Coulometric electrochemical detection of hydroxyproline using 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole.

A new method for detection of hydroxyproline has been developed. Hydroxyproline was derivatized using 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole; separation and detection were accomplished using HPLC with coulometric electrochemical detection. Derivatized hydroxyproline was initially detected using a 16 channel coulometric electrochemical array system. The assay was slightly modified for use with a simpler 2-channel coulometric electrochemical detector. Both detectors are sensitive into the upper fmol range. Derivatization of hydroxyproline occurs in 5 min and the derivative is stable for several hours at 0 degrees C. The techniques were used to quantitate hydroxyproline in purified type I collagen and other biological samples.