Selective determination of urinary free cortisol by liquid chromatography after solid-state extraction

Area of Science:

• Analytical Chemistry
• Biochemistry
• Clinical Diagnostics

Background:

• Accurate measurement of urinary free cortisol is crucial for diagnosing endocrine disorders.
• Existing methods, such as radioimmunoassay kits, may have limitations in sensitivity or sample preparation.
• Development of a more precise and efficient chromatographic method is needed.

Purpose of the Study:

• To develop and validate a selective and precise high-performance liquid chromatographic (HPLC) method for quantifying urinary free cortisol.
• To improve the sample clean-up process for enhanced accuracy and efficiency.
• To compare the detection limits of the developed method with existing radioimmunoassay kits.

Main Methods:

• Developed a selective HPLC method utilizing C18 Sep-Pak cartridges for sample clean-up.
• Employed a sequential washing protocol with acetone-water, water, and hexane.
• Utilized diethyl ether for cortisol elution, followed by evaporation and redissolution.
• Optimized the mobile phase (tetrahydrofuran-methanol-water) for rapid and well-resolved cortisol elution.
• Performed isocratic chromatography on a reversed-phase column with UV detection at 254 nm.

Main Results:

• Achieved a selective and precise method for urinary free cortisol analysis.
• Demonstrated significantly improved sample clean-up through a double extraction process.
• Obtained detection limits for urinary free cortisol that were two to three times lower than commercial radioimmunoassay kits.
• Successfully applied the method in diagnosing patients with hypercortisolism and Cushing's syndrome.

Conclusions:

• The developed HPLC method provides a sensitive, precise, and efficient approach for urinary free cortisol determination.
• The improved sample clean-up procedure enhances analytical performance.
• This chromatographic method offers a valuable diagnostic tool for conditions characterized by cortisol excess.