Otimização e validação de metodologia quantitativa para determinação de fenilalanina em farinha de trigo empregando cromatografia líquida de alta eficiência com detector UV-VIS.

Abstract

Phenylketonuria is considered an inborn error of metabolism associated with phenylalanine. It is caused by mutations in the gene that encodes the liver enzyme PAH, which is responsible for converting phenylalanine to tyrosine. The absence of this enzyme in the body leads to the accumulation of phenylalanine in the blood, brain and tissues. Excess is neurotoxic leading to defects in neuromotor and neurocognitive development. The basic treatment consists of the consumption of foods with low protein content associated with a protein-free substitute or with very low levels of phenylalanine. Therefore, it is essential to have information on the amount of phenylalanine present in foods to maintain serum phenylalanine concentrations in accordance with recommendations and individual tolerances. Based on the AOAC food composition triangle, wheat flour was selected for this study, although it is considered a restricted food in the diet of phenylketonurics. This is because it belongs to a sector of this triangle, in which foods have a protein content of 0 to 33% and in this context, a validated analytical method is applicable to other foods that belong to this same sector. In view of the above, the objective of the present work was to optimize and validate an analytical method to quantify wheat flour using high performance liquid chromatography with an ultraviolet detector. The protein present in the wheat flour was hydrolyzed in 6 M hydrochloric acid with phenol for 22 hours at a temperature of 110 °C. Free amino acids were derived with phenylisothiocyanate. Linearity found was in the range of 0.20 to 0.95 nmol/mL (corresponding to 49.97 to 237.34 mg/100g of phenylalanine in wheat flour), with significant matrix effects. The average recovery percentages for the levels studied corresponded to 62.46; 124.92 and 224.85 mg/100g of wheat flour phenylalanine were 97.56%, 93.57% and 87.49%, respectively. The relative standard deviations for these same levels studied under repeatability and intermediate precision conditions were 4.49%, 6.32% and 4.31% and 4.90%, 6.95% and 5.11%, respectively. The detection limit was 3.20 mg/100g and the quantification limit was 12.80 mg/100g. The performance parameters studied indicated the suitability of the method for monitoring and controlling phenylalanine levels in wheat flour.