Highly selective fluorometric determination of polyamines based on intramolecular excimer-forming derivatization with a pyrene-labeling reagent

We introduce a novel approach in highly selective and sensitive fluorescenc e derivatization of polyamines. This method is based on an intramolecular e xcimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyre ne)butyric acid N-hydroxysuccinimide ester (PSE), followed by reversed-phas e high-performance liquid chromatography (HPLC), Polyamines, having two to four amino moieties in a molecule, were converted to the corresponding dipy rene- to tetrapyrene-labeled derivatives by reaction (100 degrees C, 20 min ) with PSE. The derivatives afforded intramolecular excimer fluorescence (4 50-520 nm), which can clearly be discriminated from the monomer (normal) fl uorescence (360-420 nm) emitted from PSE, its hydrolysate and monopyrene-la beled derivatives of monoamines, The structures of the derivatives were con firmed by HPLC with mass spectrometry, and the emission of excimer fluoresc ence could be proved by spectrofluorometry and time-resolved fluorometry. T he PSE derivatives of four polyamines [putrescine (Put), cadaverine (Cad), spermidine (Spd), and spermine (Spm)] could be separated by reversed-phase HPLC on a Cg column with linear gradient elution, The detection limits (sig nal-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 sel ective that biogenic monoamines gave no peak in the chromatogram.