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Título: Optimization of Enzymatic Extraction for HPLC Quantification of Vitamin B2
Autor: Flores, Cristina
Santos, Mariana
Palavras-chave: Enzymatic Extraction
Vitamin B2
Composição dos Alimentos
Issue Date: Jan-2012
Editora: Instituto Nacional de Saúde Doutor Ricardo Jorge, IP
Resumo: The purpose of this study was to optimize the enzymatic extraction for HPLC quantification of vitamin B2 in foodstuffs, by testing Takadistase to replace one of the enzymes (α-amylase) employed in the implemented method and, to change the extraction incubation temperature, in order to optimize equipment utilization. For the optimization of the enzymatic extraction we first tested the replacement of the previous enzymatic mixture; α-amylase (0,5g/sample) + β-amylase (0.05g/sample) for: A -Takadistase (2g / sample) and β-amylase (0.05g / sample) and B- Takadistase (1g / sample) and β-amylase (0.05g / sample). After proving the new mixture efficiency, we tested the reduction of Takadiastase quantity to 0.5 g and 0.25 g. Afterwards, we tested the incubation temperature change from 40º to 37ºC. Chromatographic separation was performed, at 37 ºC, by reverse phase high performance liquid chromatography with fluorometric detection (excitation and emission wavelengths 422 nm and 522 nm, respectively). A chromatographic column Phenomenex Luna 5 μm C18 1000A column (250 x 4.6 mm) was empolyed. The mobile phase consisted of 0.05 mol/l acetate buffer + Methanol (70+30) with a flow rate of 1 ml/min. Quantification was made by external calibration. To compare the enzyme replacement, breakfast cereals (FAPAS) were used as test samples. We used milk powder, spiked with riboflafin-5P, to test enzyme quantities and infant formula to test different incubation temperatures. Accuracy was verified by running proficiency test samples (FAPAS). ANOVA tests proved that there are no significant differences between vitamin B2 content obtained with the different enzyme treatments (Takadistase + β-amylase; α-amylase + β-amylase) or with the different incubation temperatures. The results obtained with the different enzyme quantities, also proved to be equivalent after tested trough ANOVA. In all cases we considered p=0.05. Enzymatic extraction performed with Takadistase (0.25 g/sample) and β-amylase (0.05 g/sample) at 37ºC, proved to be efficient and contributed to optimize the existing laboratory equipment utilization and test cost.
Descrição: Abstract publicado na página 172 do livro "7º Encontro Nacional de Cromatografia", do Departamento de Química e Bioquímica da Faculdade de Ciências da Universidade do Porto; ISBN: 978-989-97667-0-9
Arbitragem científica: no
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