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Desenvolvimento de um método para determinação de Vitamina B12 em alimentos, por cromatografia líquida acoplada a espectrometria de massas (LC-MS/MS)
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Orientador(es)
Resumo(s)
O objetivo deste trabalho centrou-se na otimização do método de extração e do método
cromatográfico utilizados com o objetivo de determinar os teores de vitamina B12 em amostras
fortificadas (bebidas vegetais de soja) e não fortificadas (cavala crua).
A utilização de colunas de imunoafinidade como método de clean-up contribuiu para a otimização
do método e, consequentemente, para a determinação dos diferentes vitâmeros nas diferentes
matrizes.
Nas bebidas vegetais fortificadas com cianocobalamina, matrizes mais simples, foi possível a
identificação da cianocobalamina presente, estimando-se teores entre os 0,25 e 0,66 μg/100 mL,
resultados muito semelhantes aos obtidos por ultracentrifugação.
Para as cavalas, matrizes mais complexas, foram testados dois métodos de extração: com e sem
cianeto de sódio, com o objetivo de determinar as diferentes formas naturais de vitamina B12
bem como o seu teor total. Os resultados obtidos mostraram que a hidroxocobalamina é o
vitâmero predominante na amostra, representando entre 35 e 58% do teor total de vitamina B12,
ao contrário da cianocobalamina que se encontra em teores vestigiais. Já na determinação do
teor total de vitamina B12, expresso em teor de cianocobalamina, o valor estimado é de
aproximadamente 13,9 μg/ 100 g.
Foram ainda avaliados alguns parâmetros para a validação do método. A linearidade foi
verificada para a gama de concentração das curvas de calibração em estudo e, embora a
homogeneidade de variâncias não se tenha verificado, a análise de materiais de referência
permitiu avaliar a exatidão, tendo sido obtido um z-score < 2.
O desenvolvimento deste trabalho representa assim uma contribuição para a otimização e
desenvolvimento do método para quantificar a vitamina B12, podendo considerar-se os
resultados obtidos, embora seja necessária a realização de mais testes, promissores.
The aim of the present work was focused on the otimization of the extraction and chromatographic method used for the purpose of determining the vitamin B12 content in fortified (soy based vegetable drinks) and unfortified (raw mackerel) samples. The immunoaffinity columns used as clean-up method in the optimization process showed not only to be effective but also allowed the determination of different vitamers on matrices. In the fortified soy based vegetable drinks (simpler matrices) it was possible to identify the presence of cyanocobalamin, with levels estimated between 0,25 and 0,66 μg/100 mL, being similar to those obtained by ultracentrifugation. In the mackerel samples (more complex matrices) two extraction methods were tested: with and without sodium cyanide, in order to determine the different natural forms of vitamin B12 as well as their total content. The results show that hydroxocobalamin is the major form in this sample and it represents 35 to 58% of the total vitamin B12 content, cyanocobalamin on the contrary is found in residual levels. The total content of vitamin B12, expressed by content of cyanocobalamin is estimated to be approximately 13,9 μg/100 mL. Several parameters for the validation of the method were also evaluated. Linearity of calibration curve was verified in the range of concentrations of studied samples and, although the homogeneity of variances was not verified, the analysis of reference materials allowed to evaluate the accuracy, with a z-score < 2. In summary, the present work is a contribution to development and optimization method for the determination of vitamin B12.
The aim of the present work was focused on the otimization of the extraction and chromatographic method used for the purpose of determining the vitamin B12 content in fortified (soy based vegetable drinks) and unfortified (raw mackerel) samples. The immunoaffinity columns used as clean-up method in the optimization process showed not only to be effective but also allowed the determination of different vitamers on matrices. In the fortified soy based vegetable drinks (simpler matrices) it was possible to identify the presence of cyanocobalamin, with levels estimated between 0,25 and 0,66 μg/100 mL, being similar to those obtained by ultracentrifugation. In the mackerel samples (more complex matrices) two extraction methods were tested: with and without sodium cyanide, in order to determine the different natural forms of vitamin B12 as well as their total content. The results show that hydroxocobalamin is the major form in this sample and it represents 35 to 58% of the total vitamin B12 content, cyanocobalamin on the contrary is found in residual levels. The total content of vitamin B12, expressed by content of cyanocobalamin is estimated to be approximately 13,9 μg/100 mL. Several parameters for the validation of the method were also evaluated. Linearity of calibration curve was verified in the range of concentrations of studied samples and, although the homogeneity of variances was not verified, the analysis of reference materials allowed to evaluate the accuracy, with a z-score < 2. In summary, the present work is a contribution to development and optimization method for the determination of vitamin B12.
Descrição
Palavras-chave
Vitamina B12 Vitâmeros Imunoafinidade UHPLC LC-MS/MS