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

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 0.5-10.0 & mu;g.mL(-1) for histamine derivatized and a correlation coefficient of 0.994 was obtained for histamine derivatized. The method detection limit was 0.36 & mu;g.mL(-1) and the limit of quantification (LOQ) was 1.19 & mu;g.mL(-1). The relative standard deviation of the method was obtained for concentrations of 1.0-100.0 & mu;g.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.

Automated summary

In this study, histamine in tuna fish samples was measured using ultra-performance liquid chromatography with a phenyl isothiocyanate derivative. The experiment was designed and investigated using Minitab software, including screening and optimization steps. The effective parameters, such as triethylamine volume, phenyl isothiocyanate volume, reaction temperature, reaction time, and mobile phase pH, were examined in the screening step. The calibration curve for histamine derivatized was then drawn, with a correlation coefficient of 0.994. The method detection limit was 0.36 μg/mL and the limit of quantification was 1.19 μg/mL. The method showed a relative standard deviation between 1.06% and 2.21% for concentrations of 1.0-100.0 μg/mL, with a recovery method ranging from 90.8% to 103.1% for measuring histamine derivatized in real fish samples.