Native fluorescent detection with sequential injection chromatography for doping control analysis

Area of Science:

• Analytical Chemistry
• Chromatographic Techniques

Background:

• Sequential Injection Chromatography (SIC) is a separation technique known for reagent efficiency, speed, and simplicity.
• SIC coupled with UV detection is effective for pharmaceutical analysis.
• This study explores hyphenating SIC with a multi-wavelength fluorescence (FL) detector.

Purpose of the Study:

• To develop and validate a SIC-FL method for amiloride (AML) and furosemide (FSM) separation and quantification.
• To analyze AML and FSM in human urine and tablet formulations.
• To assess the method's sensitivity and efficiency.

Main Methods:

• Utilized a stand-alone multi-wavelength fluorescence detector coupled with an SIC system.
• Employed a C18 monolithic column for separation.
• Optimized mobile phase composition (phosphate buffer: acetonitrile, 35:65 v/v, pH 4.0).

Main Results:

• Achieved separation of AML and FSM in under 2.0 minutes.
• Determined optimal excitation/emission wavelengths for AML (380/413 nm) and FSM (270/470 nm).
• Established detection limits of 12 ng/mL for AML and 470 ng/mL for FSM.

Conclusions:

• The SIC-FL method demonstrates adequate sensitivity for AML and FSM in urine, meeting World Anti-Doping Agency standards.
• The method conserves reagents and offers high speed for industrial pharmaceutical analysis.
• This hyphenated technique provides an efficient analytical solution.