Liquid chromatography/luminescence techniques.

Area of Science:

• Analytical Chemistry
• Biochemistry

Background:

• High-performance liquid chromatography (HPLC) with fluorimetric detection is used for catecholamine (CA) analysis.
• The trishydroxyindole (THI) post-column reaction is common but has limitations for dopamine (DA) detection in plasma due to weaker fluorescence intensity.

Purpose of the Study:

• To describe and evaluate improved HPLC techniques for sensitive and selective catecholamine (CA) analysis in biological samples.
• To address the challenge of detecting dopamine (DA) in plasma using HPLC.

Main Methods:

• Evaluation of post-column reaction techniques, specifically trishydroxyindole (THI) formation.
• Development and application of a novel pre-column derivatization method using 1,2-diphenylethylenediamine (DPE) after cation-exchange clean-up.
• Modification of the THI technique with electrochemical oxidation.

Main Results:

• The DPE pre-column method achieved detection limits of approximately 2 fmol for norepinephrine (NE), epinephrine (E), and dopamine (DA) at a signal-to-noise ratio of 2.
• This DPE method enables the determination of DA in plasma, which was previously difficult.
• Both evaluated methods demonstrate high sensitivity and selectivity for CA measurement.

Conclusions:

• The 1,2-diphenylethylenediamine (DPE) pre-column derivatization method offers a sensitive and selective approach for analyzing catecholamines (CAs), including dopamine (DA), in biological samples like plasma.
• Modified trishydroxyindole (THI) techniques also provide sensitive and selective CA measurements.
• These advanced HPLC techniques enhance the routine analysis of critical biomolecules.