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Rapid Analysis of NAD and Other Phosphorylated Metabolites in Complex Biological Samples by Hydrophilic Interaction Liquid Chromatography Coupled with Tandem Mass Spectrometry

Pravdova, Adela ; Kleinert, Maximilian ; Henderson, John ; Kafkia, Eleni ; Pladevall-Morera, David ; Yonamine, Caio Y ; Treebak, Jonas T ; Brodiazhenko, Tetiana ; Terenin, Ilya LU orcid and Zylicz, Jan Jakub , et al. (2026) In Analytical Chemistry 98(15). p.11428-11437
Abstract

Nucleotides and coenzymes play critical roles in energy metabolism and cellular signaling and as building blocks of nucleic acids. This work addresses the challenges in the measurement of the phosphorylated metabolites using hydrophilic interaction liquid chromatography coupled with mass spectrometry, which facilitates the separation and detection of polar metabolites. Here, we present optimized HILIC-MS/MS methods for rapid analysis of polar metabolites including nucleotides and their derivatives in complex biological matrices, such as murine adipose, skeletal, and liver tissues, human plasma, and bacteria. The developed methodologies enable separation of key nucleotides and other phosphorylated metabolites within 6 min and cofactors... (More)

Nucleotides and coenzymes play critical roles in energy metabolism and cellular signaling and as building blocks of nucleic acids. This work addresses the challenges in the measurement of the phosphorylated metabolites using hydrophilic interaction liquid chromatography coupled with mass spectrometry, which facilitates the separation and detection of polar metabolites. Here, we present optimized HILIC-MS/MS methods for rapid analysis of polar metabolites including nucleotides and their derivatives in complex biological matrices, such as murine adipose, skeletal, and liver tissues, human plasma, and bacteria. The developed methodologies enable separation of key nucleotides and other phosphorylated metabolites within 6 min and cofactors such as NAD+, NADH, NADP+, and NADPH within 4 min. Validation of these methods demonstrated high accuracy, precision, and sensitivity and stresses the substantial impact of matrix effects. The applicability of the methods was also tested on 13C-labeling experiments with mouse pluripotent stem cells. Additionally, sample pretreatment techniques, such as liquid-liquid extraction and solid-phase extraction, were evaluated as a tool to decrease the negative impact of matrix effects in complex samples. This work enhances the analytical capabilities for nucleotide quantification in metabolomics, facilitating the study of metabolic pathways and disease markers.

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organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Analytical Chemistry
volume
98
issue
15
pages
11428 - 11437
publisher
The American Chemical Society (ACS)
external identifiers
  • pmid:41949818
  • scopus:105036549666
ISSN
1520-6882
DOI
10.1021/acs.analchem.6c00721
language
English
LU publication?
yes
id
9216b968-0b05-46dc-a467-3d2c7e574fcb
date added to LUP
2026-04-14 07:11:05
date last changed
2026-06-10 14:26:36
@article{9216b968-0b05-46dc-a467-3d2c7e574fcb,
  abstract     = {{<p>Nucleotides and coenzymes play critical roles in energy metabolism and cellular signaling and as building blocks of nucleic acids. This work addresses the challenges in the measurement of the phosphorylated metabolites using hydrophilic interaction liquid chromatography coupled with mass spectrometry, which facilitates the separation and detection of polar metabolites. Here, we present optimized HILIC-MS/MS methods for rapid analysis of polar metabolites including nucleotides and their derivatives in complex biological matrices, such as murine adipose, skeletal, and liver tissues, human plasma, and bacteria. The developed methodologies enable separation of key nucleotides and other phosphorylated metabolites within 6 min and cofactors such as NAD+, NADH, NADP+, and NADPH within 4 min. Validation of these methods demonstrated high accuracy, precision, and sensitivity and stresses the substantial impact of matrix effects. The applicability of the methods was also tested on 13C-labeling experiments with mouse pluripotent stem cells. Additionally, sample pretreatment techniques, such as liquid-liquid extraction and solid-phase extraction, were evaluated as a tool to decrease the negative impact of matrix effects in complex samples. This work enhances the analytical capabilities for nucleotide quantification in metabolomics, facilitating the study of metabolic pathways and disease markers.</p>}},
  author       = {{Pravdova, Adela and Kleinert, Maximilian and Henderson, John and Kafkia, Eleni and Pladevall-Morera, David and Yonamine, Caio Y and Treebak, Jonas T and Brodiazhenko, Tetiana and Terenin, Ilya and Zylicz, Jan Jakub and Moritz, Thomas and Hodek, Ondrej}},
  issn         = {{1520-6882}},
  language     = {{eng}},
  number       = {{15}},
  pages        = {{11428--11437}},
  publisher    = {{The American Chemical Society (ACS)}},
  series       = {{Analytical Chemistry}},
  title        = {{Rapid Analysis of NAD and Other Phosphorylated Metabolites in Complex Biological Samples by Hydrophilic Interaction Liquid Chromatography Coupled with Tandem Mass Spectrometry}},
  url          = {{http://dx.doi.org/10.1021/acs.analchem.6c00721}},
  doi          = {{10.1021/acs.analchem.6c00721}},
  volume       = {{98}},
  year         = {{2026}},
}