Selective Determination of Native Fluorescent Bioamines through Precolumn Derivatization and Liquid Chromatography Using Intramolecular Fluorescence Resonance Energy Transfer Detection

Abstract

In this paper, we introduce a novel approach for the highly selective and sensitive analysis of native fluorescent bioamines (indoleamines and catecholamines). This method is based on intramolecular fluorescence resonance energy transfer (FRET) detection in a liquid chromatography (LC) system following precolumn derivatization of the bioamines' amino groups. In this detection process, we monitored the FRET from the native fluorescent moieties (donor) to the derivatized fluorophore (acceptor). From a screening study involving 15 fluorescent reagents, we found that o-phthalaldehyde (OPA) generated the FRET most effectively. The OPA derivatives of the native fluorescent bioamines emitted OPA fluorescence (445 nm) through an intermolecular FRET process when they were excited at the excitation maximum wavelengths of the native fluorescent bioamines (280 nm). The generation of FRET was confirmed through comparison with the analysis of a nonfluorescent amine (isoleucine) performed using LC and a three-dimensional fluorescence detection system. We were able to separate the OPA derivatives of the indoleamines and catecholamines when performing LC on an ODS column. The detection limits (signal-to-noise ratio, 3) for the indoleamines and catecholamines, at a 20-muL injection volume, were 17-120 and 28-200 fmol, respectively. The sensitivity of the intramolecular FRET-forming derivatization method is higher than those of systems that take advantage of both native fluorescence detection (i.e., without derivatization) and the conventional detection of OPA derivatives. Furthermore, this method provides enough selectivity and sensitivity for the determination of the indoleamines present in the urine of healthy humans.

Development of Analytical Methods Utilizing Selectivity of Fluorous Affinity and Their Applications

“…The determination results (mean ± SD) of the examined biogenic amines in human urine from healthy subjects (n =7) were as follows: DOPA 5.4 ± 2.4, DA 5.0 ± 1.6, NE 4.4 ± 1.6, E 1.9 ± 1.1, MN 3.6 ± 1.0, Trp 32 ± 6.5, 5-HTrp 1.4 ± 0.69, and 5-HT 2.6 ± 0.81 nmol/mL urine. These results agree well with previously reported data [8][9][10].…”

Development and Validation of a Specific RP-HPLC Method for the Estimation of γ-Aminobutyric Acid in Rat Brain Tissue Samples Using Benzoyl Chloride Derivatization and PDA Detection

“…Based on the hippuric acid (n-benzoyl glycine) synthesis, we have conceived to synthesize the n-benzoyl GABA derivative since the benzoyl derivatives are quite stable for nearly 2 months and showed characteristic UV absorbance. The UV-PDA detection has other advantages like wide availability because of comparative lower cost, free from the probability of coulometric or amperometric electrode degradation usually present in electrochemical detectors, and therefore, good reproducibility can be obtained with derivatives detectable by UV-PDA detectors [14,16].…”

“…Moreover, this economical and specific method facilitates the UV-PDA detection of stable GABA derivative in biological samples with enhanced throughput. Moreover, the simple UV detection was reported to be helpful for the detection of amino acids like GABA, and the electrochemical detection tends to lack reproducibility mainly because of hysteretic degradation of the electrode [14,16].…”

Selective Determination of Tryptophan-containing Peptides through Precolumn Derivatization and Liquid Chromatography Using Intramolecular Fluorescence Resonance Energy Transfer Detection

“…10 This method is based on the FRET-inducing derivatization of native fluorescent indoleamines and follows the detection of FRET from indoleamines (native fluorescent amines) to the derivatized fluorophore. FRET is the nonradiative transfer of excited-state energy from an initially excited donor fluorophore to an acceptor fluorophore.…”