Desenvolvimento de um método para determinação de Vitamina B12 em alimentos, por cromatografia líquida acoplada a espectrometria de massas (LC-MS/MS)

Dissertação de mestrado em Tecnologia e Segurança Alimentar, apresentado à Faculdade de Ciências e Tecnologia da Universidade NOVA de Lisboa, 2020.

Orientação: Professora Doutora Ana Lúcia Monteiro Durão Leitão, Professora Auxiliar, FCT/UNL ( orientadora); Doutora Carla Alexandra Fino Alberto da Motta, colaboradora do Departamento de Alimentação e Nutrição (DAN), Instituto Ricardo Jorge (co-orientadora).

The aim of the present work was focused on the otimization of the extraction and chromatographic method used with the purpose of determining the B12 vitamin content in fortified samples (soy based vegetable drinks) and natural (raw mackerel). The immunoaffinity columns use as the clean-up method contributed to the optimization of the method and, consequently, to the determination of the diferente vitamers in the different matrices. In the fortified soy based vegetable drinks, simpler matrices, it was possible to identify the present cyanocobalamin, with levels estimated between 0,25 and 0,66 μg/100 mL, values very similar to those obtained by ultracentrifugation. In the mackerel samples, more complex matrices, it was tested two extraction methods: with and without sodium cyanide, in order to determine the different natural forms of vitamin B12 as well as their total content. The obtained results show that hydroxocobalamin is the major present form in this natural sample and it represents 35 to 58% of the total vitamin B12 content, unlike cyanocobalamin which is found in residual levels. The total content of vitamin B12, express by content of cyanocobalamin, the average value is estimated to be 13,9 μg/100 mL. Some parameters were also evaluated for the validation of the method. Linearity was verified for the concentration range of the calibration curves under study and, although the homogeneity of variances was not verified, the analysis of reference materials allowed to evaluate the accuracy, having obtained a z-score < 2. The development of this work thus represents a contribution to the optimization and development of the method, and the results obtained can be considered, although it is necessary to carry out more tests, promising.

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