Abstract: OBJECTIVE To establish a general LC-MS method for the separation and identification of nucleoside and nucleotide compounds.METHODS The ODS-AQ column was picked, the mobile phase A was buffer 1(120 mmol·L^-1 Na₂HPO₄, 120 mmol·L^-1 KH₂PO₄ and 3 mmol·L^-1 TBAOH), the mobile phase B was water, and the mobile phase C was methanol, gradient elution. The 16 nucleoside and nucleotide compounds were separated and analyzed. Electrospray ionization (ESI) detection was adopted, spray voltage was 4 000 V (positive ion mode)/3 500 V(negative ion mode), atomizing gas pressure was 45 psi, drying gas flow rate was 10 L·min^-1, and solvent removal temperature was 350 ℃. The fragmentation voltage was 180 V, and the collision energy was set to 5-30 eV. Mass spectrometry fragments of 4 different types of nucleoside and nucleotide compounds were analyzed.RESULTS The established LC method achieved baseline separation(R>2.0) for 16 representative nucleoside and nucleotide compounds. Based on the mass spectrum fragmentation rule, a mass spectrometry platform for nucleoside and nucleotide compounds was constructed.CONCLUSION The established LC-MS method can provide reference for the separation and identification of unknown nucleoside and nucleotide compounds.