Nucleotide bases are parts of DNA and RNA. Adenine and guanine are purine-bases; uracil, thymine and cytosine are pyrimidine-bases. In the view of chromatography these compounds are very polar and similar in properties. It is impossible to retain and obtain base-line separation for this compounds on traditional reversed-phase core-shell column. In this application, nucleobases are well retained and separated on Coresep 100 core-shell mixed-mode column. Compounds are retained by weak reverse phase and weak ion-exchange mechanisms, but synergy of two mechanism provides efficient separation with excellent peak shape, symmetry and efficiency. All five compounds are separated within one minute. Method can utilize UV, ELSD, and LC/MS detection and can be applied for fast and efficient method using regular HPLC system.