Highly Selective Fluorometric Determination of Polyamines Based on Intramolecular Excimer-Forming Derivatization with a Pyrene-Labeling Reagent

Abstract

We introduce a novel approach in highly selective and sensitive fluorescence derivatization of polyamines. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester (PSE), followed by reversed-phase high-performance liquid chromatography (HPLC). Polyamines, having two to four amino moieties in a molecule, were converted to the corresponding dipyrene- to tetrapyrene-labeled derivatives by reaction (100 degrees C, 20 min) with PSE. The derivatives afforded intramolecular excimer fluorescence (450-520 nm), which can clearly be discriminated from the monomer (normal) fluorescence (360-420 nm) emitted from PSE, its hydrolysate and monopyrene-labeled derivatives of monoamines. The structures of the derivatives were confirmed by HPLC with mass spectrometry, and the emission of excimer fluorescence could be proved by spectrofluorometry and time-resolved fluorometry. The PSE derivatives of four polyamines [putrescine (Put), cadaverine (Cad), spermidine (Spd), and spermine (Spm)] could be separated by reversed-phase HPLC on a C8 column with linear gradient elution. The detection limits (signal-to-noise ratio of 3) for the polyamines were 1 (Put), 1 (Cad), 5 (Spd), and 8 (Spm) fmol on the column. Furthermore, the present method was so selective that biogenic monoamines gave no peak in the chromatogram.

Simultaneous Determination of Histamine and Histidine by Liquid Chromatography Following Intramolecular Excimer-forming Fluorescence Derivatization with Pyrene-labeling Reagent

“…19,20 In the methods, the primary and secondary amino moieties in histamine were labeled with a pyrene reagent, 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester (PSE), and the excited dipyrenelabeled derivative afforded an intramolecular excimer to fluoresce in a wavelength region (440 -540 nm) longer than that for the usual pyrene derivatives (360 -420 nm). By using these characteristics, not only histamine, but also other polyamines, [21][22][23][24] were determined with high selectivity and sensitivity, even though the sample was contaminated by monoamino compounds.…”

Triethylenetetramine Pharmacology and Its Clinical Applications

“…One solution, inspired by aqueous polyamine analytic methods (18), is to use fluorescence-labeling reagents to derivatize TETA and detect its derivatives by using a fluorimetric detector. A number of fluorescence-labeling reagents have been tried, including m-toluoyl chloride, fluorescamine, dansyl chloride, O-phthalaldhyde, 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester, and 9-flouorenylmehylchlorofomate (19)(20)(21)(22)(23)(24)(25). However, fluorimetric methods are associated with challenges, such as whether the analyte is fully or partially labeled, and whether detected peaks are separated from other known or unknown metabolites, polyamines, and their metabolites.…”

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.…”