Liquid chromatographic determination of polyamines in human urine based on intramolecular excimer‐forming fluorescence derivatization using 4‐(1‐pyrene)butanoyl chloride

Abstract

A liquid chromatographic method for highly sensitive and selective fluorometric determination of polyamines (putrescine, cadaverine, spermidine and spermine) in human urine is described. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butanoyl chloride (PBC), followed by reversed-phase liquid chromatography. The method offers higher sensitivity for determination of spermidine and spermine than previously reported method utilizing 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester as a derivatization reagent. Samples containing free polyamines in diluted human urine were directly derivatized with PBC and separated on an octyl column. The derivatives were detected at excitation 345 and emission 475 nm wavelengths. For determination of total polyamine content, the conjugated polyamines were first hydrolyzed in 4 M HCl. The detection limits (signal-to-noise ratio = 3) for polyamines in urine were 1.1-3.4 pmol/mL. At optimized derivatization and chromatographic conditions, interferences such as biogenic monoamines gave no peaks or the peaks did not interfere with the peaks of polyamine derivatives. In conclusion, the present derivatization method allows direct determination of polyamines in human urine samples without the need for sample clean-up procedures.

Determination of Catecholamines and Indoleamines in Human Urine Based on Intramolecular Excimer-forming Derivatization and Fluorescence Detection

“…In our previous method, 13 Tyr and Tyra in urine could be determined without any pretreatments, owing to the selectivity of intramolecular excimer-forming fluorescence derivatization. [14][15][16] In this study, however, deproteinization with perchloric acid and solid-phase extraction were applied as a pretreatment of urine samples to obtain simpler chromatograms and to prevent any damage of the column. As shown in Table 2, when we applied the pretreatment procedure described in 487 ANALYTICAL SCIENCES APRIL 2007, VOL.…”

“…The resulting dipyrene-labeled derivatives provided intramolecular excimer fluorescence at the wavelength region of 440 -520 nm, which was shifted markedly to higher emission wavelengths, as compared to the wavelengths of the monopyrene-labeled monofunctional compounds (360 -420 nm). This chemistry allowed up to perform the selective analysis of tyrosine and tyramine, even in complex samples containing monoaminergic and monophenolic compounds, as is the cases with polyamine 14,15 and bisphenol 16 analyses.…”

Liquid Chromatographic Determination of Ethambutol in Serum Samples Based on Intramolecular Excimer-forming Fluorescence Derivatization

“…In previous studies for the determinations of polyamino compounds, PSE [15][16][17][18][19][20] and PBC 22 were used as pyrene-labeling reagents for amino groups. EMB was reacted only with highly reactive PBC, probably due to hindrance of the bulky alkyl groups with the derivatization reaction.…”

“…All of these polyamino compounds were converted to the corresponding polypyrene-labeled derivatives by a reaction with 4-(1-pyrene)butyric acid Nhydroxysuccinimide ester (PSE), and the derivatives generated an intramolecular excimer fluorescence. Recently, we have developed the intramolecular excimer-forming fluorescence determination methods for bisphenols 21 and polyamines 22 by using 4-(1-pyrene)butanoyl chloride (PBC), whose reactivity to the amino group is higher than that of PSE. In this study, we applied the derivatization technique with PBC to the determination of EMB.…”

Multi-Perfluoroalkyl Derivatization of Polyamines for Selective Liquid Chromatography-Tandem Mass Spectrometric Analysis Utilizing Fluorous Affinity

“…Previously, we developed an excimer fluorescence derivatization method for selective LC analysis of polyfunctional compounds including polyamines [15][16][17]. The pyrene-derivatized poly-functional compounds could be selectively detected by intramolecular excimer fluorescence (440-540 nm), whereas mono-functional compounds and reagent blanks, which emitted monomer fluorescence (360-420 nm), did not affect the analysis of the poly-functional compounds.…”