Reversed-phase high-performance liquid chromatographic analysis of hydroxyproline and proline from collagen by derivatization with dabsyl chloride.
A high-performance liquid chromatographic method for the analysis of hydroxyproline and proline has been developed. The method is based on the derivatization of the secondary amino group with dabsyl-chloride after blocking of the primary amino group with o-phthalaldehyde. Dabsyl-hydroxyproline and dabsyl-proline were separated from other amino acids by high-performance liquid chromatography in the gradient elution mode, and eluted at 10.27 and 16.02 min, respectively. The correlations between the peak areas of dabsyl-hydroxyproline and dabsyl-proline were linear in the range from 20-200 pmol, with equations y = 1.10x - 0.80 (r = 0.999) and y = 1.12x - 0.52 (r = 0.999), respectively. The method was applied to the analysis of rat tail collagen, and the contents of hydroxyproline and proline were 1.55 +/- 0.04 and 2.03 +/- 0.04 nmol/micrograms, respectively.
Ikeda
M
M
Laboratory of Medical Science, Dokkyo University School of Medicine, Tochigi, Japan.
Sorimachi
K
K
Akimoto
K
K
Yasumura
Y
Y
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003094:Collagen / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D006909:Hydroxyproline / Q000032:analysis*; D007202:Indicators and Reagents; D011392:Proline / Q000032:analysis*; D051381:Rats; D013623:Tail; D013710:Tendons / Q000737:chemistry; D009764:o-Phthalaldehyde / Q000008:administration & dosage / Q000494:pharmacology; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives*
D007202:Indicators and Reagents; C010572:4-N,N-dimethylaminoazobenzene-4'-sulfonyl chloride; D009764:o-Phthalaldehyde; D003094:Collagen; D011392:Proline; D004124:p-Dimethylaminoazobenzene; D006909:Hydroxyproline