneesadowski mixedmodelcmsbasedmethodforcomprehensiveanalysisofnadandrelatedmetabolitesfrombiologicalsamplematrices AT chrisling mixedmodelcmsbasedmethodforcomprehensiveanalysisofnadandrelatedmetabolitesfrombiologicalsamplematrices AT naritalaty mixedmodelcmsbasedmethodforcomprehensiveanalysisofnadandrelatedmetabolitesfrombiologicalsamplematrices AT robertjohnson mixedmodelcmsbasedmethodforcomprehensiveanalysisofnadandrelatedmetabolitesfrombiologicalsamplematrices AT andrewmswensen mixedmodelcmsbasedmethodforcomprehensiveanalysisofnadandrelatedmetabolitesfrombiologicalsamplematrices |