A fluorometric determination of urinary 17-hydroxycorticosteroids using benzamidine

Area of Science:

• Biochemistry
• Analytical Chemistry
• Clinical Chemistry

Background:

• Urinary 17-hydroxycorticosteroids are important biomarkers for adrenal function.
• Accurate and sensitive measurement methods are crucial for clinical diagnosis.
• Existing methods like the Porter-Silber reaction have limitations.

Purpose of the Study:

• To develop and validate a novel fluorometric method for quantifying urinary 17-hydroxycorticosteroids.
• To compare the performance of the new method with the established Porter-Silber reaction.
• To assess the sensitivity, recovery, and precision of the fluorometric assay.

Main Methods:

• Fluorometric determination using benzamidine reaction with dihydroxyacetone side chain compounds.
• Enzymatic hydrolysis with beta-glucuronidase and extraction with methylene chloride.
• Fluorescence generation in alkaline solution (pH 13.4) with excitation/emission maxima at 370/480 nm.

Main Results:

• High correlation (0.945) with the Porter-Silber reaction, indicating good agreement.
• Sensitivity of 0.5 microgram/ml and mean cortisol recovery of 95%.
• Low coefficient of variation (6.2%) demonstrating high precision.

Conclusions:

• The described fluorometric method provides a sensitive, specific, and precise alternative for urinary 17-hydroxycorticosteroid measurement.
• This assay shows excellent correlation with the Porter-Silber reaction, suggesting clinical utility.
• The method's performance characteristics support its application in routine clinical analysis.