The product of the first three steps of de novo pathway of pyremidine biosynthesis in mammalian cell catalyzed by carbamoyl phosphate synthetase- asparate transcarbamoylase - dihyroorotase are adenosine diphosphate (ADP) and dihydroorotate (DHO. ). And one of reactant is adenosine tiphosphate(ATP). A HPLC method for separation of ADP, ATP and DHO was developed. A Zarbox column (150mm×4.6mmI.D.) was used. The mobile phase were 0.01mol·L^-1 potassium dihydrogen phosphate buffer(pH4.7) and 0.5mol·L^-1potassium dihydrogen phosphate (pH4.6). The gradient was programmed at 0～5min, (φ=1, volume ratio)0.01mol ·L^-1KH~2PO~4 then 5～50 min, (φ=1,volume ratio) 0.01molL^- 1KH~2PO~4 to (φ=1,volume ratio) 0.5molL^-1KH~2PO~4. The flow rate was 0.7 mL·min^-1. The optimum deteation wavelenght and the sequence of ATP, ADP and DHO peaks were detected. The working cursor was plotted and then the corresponding concentration of ATP, ADP and DHO in the catalytic reaction were calculated.

Key words: ADENOSINE TRIPHOSPHATE (ATP), HIGH SPEED LIQUID CHROMATOGRAPHY, POLYPEPTIDE