Selective determination of hydroxyproline in urine by high-performance liquid chromatography using precolumn...

Area of Science:

• Biochemistry
• Analytical Chemistry
• Clinical Chemistry

Background:

• Hydroxyproline is a key component of collagen, and its urinary excretion reflects collagen turnover.
• Accurate measurement of urinary hydroxyproline is crucial for diagnosing and monitoring conditions involving altered collagen metabolism.
• Existing methods may lack the sensitivity or precision required for comprehensive analysis.

Purpose of the Study:

• To develop and validate a novel, highly sensitive method for quantifying total hydroxyproline in human urine.
• To establish the reliability and precision of the developed analytical technique.
• To determine reference ranges for urinary hydroxyproline excretion in healthy adults.

Main Methods:

• Development of a dual derivatization strategy: ortho-phthaldialdehyde for primary amines and 9-fluorenylmethyl chloroformate for imino acids.
• Separation and quantification of fluorescent derivatives using reversed-phase high-performance liquid chromatography (RP-HPLC).
• Utilization of 3,4-dehydroproline as an internal standard for enhanced accuracy and precision.

Main Results:

• The method demonstrated linear calibration for hydroxyproline and internal standard from 1 to 200 pmol.
• Excellent precision was achieved, with within- and between-run variability below 3.2%.
• High analytical recovery (99.2 +/- 2.6%) confirmed the method's accuracy for urine samples. The mean hydroxyproline/creatinine ratio in adult volunteers was 15.8 +/- 4.6 mmol/mol, with no significant sex difference.

Conclusions:

• A robust and precise HPLC-based method for total urinary hydroxyproline measurement has been successfully developed.
• The validated method offers high sensitivity and accuracy, suitable for clinical applications.
• Established reference values provide a baseline for assessing collagen metabolism in human populations.