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.

Ornithine and lysine, both basic amino acids, are closely related with Krebs-Henseleit cycle and polyamine synthesis. [1][2][3] It was reported that their high concentration levels in plasma and urine were indicative in diagnoses of some congenital metabolic disorders like cystinuria or hyperlysinemia. [2][3][4] Thus, the selective determination of ornithine and lysine might be more valuable for the diagnoses of these disorders. An amino acids analyzer based on derivatization has been used for the determination of the two amino acids. However, the analyzer method allows the quantification of many amino acids including these two amino acids. Accordingly, the method is timeconsuming for the liquid chromatographic (LC) separation. Therefore, a simple and selective determination method for ornithine and lysine is required in biomedical and clinical fields.A large number of fluorescence derivatization reagents for amino acids in LC have been reported. 5-14 Some fluorogenic Edman-type derivatization reagents for the sequencing of peptides have also been developed. [15][16][17][18][19] All of these reagents react with almost all amino acids to form the corresponding fluorescence derivatives.They are thus useful for the simultaneous determination of many amino compounds including amino acids analysis. However, when they were used for the determination of specific amino compound(s), troublesome clean-up procedure(s) like liquid-liquid or solidphase extraction and/or elegant LC separation conditions like gradient elution or column-switching technique are needed to avoid the interferences from impurities having amino moiety and/or the excess reagents.Only a few fluorogenic derivatization methods for specific amino acid(s) have been introduced: i.e., ninhydrin, 20 9,10-phenanthraquinone, 21 and benzoin-type reagents 22,23 for arginine, o-phthalaldehyde 24 in the absence of a thiol for cysteine, 1,2-diamino-4,5-dimethoxybenzene 25 and borate-hydroxylamineCo(II) reagent 26 for tyrosine related compounds, and glyoxaltype reagents 27,28 for tryptophan. These methods are so selective that they do not require complicated separations of analytes from other amino concomitants and reagent components. However, selective derivatization methods for ornithine and lysine have not been reported.In our previous research, we developed a highly selective and sensitive determination method for polyamines in LC based on intramolecular excimer-forming fluorescence derivatization. 29 In this method, all the primary and secondary amino moieties in polyamines were labeled with a pyrene reagent, 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester (PSE), and the excited polypyrene-labeled derivative afforded an intramolecular excimer to fluoresce in the wavelength region (450 -520 nm) longer than that for usual pyrene derivatives (360 -420 nm). By using these characteristics, polyamines were determined highly selectively, even though the sample was contaminated with monoamino compounds.In this paper, we have reported the determination method for orn...