A quantitative gas-liquid chromatographic (GLC) assay of hydroxyproline was developed. Sample analysis was readily achieved by adding blood serum or urine acid hydrolyzate into a micro-vial containing a known amount of an internal standard for the direct formation of N-trifluoroacetyl, n-butyl ester derivatives and GLC injection without laborious cleanup. Specificity (using the nitrogen detection system), sensitivity (detection in the nanogram range), and speed (less than 30 min) of the developed procedure can be used for the rapid determination of hydroxyproline in physiological fluids. An accelerated hydrolysis method (145° for 4 h) was evaluated for the determination of total urinary hydroxyproline. A low hydroxyproline yield resulting from incomplete hydrolysis of some segments of the collagen peptides present in the urine by the "overnight" hydrolysis process was demonstrated. Recovery of hydroxyproline added to various urine samples and analyzed after acid hydrolysis exceeded 90%. © 1973.
Mee, J. M. L. (1973). Specific assay of hydroxyproline by gas chromatography. Journal of Chromatography A, 87(1), 155–161. https://doi.org/10.1016/S0021-9673(01)91528-6