Highly Selective Fluorometric Determination of Dicarboxylic Acids Based on Intramolecular Excimer-Formfng Derivatization with a Pyrene-Labeling Reagent.

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A highly selective and sensitive method was developed to determine dicarboxylic acids, based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(l-pyrene)butanoic acid hydrazide (PBH) in the presence of l-ethyl-3-(3-dimrthylaminopropyDcarbodiimide, followed by reversed phase high performance liquid chromatography (HPLC). Dicarboxylic acids, having two carboxylic acid moieties in a molecule, were converted to the corresponding dipyrene-labeled derivatives by reaction (40ーC, 60 min) with PBH. The derivatives afforded intramolecular excimer fluorescence (450-520 nm) which can clearly be discriminated from the monomer (normal) fluorescence (360-420 nm) emitted from PBH and monopyrene-labeled derivatives of monocarboxylic acid. 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 PBH derivatives of four dicarboxylic acids [glutaric acid (GA), adipic acid (AA), suberic acid (SubA) and sebacic acid (SebA)] 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 dicarboxylic acids were 22 (GA), 25 (AA), 31 (SubA) and 54 (SebA) fmol on column. Furthermore, the present method was so selective that biogenic monocarboxylic acids gave no peak in the chromatogram.