Abstract: The reverse phase ion pair chromatography was optimized to detect 4-purine bases and 3-purine nucleosides in the wort. The average recovery of the seven compounds was 95. 13% ～ 101. 12% and the relative standard deviation was 0. 10% ～ 6. 55%. The effect of mashing process on the content of purine bases were investigated by the orthogonal experimental design on SPSS 18. 0. The results showed that ratio of material to water (A) , mashing temperature (B) , proteolysis time (C) and proteolysis temperature (D) significantly influenced the conent of total free purine bases in wort. And their order of importance was B>A>C>D. The optimal conditions were ratio of material to water 1:2. 5, proteolysis temperature 45℃, proteolysis time 50min and mashing temperature 62℃. The content of total free purine bases increased from 43. 82mg/L to 61. 39mg/L, meanwhile, the content of purine nucleosides reduced from 55. 46mg/L to 29. 21mg/L at the optimum process.