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.
Welch
R W
RW
Laboratory of Cell Biology and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Acworth
I
I
Levine
M
M
D009327:4-Chloro-7-nitrobenzofurazan; D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D003094:Collagen / Q000302:isolation & purification; D004563:Electrochemistry / Q000379:methods* / Q000706:statistics & numerical data; D005069:Evaluation Studies as Topic; D005347:Fibroblasts / Q000378:metabolism; D006909:Hydroxyproline / Q000032:analysis* / Q000378:metabolism; D012680:Sensitivity and Specificity
D003094:Collagen; D009327:4-Chloro-7-nitrobenzofurazan; D001205:Ascorbic Acid; D006909:Hydroxyproline