Development of an Assay for Histamine Using Automated High-performance Liquid Chromatography with Electrochemical Detection

Previous studies have measured histamine by derivatization with o-phthaldialdehyde (OPA) and mercaptoethanol (ME), followed by reversed-phase HPLC separation and electrochemical detection. The derivatization product, however, was very unstable. In the present study, inclusion of less polar solvents (e.g., acetonitrile or tetrahydrofuran) in the OPA/ME derivatization reaction produced an OPA/ME-histamine product that was stable for many hours. Changes of the HPLC mobile phase (increasing its ionic strength and pH and including triethylamine) dramatically improved the chromatography and reduced the histamine detection limit to < 0.1 pmol. The modified assay was suitable for batchwise manual derivatization of histamine samples followed by their automated analysis by HPLC with an automatic injector.