Rapid Analysis of NAD and Other Phosphorylated Metabolites in Complex Biological Samples by Hydrophilic Interaction...

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.