Experimental Design Application for Measuring Histamine in Tuna Fish Samples by Phenyl Isothiocyanate Derivation Method Using Ultra-Performance Liquid Chromatography.

Abstract

Histamine as an important biogenic amino acid was measured in tuna fish samples by ultra-performance liquid chromatography using a phenyl isothiocyanate derivative. Minitab software was used to design the experiment and investigate the effective factors during the process, which includes screening and optimization steps. A partial factorial design was used in the screening stage and a central composite design was used in the optimization. Effective parameters in histamine derivatized were examined in the screening step including triethylamine volume, phenyl isothiocyanate volume, reaction temperature, reaction time and mobile phase pH. Then, in the optimization, effective parameters were identified and finally, the calibration curve was drawn from a concentration of 0.5-10.0μg.mL-1 for histamine derivatized and a correlation coefficient of 0.994 was obtained for histamine derivatized. The method detection limit was 0.36μg.mL-1 and the limit of quantification (LOQ) was 1.19μg.mL-1. The relative standard deviation of the method was obtained for concentrations of 1.0-100.0μg.mL-1 in the range between 1.06 and 2.21%. The recovery method was obtained from 90.8 to 103.1% for measuring histamine derivatized in real fish samples.