Simultaneous Quantification of Nucleosides and Nucleotides from Biological Samples

Area of Science:

• Biochemistry
• Analytical Chemistry
• Metabolomics

Background:

• Nucleosides and nucleotides are vital biomolecules involved in numerous cellular processes.
• Accurate quantification of these metabolites is crucial for biomarker discovery and understanding disease mechanisms.
• Existing methods may lack the sensitivity or scope for comprehensive analysis of these compounds in complex biological matrices.

Purpose of the Study:

• To develop and validate a robust liquid chromatography-mass spectrometry (LC-MS) method for the simultaneous quantification of a wide range of nucleosides and nucleotides.
• To establish a platform for high-throughput analysis of these metabolites in various biological samples.
• To demonstrate the utility of the method in identifying and quantifying these compounds for biomarker discovery.

Main Methods:

• Development of a reverse-phase liquid chromatography-mass spectrometry (LC-MS) method.
• Utilized a tier-wise approach for compound identification and external calibration for quantification.
• Validated the method using 65 authentic standards, determining limits of detection (LOD) and quantification (LOQ).

Main Results:

• The developed LC-MS method achieved sensitive detection and quantification of nucleosides and nucleotides.
• Successfully quantified 28, 22, and 23 nucleosides/nucleotides from human urine, human plasma, and rat liver, respectively.
• Identified a broad spectrum of these metabolites, with concentrations ranging from nmol/L to μmol/L levels across different sample types.

Conclusions:

• The developed LC-MS method provides a reliable and sensitive approach for simultaneous quantification of nucleosides and nucleotides in biological samples.
• This method is suitable for large-scale metabolomic studies, facilitating biomarker discovery and the elucidation of disease mechanisms.
• The platform enables comprehensive analysis of the metabolome, contributing to a deeper understanding of biological systems.