Development and Validation of an RP-HPLC-FLD Method for the Determination of Biogenic Amines after Pre-column Derivatization with a New Pyrazoline Based Reagent, 3- Naphthyl-1-(4-trifluoromethyl)-5-(4-carboxy phenyl)-2-pyrazoline

Abstract

Application of 3-Naphthyl-1-(4-trifluoromethyl)-5-(4-carboxy phenyl)-2-pyrazoline (NFCP) as a novel fluorigenic label for the derivatization of biogenic amines (BAs) and separation by liquid chromatography (LC) was investigated. Optimum conditions for the pre-column derivatization and chromatographic separation were investigated. The resulting derivatives are fluorescent at λex/λem: 380/460 nm. For the sake of evaluating the efficacy of the proposed analysis of BAs of different nature, the formation of nine NFCP-BAs derivatives was tested individually. Simultaneous derivatization and separation of four BAs (histamine, tyramine, tryptamine, and phenylethylamine) were achieved under the isocratic elution mode. Furthermore, the derivatives were identified by the characteristic product ion obtained during LC-MS/MS analysis. The viability of the method was established by measuring levels of histamine in fresh and spoiled fish samples. Histamine concentrations were found to be 0.4 µgg^-1 and 4.1 µgg^-1 in fresh and spoiled tuna fish samples respectively. As a simple, reliable, and sensitive pre-column derivatization method, this work provides for future research to develop a novel HPLC method for the analysis of BAs from food samples such as fish and various other matrices by utilizing the new pyrazoline compound.