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

1999 Fiscal Year Final Research Report Summary

• Project/Area Number: 10672033
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Physical pharmacy
• Research Institution: Fukuoka University
• Principal Investigator: YAMAGUCHI Masatoshi Fukuoka Univ., Fac. Pharm. Sci., Prof., 薬学部, 教授 (50117280)
• Project Period (FY): 1998 – 1999
• Keywords: Excimer Fluorescence / Derivatization / Pyrene reagent / HPLC / Polyamine

Research Abstract

A highly selective and sensitive method was developed to determine polyamines, based on 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℃ ,20min) with PSE. The derivatives afforded intramolecular excimer fluorescence (450-520 nm) which can clearly be discriminated from 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 phased HPLC on C8 column with linear gradient elution. The detection limits (signal-to-noise ratio of 3) for the polyamines were 1.0 (Put), 1.0 (Cad), 4.7 (Spd) and 7.0 (Spm) fmol on column. Furthermore, the present method was so selective that biogenic monoamines gave no peak in the chromatogram.