title: 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.
authors.affiliation: Laboratory of Molecular Biophotonics, Hamakita, Shizuoka, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Nohta
authors.forename: H
authors.initials: H
authors.lastname: Satozono
authors.forename: K
authors.initials: K
authors.lastname: Koiso
authors.forename: H
authors.initials: H
authors.lastname: Yoshida
authors.forename: J
authors.initials: J
authors.lastname: Ishida
authors.forename: M
authors.initials: M
authors.lastname: Yamaguchi
mesh_terms: D002851:Chromatography, High Pressure Liquid; D005470:Fluorometry; D013058:Mass Spectrometry; D011073:Polyamines / Q000032:analysis*; D011721:Pyrenes
chemical_list: D011073:Polyamines; D011721:Pyrenes