Quantificação do ácido fólico em farinha de trigo adotando o método de cromatografia líquida de alta eficiência com detector UV: validação do método

Abstract

The lack of vitamin B9 in daily diet is mainly related to abnormalities in the fetus development and heart and bone marrow diseases. In order to avoid folic acid deficiency, some countries adopted the mandatory fortification of foods with folic acid. Wheat and cornflour were chosen as the main source due to their presence in practically all breakfasts. In order to ensure quality control for the folic acid fortification on the commercialized products, adequated methodologies are necessary. Thus, this work aimed to evaluate the method for folic acid extraction from samples of type I wheat flour and the development of the analytical conditions and method validation by high-performance liquid chromatography. The analyte extraction was carried out with a 0,1 mol/L potassium hydroxide solution, followed by ultrasound dispersion. To reduce interferences, the extract was clarified with phosphoric acid and trichloroacetic acid solutions, both at a concentration of 0,1 mol/L. Prior to the CLAE analysis, the extract was centrifuged at 3600 rpm for 10 min and subsequently filtered through a polyvinylidene fluoride membrane. For chromatographic analysis, 20 uL of the sample was injected using a C18 column, with isocratic elution of 85% acetate buffer with pH adjusted to 2,8 and 15% acetonitrile, flow rate of 0,5 mL/min and UV/Vis detector at 290 nm. The retention time of folic acid under the mentioned conditions was 7,6 min. Afterward, the selectivity of the method was evaluated, obtaining an Fcalculated 9,012, lower than the Ftabled, criterion to confirm that the method is selective. The working range and linearity of the calibration curve were determined, meeting the acceptance criteria for an R2 greater than 0,9945 between 9 and 500 ug/L, in addition to the random verification of the calibration curve residues. The limit of quantification and detection for the method was 17,53 g and 5,79 ug for 100 g of type I wheat flour, respectively. The recovery rate of the fortified analyte with known concentration was evaluated, obtaining an average of 101,80%. The relative standard deviation for the repeatability of the method was evaluated with 3 standard concentrations of folic acid analyzed in triplicate, obtaining a DPR 0,49%, lower than the 15% criterion. Intermediate precision was evaluated with the same repeatability concentrations, but with an interval of 10 days, obtaining a result of 0,52%, less than 15%. The quantification of folic acid from 5 commercial samples of type I wheat flour was determined by the method defined in this work and all of which presented adequate folic acid content according to the ANVISA resolution N° 150 of April 13, 2017. The evaluated statistical parameters showed that the method proposed for validation is suitable for the determination of folic acid in type I, wheat flour since the results were within the standards established as acceptance criteria. [resumo fornecido pelo autor]