Desenvolvimento de método rápido de análise de histamina por cromatografia líquida de alta eficiência em atum

Abstract

Histamine is an important biogenic amine found in fish like tuna, mackerel, bonito and sardines, and in fermented foods like cheese, salami and sauerkraut. Its presence in food should not exceed certain limits, because the intake of this substance in quantities that vary from person to person, can cause scombroid poisoning, whose main symptoms are: abdominal pain, vomiting, diarrhea, headache, erythema, urticaria and hypotension, the latter may lead to death, especially in elderly, children and people with heart disease. Health authorities have been increasingly engaged in ensuring that foods with hazardous potential to cause scombroid poisoning are vetoed the trade. From March 2003 to August 2007, in European Union, 223 notifications were made from RASFF for tuna or its derivatives, of which 99 concerned to high levels of histamine. Brazil is one of the world s tuna exporters, and so, should be concerned about the quality of fish it sends to its customers. If the country does not guarantee the quality of the fish it exports, technical barriers to Brazilian fish may be imposed. With the advances in analytical methods for detection and quantification of histamine in Europe, using more accurate, precise and sensitive methods, the trend is that the number of notifications or even a ban on Brazilian imports of tuna grows, if the country does not modernize its methods of analysis. The EU hoped that the tests are made using high performance liquid chromatography. Considering that our proposal was to develop a method that was adapted to Brazilian laboratories conditions, and using a derivatizing reagent easy to handle and with few steps, to speed and convenience, introducing less error in analysis, and generating a stable derivative of histamine to room temperature, allowing an over night analysis in equipment with an autosampler. The method developed offers good resolution and limits of detection and quantification are far below the limit set by law (100mg/Kg), 1.6 mg/kg and 33.84 mg/kg, respectively. The extraction is made with 6% perchloric acid using a sample of approximately 5g and does not requires cleaning prior or posterior derivatization, just a previous centrifugation, filtration and drying in desiccator. Derivatization with AQC is fast and simple handling. The reagent is also exempted later stages of cleaning. Elution is performed with mobile phase ACN: H2O, with 0001% of triethylamine in water, performing in gradient, which starts with 1% AcN, reaching 60% within 25 minutes of analysis, after complete elution of the matrix components. Total time of analysis, including final stabilization of the column with 1% AcN, is 30 minutes. The average RT of derivatized histamine is 18,339 minutes. The efficiency of this method was evaluated by some intralaboratorial validation parameters, and this methodology may become an INMETRO official reference method for analysis of histamine in Brazil.