Aim Phenylketonuria is an inherited metabolic disorder characterized by a deficiency in phenylalanine hydroxylase. However, the impact of this deficit on the patient's overall metabolism is not fully known. Studying this pathology through untargeted metabolomics requires to determine a method for metabolites extraction, here applied to Dried Blood Spot (DBS), a matrix offering several practical advantages. Methodology The DBS of 30 phenylketonuric patients and 30 healthy controls were used for the study. Following a literature review, different extraction protocols and solvents were investigated, with or without an evaporation step, and compared to identify the most appropriate protocol to extract metabolites from the DBS for metabolomics analysis of phenylketonuria by LC-MS/MS, then applied to the patients and controls to validate its application to phenylketonuria.

The most promising extraction method is a gentle agitation overnight at 4 °C, with an evaporation step, and an extraction solvent composed by 80%/20% acetonitrile and water. This method extracted 2 to 6 times more metabolites than other protocols tested with a better extraction of amino acids and derivatives. This protocol enabled us to identify metabolic pathways that were disrupted in phenylketonuric patients, as well as differences in metabolite abundance between the different cohorts. Metabolic profiles differed both between patients and controls, and between patients according to their phenylalanine concentration. These differences were independent of the amino acid supplementation in some patients.

The results obtained on the phenylketonuria patients cohort compared to controls, validated the extraction protocol for studying the systemic metabolic impact of phenylketonuria.