High-performance liquid chromatographic determination of histamine in biological samples after derivation with o-phthalaldehyde

Abstract

A high-performance liquid chromatographic (HPLC) method for the determination of histamine in tissues, based on precolumn derivation with o-phthalaldehyde, is described. Trichloroacetic acid extracts of rat brain, but not of rat stomach or of rat peritoneal mast cells, had to be cleaned-up by a chromatographic step before HPLC. The extracts were allowed to react with o-phthalaldehyde at pH 12.5 and -20 degrees C for 12 h, followed by acidification to pH 2.0. HPLC was performed on a reverse-phase column with isocratic elution using sulfuric acid in methanol as solvent system. A fluorescence detection system was used; excitation was set at 353 nm and emission was read at 451 nm. One chromatographic run was completed in 20 min. The detection limit with the conventional procedure was 1.5 ng histamine per sample, with a scaled-down procedure it was 250 pg per sample. With extracts of rat gastric mucosa the within-run variation was 2.7% and the day-to-day variation 4.5%.