Application of the simplex method for optimization of chromatographic analysis of biogenic amines in fish

Abstract

The aim of this study was to optimize the separation of biogenic amines with ion-exchange high-performance liquid chromatography, using the modified simplex method. A modified chromatographic response function was used, and two methodological parameters were selected as simplex variables: the proportion of the organic solvent 2-propanol in the mobile phases, added as a new mobile phase, and the variation of the gradient curve at one specific step in the elution program. In optimum conditions the correct resolution and separation of the biogenic amines (tyramine, β-phenylethylamine, histamine, tryptamine, putrescine, cadaverine, agmatine, spermidine and spermine) were completed in 16 min, reducing the analysis time by 9 min compared with the original method. The regression coefficients were higher than 0.99 in all the amines, except for spermine (0.989). The detection limits varied between 0.06 mg/L, for histamine and agmatine, and 0.22 mg/L, for tryptamine, while retention time repeatability, determined as a relative standard deviation, was between 4.64% for histamine and 11.95% for tryptamine. The repeatability variation for the amine concentrates was found between 0.71% in cadaverine and 3.23% for tryptamine. Applying the optimized method to samples of refrigerated sardine and sea bream demonstrated the effectiveness of the method in these fish species.