Analysis for indole compounds in urine by high-performance liquid chromatography with fluorometric detection

Area of Science:

• Analytical Chemistry
• Biochemistry

Background:

• Indole compounds are crucial biomarkers in various physiological and pathological processes.
• Accurate and sensitive measurement of indoles in biological samples like urine is essential for clinical diagnostics and research.

Purpose of the Study:

• To develop and validate a robust chromatographic method for the quantitative analysis of indoles in human urine.
• To enhance the sensitivity and selectivity of indole detection using fluorescence detection.

Main Methods:

• Utilized a high-performance chemically-bonded reverse-phase column for indole separation.
• Optimized mobile phase composition (methanol content and pH) to control retention and selectivity.
• Employed fluorescence detection for sensitive quantification of indoles.
• Implemented a single urine-deproteinization step for sample preparation.

Main Results:

• Achieved separation of six reference indoles within 20 minutes.
• Successfully eluted three 5-hydroxyindole derivatives in under 7 minutes.
• Demonstrated a detection limit of approximately 5-15 ng for aqueous indole solutions.
• Enabled the analysis of five out of six target indoles in urine samples post-deproteinization.

Conclusions:

• The developed chromatographic system offers efficient and selective analysis of indoles in urine.
• The combination of reverse-phase chromatography and fluorescence detection provides a sensitive method for indole biomarker studies.
• This method simplifies sample preparation, making it suitable for routine clinical and research applications.