Abstract: A method for determining collagen in tendon by reversed-phase high performance liquid chromatography (RP-HPLC) was developed. After hydrolysis with hydrochloric acid, the collagen in samples was decomposed into hydroxyproline which hydroxyproline can be derivatized with 2，4-dinitrochlorobenzene for the determination by HPLC (reversed-phase C18 column, 0.01 mol/L NaAc-HAc（pH 6.0 ）-CH3CN （80∶20, v/v） as mobile phase, detection at 360 nm). The factors influencing hydrolysis, derivatization and HPLC analysis were studied and optimized. Sixty samples were analyzed with the proposed method. The linear range was from 3 μg/L to 100 mg/L and the detection limit was 3 μg/L. The relative standard deviation (RSD) of determination was 1.95%. The recoveries of spiked samples were 98.4%-110.8%. The results show that the method is sensitive, accurate and suitable for tendon determination.

Key words: collagen, hydroxyproline, tendon , high performance liquid chromatography (HPLC)