Analysis of Gizzerosine in Foodstuffs by HPLC Involving Pre-column Derivatization with o-Phthaldialdehyde

GizzerosineGizzerosine [2-amino-9-(4-imidazolyl)-7-azanonanoic acid] is a toxicToxic amino acidAmino acid formed from histamine and lysineLysine at high temperatures, and may be present in foodstuffsFoodstuff (e.g., fishmealFishmeal and meat-boneBone mealMeal) for animalsAnimal including catsCat and dogsDog. Here we developed a simple, rapid, sensitive, specific, and automated method for the analysisAnalysis of gizzerosineGizzerosine in foodstuffsFoodstuff by high-performance liquid chromatography (HPLC) involving pre-column derivatizationDerivatization with o-phthaldialdehyde (OPA) in the presence of N-acetylcysteine (instead of the usual 2-mercaptoethanol or ethanethiol reagent). OPA reacted immediately (within 1 min) with gizzerosineGizzerosine in an autosampler at room temperatures (e.g., 20–25 °C), and their derivative was directly injected into the HPLC column. The highly fluorescent gizzerosineGizzerosine-OPA derivative was well separated from the OPA derivatives of all natural amino acidsAmino acid known to be present in physiological fluids (e.g., plasmaPlasma), proteinsProtein and foodstuffsFoodstuff, and was detected at an excitation wavelength of 340 nm and an emission wavelength of 450 nm. The total time for chromatographic separation (including column regeneration) was 20 min per sample rather than 40 min and longer in previous HPLC methods. The detection limit for gizzerosineGizzerosine was at least 6 pmol/ml in an assay solution (HPLC vial) or at least 0.09 pmol per injection into the HPLC column. The analysisAnalysis of gizzerosineGizzerosine was linear between 1 and 100 pmol per injection. When gizzerosineGizzerosine was extracted from foodstuffsFoodstuff, its detection limit was at least 875 pmol/g foodstuff or at least 0.21 mg/kg foodstuff. Our routine HPLC techniqueTechnique does not require any cleanup of samples or the OPA derivatizationDerivatization products (including the OPA-gizzerosineGizzerosine adduct), and is applicable for the analysisAnalysis of gizzerosineGizzerosine in both foodstuffsFoodstuff and animalAnimal tissues.