Eficiência e seletividade de diferentes métodos de extração, purificação e detecção de histamina e tiramina em queijo ralado

Abstract

Efficiency and selectivity of different methods of extraction, purification and detection of histamine and tyramine in grated cheese. The aim of this work was to evaluate the efficiency and selectivity of extraction, purification and detection steps of histamine (HIM) and tyramine (TYM) determination in grated cheese with a simple analysis by molecular absorption spectrophotometry. The profile and levels of HIM, TYM and eightamines (putrescine, cadaverine, agmatine, spermine, spermidine, phenylethylamine, serotonine, tryptamine) were determined in 13 brands of grated cheese. Ten bioactive amines were detected, including HIM and TIM at levels up to 78.27 and 34.42 mg/100 g, respectively. The extraction of bioactive amines was evaluated with five acid solutions, borate buffer, methanol and ethanol, as solvents. HCl 1.0 mol/L and ethanol provided the best results. None of the extractors was selective for HIM and TYM. HIM and TYM derivatized with ninhydrin and dansyl chloride showed the same absorption profile compared to those exhibited by the eight other amines found in grated cheese. Since these amines, as well as amino acids, might show molecular spectrophotometric absorption which would interfere on HIM and TYM quantification, purification proceduresare necessary. HIM and TYM purification by liquid-liquid extraction with four organic solvents was evaluated. Appropriate HIM and TYM recoveries were observed with ethyl ether at pH 10.0. However none of the procedures was specific for these amines. Solid phase extraction with SEP-PAK C18, bentonite, anion and cation exchange wereevaluated. SEP-PAK C18 and bentonite purification were not selective and provided inappropriate recoveries for HIM and TYM. When using anion exchange, TIM, serotonin and amino acids were strongly retained and did not elute efficiently. The bioactive amines were strongly retained on the cation exchange resin, and high ionic strength was necessary to elute them. Selective HIM and TYM analysis was not viable in the evaluated conditions. However, it is possible to evaluate biogenic amines, except TYM, by HCl 1.0 mol/L extraction, anion exchange purification and quantification by molecular absorption with ninhydrin derivatization.Key-words: histamine; tyramine; grated cheese; liquid-liquid extraction; solid phaseextraction; spectrophotometry.