Selective determination of hydroxyproline in urine by high-performance liquid chromatography using precolumn derivatization.
A method to measure total hydroxyproline in human urine was developed. Primary amino acids were derivatized with ortho-phthaldialdehyde, followed by derivatization of imino acids with 9-fluorenylmethyl chloroformate. The fluorescent 9-fluorenylmethyl chloroformate derivatives were separated by reversed phase high-performance liquid chromatography. 3,4-Dehydroproline was used as internal standard. Calibration curves for hydroxyproline and internal standard were linear from 1 to 200 pmol injected. Both within- and between-run precision were below 3.2%. Analytical recovery of hydroxyproline added to urine samples was 99.2 +/- 2.6%. Values for excreted hydroxyproline were determined by analysis of urine samples from adult volunteers. The hydroxyproline/creatinine ratio was found to be 15.8 +/- 4.6 mmol/mol (range 7.0-27.3) with no significant sex-related difference.
Teerlink
T
T
Department of Clinical Chemistry, Free University Hospital, Amsterdam, The Netherlands.
Tavenier
P
P
Netelenbos
J C
JC
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005449:Fluorenes; D006909:Hydroxyproline / Q000031:analogs & derivatives / Q000652:urine*; D007202:Indicators and Reagents
D005449:Fluorenes; D007202:Indicators and Reagents; C052812:1-(9-fluorenyl)ethyl chloroformate; D006909:Hydroxyproline