Determination of Catecholamines and Indoleamines in Human Urine Based on Intramolecular Excimer-forming Derivatization and Fluorescence Detection

Abstract

Experimental Reagents, solutions, and materialAll of the chemicals and solvents were of highest purity available, and were used as received. Distilled water, which was further purified with a MilliQ gradient system (Millipore, Milford, MA, USA), was used to prepare all aqueous solutions. PBC was obtained from Toronto Research Chemicals (North York, Ontario, Canada), and was used without further purification.Stock solutions (1.0 mM) of bioactive amines were prepared in water and stored at 4˚C. These solutions were stable for at least one week, and were diluted further with acetonitrile to the required concentrations before use. A 5 mM solution of PBC A liquid chromatographic (LC) determination of catecholamines and indoleamines is described. This is based on intramolecular excimer-forming fluorescence derivatization with 4-(1-pyrene)butanoyl chloride, followed by reversedphase LC. The analytes, containing an amino moiety and phenolic hydroxyl moieties in a molecule, were converted to the corresponding polypyrene-labeled derivatives by one-step derivatization. They afforded intramolecular excimer fluorescence, which can clearly be discriminated from the normal fluorescence emitted from reagent blanks. The detection limits (S/N = 3) for catecholamines and indoleamines were femto-mole levels per 20-μL injection. Furthermore, this method was applied to a urine assay.

Highly sensitive and selective derivatization-LC method for biomolecules based on fluorescence interactions and fluorous separations

“…An intramolecular excimer-forming derivatization method for the determination of heterofunctional compounds, which have an amino and a phenolic hydroxyl group in a molecule, based on their one-step derivatization with PBC has been described [87,88]. This method was successfully applied to the determination of tyrosine and tyramine [87] and catecholamines and indoleamines [88] in human urine.…”

“…This method was successfully applied to the determination of tyrosine and tyramine [87] and catecholamines and indoleamines [88] in human urine.…”

Liquid chromatographic determination of microcystins in water samples following pre-column excimer fluorescence derivatization with 4-(1-pyrene)butanoic acid hydrazide

“…Previously, we developed the selective detection-oriented derivatization methods to detect poly-functional compounds such as polyamines [13][14][15][16][17], polycarboxylic acids [18][19][20], and polyphenols [21,22]. These methods worked by derivatization with pyrene followed by reversed-phase LC-separation and fluorescence detection.…”

Highly Selective and Simple Method for Determination of Polythiols Based on Liquid Chromatography with Postcolumn Excimer Fluorescence Derivatization

“…[11][12][13][14][15][16][17][18][19][20][21][22] Pyrene-containing precolumn derivatization reagents such as 4-(1-pyrene)butanoic acid N-hydroxysuccinimide ester, [11][12][13]18 4-(1-pyrene)butanoyl chloride, 14,17,19,21 4-(1-pyrene) butanoic acid hydrazide, 15,16,20 N-(1-pyrene)iodoacetamide, 22 and N-(1-pyrenemethyl)iodoacetamide 22 were reacted with corresponding reactive functional groups of target molecules. The resulting polypyrene-labeled derivatives exhibited intramolecular excimer fluorescence at wavelength of 440 -520 nm, which was a significant shift towards longer wavelengths when compared to those of derivatization reagents and monopyrene-labeled derivatives (360 -420 nm).…”

“…E-mail: masayama@fukuoka-u.ac.jp selective determination of polyfunctional compounds was possible even in complex samples containing monofunctional compounds. [11][12][13][14][15][20][21][22] However, precolumn derivatization procedures, such as the addition of reagents, heating, and neutralization before injection, are required, even though the excimer fluorescence derivatization method is selective and sensitive. Therefore, a simple and convenient procedure is desired for analysis.…”