An aqueous normal phase HPLC method coupled with UV or ESI/MS detection was used for the determination of a wide variety of nucleotides, essential in metabolomics studies. Fifteen nucleotides were tested in clinically relevant mixtures at levels of 100 microg/mL for UV detection and 1 microg/mL for ESI-MS detection. Analysis times for all protocols developed were less than 20 min. The chromatographic conditions were changed to achieve optimized retention and separation of the nucleotides studied. The aqueous normal phase-HPLC methods were developed utilizing two columns, one having a minimally modified hydride surface another having an undecanoic acid moiety on a hydride surface. Volatile, low ionic strength mobile phases were used. Negative ion mode ESI-MS at near neutral pH mobile phase, combined with a TOF detector provided a highly sensitive and specific method, which is equally suitable for quadrupole and ion trap instruments.