Hydroxyproline Measurement by High Performance Liquid Chromatography: An Improved Method of Derivatization

Abstract

Hydroxyproline is a postsynthetic derivative of proline which is commonly used to estimate the collagen content of tissues. Values (weight %) of hydroxyproline range from ten to twenty percent in most collagen types, with Type I having 11.4 percent hydroxyproline. This fact is useful for estimating the collagen content in various acid hydrolyzed tissue samples. In this paper, the authors describe a technique which produces linear hydrolysis of collagen coupled with a sensitive ultraviolet detection scheme for the 9-Fluorenylmethoxycarbonyl Chloride(FMOC-Cl) derivative of hydroxyproline. The separation itself employs reverse phase chromatography with a sensitivity of approximately 0.12 nmoles of hydroxyproline or 0.14 μg Type I collagen.