Abstract: Isosorbide dinitrate (ISDN) and its degradation products. isosorbide-5-mononitrate(5-ISMN) and isosorbide-2-mononitrate (2-ISMN), in a spray formulation were separated and determined by step gradient RPHPLC Within 8 minutes. A Shimadzu LC-4A liquid chromatograph, Nucleosil C₁₈ 5μm 150×4.6 mm (ID)column, SPD 2AS spectrophotometric detector at 220 nm With variable attenuation were used, the sensitivity was kept at 0.16 AUFS in the first 4.5 min and changed to 2.56 AUFS during 4.5～12 min. Solution A (70% methanol) and solution B (5% methanol) were emp loyed as mobile phase at a flow rate of 1.0 ml/min in gradient elution. Good results could be obtained by CR 3A data module using external standard method. The recoveries were 98.20% (ISDN), 96.92% (5-ISMN) and 95.83% (2-ISMN), and the coefficients of correlation were 0.9990(ISDN), and 0.9994 (5-ISMN and 2-ISMN).