chapter  Section II.X
The Determination of Proline and Hydroxyproline By Derivatization with 4-Chloro- or 4-Fluoro-7-Nitrobenzofurazan (NBD-C1 and NBD-F)
WithStanley Blackburn
Pages 4

The hydroxyprolines are the most characteristic amino acids found in collagens. 4-Hydroxyproline is a marker for all types of collagens, while 3-hydroxyproline is a marker for basement membrane collagen. The reaction rates of proline and hydroxyproline with 4-Chloro-7-nitrobenzofurazan (NBD-C1) are one order of magnitude higher than those of primary amino acids; hydroxyproline reacts quantitatively with NBD-C1 under milder conditions than do primary amines. The high quantum efficiency in medium polar solvents and the high absorptivity means that fluorescence is a very suitable method for the detection of compounds such as NBD-hydroxyproline. The NBD technique for the determination of hydroxyproline isomers is far more sensitive than many ion exchange methods, allowing for the accurate measurement of picomole amounts of amino acids. H. Yoshida et al. have described the application of NBD-C1 to the postcolumn detection of picomole quantities of primary and secondary amino acids.