A sensitive and selective determination method of histamine by HPLC with intramolecular excimer‐forming derivatization and fluorescence detection

Abstract

Abstract A highly sensitive, selective and simple method is described for the determination of histamine by high‐performance liquid chromatography (HPLC) with fluorescence detection. The method is based on an intramolecular excimer‐forming fluorescence derivatization of histamine with 4‐(1‐pyrene)butyric acid N ‐hydroxysuccinimide ester (PSE), followed by reversed‐phase HPLC. Histamine, having two amino moieties in a molecule, was converted to the dipyrene‐labeled derivative by reaction with PSE. The derivative afforded intramolecular excimer fluorescence (450–540 nm), which can clearly be discriminated from the monomer fluorescence (370–420 nm) emitted from PSE. Typically, a 10 µL sample solution was mixed with 100 µL of derivatization reagent solution, which was a mixture of 0.5 m m PSE in acetonitrile and 0.5 m m potassium carbonate in water (8:2, v/v). The derivatization was carried out at 100°C for 90 min. The PSE derivative of histamine could be separated by reversed‐phase ODS column with isocratic elution using acetonitrile:water (82:18, v/v) containing 0.03% triethylamine. The detection limit (singnal‐to‐noise ratio = 3) of histamine was 0.5 fmol for a 30 µL injection. The method was successfully applied to the determination of histamine in human urine, and had enough selectivity and sensitivity for urinary histamine quantification. Copyright © 2003 John Wiley & Sons, Ltd.