Simultaneous Quantification of Nucleosides and Nucleotides from Biological Samples

We report a reverse phase chromatography mass spectrometry (LC-MS) method for simultaneous quantification of nucleosides and nucleotides from biological samples, where compound identification was achieved by a tier-wise approach and compound quantification was achieved via external calibration. A total of 65 authentic standards of nucleosides and nucleotides were used for the platform development. The limit of detection (LOD) of those compounds ranged from 0.05 nmol/L to 1.25 μmol/L, and their limit of quantification (LOQ) ranged from 0.10 nmol/L to 2.50 μmol/L. Using the developed method, nucleosides and nucleotides from human plasma, human urine, and rat liver were quantified. Seventy-nine nucleosides and nucleotides were identified from human urine and 28 of them were quantified with concentrations of 13.0 nmol/L–151 μmol/L. Fifty-five nucleosides and nucleotides were identified from human plasma and 22 of them were quantified with concentrations of 1.21 nmol/L–8.54 μmol/L. Fifty-one nucleosides and nucleotides were identified from rat liver and 23 were quantified with concentrations of 1.03 nmol/L–31.7 μmol/L. These results demonstrate that the developed method can be used to investigate the concentration change of nucleosides and nucleotides in biological samples for the purposes of biomarker discovery or elucidation of disease mechanisms.