Abstract: We established a quantitative method to determine the content of the triple helix conformation of collagen.This method based on using High Performance Liquid Chromatography (HPLC) coupled with precolumn derivatization assay to determine the variation of hydroxyproline (Hyp) content subsequent to digestion with trypsin.It was evaluated the effect of digestion time (0~48 h),enzyme/substrate ratios (1∶100,1∶50 and 1∶20) and temperature (20,25,30 and 37 ℃) on the degradation of gelatin.The optimal enzymatic hydrolysis conditions were obtained with 1∶50 ratio of trypsin/substrate,being agitated at 25 ℃ for 3 h.Application of the method to determine mixture of gelatin and collagen solution showed that the method was precise(<10% RSD) for determining the content of triple helix conformation.This method is useful not only in the field of native tissue research,but also the quality assessment of collagen food,health food and bioengineered tissues.

Key words: active, collagen, quantitative determination, triple helix conformation, enzymatic sensitivity