title: Validation of amino-acids measurement in dried blood spot by FIA-MS/MS for PKU management.
abstract: Phenylketonuria (PKU) is a metabolic disorder leading to high concentrations of phenylalanine (Phe) and low concentrations of tyrosine (Tyr) in blood and brain that may be neurotoxic. This disease requires a regular monitoring of plasma Phe and Tyr as well as branched-chain amino-acids concentrations to adapt the Phe-restricted diet and other therapy that may be prescribed in PKU. We validated a Flow Injection Analysis tandem Mass Spectrometry (FIA-MS/MS) to replace the enzymatic method routinely used for neonatal screening in order to monitor in parallel to Phe, Tyr and branched-chain amino-acids not detected by the enzymatic method.
  
  
  
  We ascertained the performances of the method: linearity, detection and quantification limits, contamination index, accuracy. We cross validated the FIA-MS/MS and enzymatic methods and we evaluated our own reference ranges to monitor Phe, Tyr, Leu, Val on 59 dried blood spots of normal controls. We also evaluated Tyr, Leu and Val concentrations in PKU patients to detect some potential abnormalities, not evaluated by the enzymatic method.
  
  
  
  We developed a rapid method with excellent performances including precision and accuracy <15%. We noted an excellent correlation of Phe concentrations between FIA-MS/MS and enzymatic methods (p<0.0001) based on our database which are similar to references ranges published. We observed that 50% of PKU patients had lower concentrations of Tyr, Leu and/or Val that could not be detected by the enzymatic method.
  
  
  
  Based on laboratory accreditation recommendations, we validated a robust, rapid and reliable FIA-MS/MS method to monitor plasma Phe concentrations but also Tyr, Leu and Val concentrations, suitable for PKU management. We evaluated our own reference ranges of concentration for a routine application of this method.
authors.affiliation: CHRU de Tours, Laboratoire de Biochimie et Biologie Moléculaire, Tours, France; UMR INSERM U930, Université François Rabelais de Tours, France.
authors.forename: C
authors.initials: C
authors.lastname: Bruno
authors.affiliation: UMR INSERM U930, Université François Rabelais de Tours, France; CHRU de Tours, Laboratoire de Médecine Nucléaire In Vitro, Tours, France.
authors.forename: D
authors.initials: D
authors.lastname: Dufour-Rainfray
authors.affiliation: CHRU de Tours, Laboratoire de Biochimie et Biologie Moléculaire, Tours, France; UMR INSERM U930, Université François Rabelais de Tours, France.
authors.forename: F
authors.initials: F
authors.lastname: Patin
authors.affiliation: CHRU de Tours, Laboratoire de Biochimie et Biologie Moléculaire, Tours, France; UMR INSERM U930, Université François Rabelais de Tours, France.
authors.forename: P
authors.initials: P
authors.lastname: Vourc'h
authors.affiliation: UMR INSERM U930, Université François Rabelais de Tours, France; CHRU de Tours, Laboratoire de Médecine Nucléaire In Vitro, Tours, France.
authors.forename: D
authors.initials: D
authors.lastname: Guilloteau
authors.affiliation: CHRU de Tours, Service de Médecine Interne, Tours, France; UMR INSERM U1069, Université François Rabelais de Tours, France; Centre de Compétences des Maladies Héréditaires du Métabolisme de l'Enfant et de l'Adulte, Tours, France.
authors.forename: F
authors.initials: F
authors.lastname: Maillot
authors.affiliation: UMR INSERM U1069, Université François Rabelais de Tours, France; Centre de Compétences des Maladies Héréditaires du Métabolisme de l'Enfant et de l'Adulte, Tours, France; CHRU de Tours, Service de Médecine Pédiatrique, Tours, France.
authors.forename: F
authors.initials: F
authors.lastname: Labarthe
authors.affiliation: UMR INSERM U1069, Université François Rabelais de Tours, France; Centre de Compétences des Maladies Héréditaires du Métabolisme de l'Enfant et de l'Adulte, Tours, France; CHRU de Tours, Service de Médecine Pédiatrique, Tours, France.
authors.forename: M
authors.initials: M
authors.lastname: Tardieu
authors.affiliation: CHRU de Tours, Laboratoire de Biochimie et Biologie Moléculaire, Tours, France; UMR INSERM U930, Université François Rabelais de Tours, France; CHRU de Tours, Service de Médecine Pédiatrique, Tours, France.
authors.forename: C R
authors.initials: CR
authors.lastname: Andres
authors.affiliation: UMR INSERM U930, Université François Rabelais de Tours, France; CHRU de Tours, Laboratoire de Médecine Nucléaire In Vitro, Tours, France.
authors.forename: P
authors.initials: P
authors.lastname: Emond
authors.affiliation: CHRU de Tours, Laboratoire de Biochimie et Biologie Moléculaire, Tours, France; UMR INSERM U930, Université François Rabelais de Tours, France; CHRU de Tours, Service de Médecine Pédiatrique, Tours, France. Electronic address: helene.blasco@univ-tours.fr.
authors.forename: H
authors.initials: H
authors.lastname: Blasco
mesh_terms: D000596:Amino Acids / Q000097:blood*; D059788:Dried Blood Spot Testing; D017022:Flow Injection Analysis / Q000379:methods*; D006801:Humans; D010661:Phenylketonurias / Q000097:blood* / Q000628:therapy*; D012016:Reference Values; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000596:Amino Acids
keywords: Mass spectrometry; Method validation; Phenylalanine; Phenylketonuria