Two nucleotides containing different sugar fragments were separated on Primesep SB mixed-mode HPLC column using LC/MS compatible conditions. Method can be used for analysis and isolation of guanosine diphosphate mannose and guanosine diphosphate fucose. Mechanism of retention is weak reversed-phase and strong anion-exchange. retention is controlled by buffer concentration and buffer pH. Both compounds exhibit strong interaction providing good retention and separation.