Determination of Free 4-hydroxyproline with Dansylchloride by HPLC in Human Urine

Lee, Keou Weon; Lee, Kyung Jong; Cho, Young Bong

Abstract

OBJECTIVES
The level of 4-hydroxyproline (4-Hyp) in human urine was measured using high performance liquid chromatography (HPLC) with a fluorescence detector. This method is useful for medical examinations and investigating the radicals induced by physical, chemical, mental stresses. This method is superior to many published several methods in terms of its low cost and ability to analyze many samples.
METHODS
The urine from workers in a tire manufacturing company (22 male pre- and post-shift workers) and 18 office-workers as controls were analyzed. Data concerning age, the cumulative drinking amount and the cumulative smoking amount was collected with a questionnaire. The optimum applied amount of dansyl-Cl, the optimum reaction temperature and time, the recoveries and the optimum pH of the eluent and buffer were determined. 4-Hyp from human urine was derivatized with dansyl-Cl (dimethylamino-naphthalene-1-sulfonyl chloride) after removing the alpha-amino acid by a treatment with phthalic dicarboxaldehyde (OPA) and cleaned with Bond Elut C18 column. The 4-Hyp derivatives were separated on a reversed phase column by gradient elution with a phosphate buffer (5 m mol, pH 8.0) and acetonitrile, and detected by fluorescence measurements at 340 nm (excitation) and 538 nm (emission). RESUJLTS: The detection limit for the urinary free 4-Hyp was 0.364 micro mol/l. The recovery rate of 4-Hyp was 99.7 %, and the effective pH of the phosphate buffer and borate buffer were 3.0 and 8.0, respectively. From statistical analysis, age, drinking and smoking did not affect the urinary free 4-Hyp in both the controls and workers. The range of urinary 4-Hyp in the controls, pre-shift, and post-shift workers were 0.33-16.44, N.D-49.06, and 0.32-56.27 micro mol/l. From the pared-sample t-test, the urinary 4-Hyp levels in post-shift workers (11.82+/-16.73 n mol/mg Cre) were 2-fold higher than in pre-shift workers (5.36+/-5.53 n mol/mg Cre) and controls (4.91+/-4.89 n mol/mg Cre).
CONCLUSIONS
This method was developed with high sensitivity, accuracy, and precision. The present method was effectively applied to analyze the urinary free 4-Hyp in both controls and workers.