title: Development of a new UPLC-ESI-MS/MS method for the determination of biopterin and neopterin in dried blood spot.
abstract: BACKGROUND
  (6R)-5,6,7,8-tetrahydrobiopterin (BH4) deficiencies are rare inherited defects of synthesis or regeneration of BH4. Due to the resulting hyperphenylalaninemia (HPA), some of them are detected by newborn screening and require the assessment of the pattern of neopterin (Neo) and biopterin (Bio) excretion in urine to be confirmed. Aim of present study was to develop a method for the measurement of these diagnostic biomarkers in dried blood spot (DBS).
  
  
  METHODS
  After DBS extraction, samples were filtered and injected into the UPLC column coupled with a tandem mass spectrometer working in positive electrospray ionization.
  
  
  RESULTS
  The chromatographic separation was accomplished in 6min. The LoQ was 0.57 and 1.45nmol/l of blood for Neo and Bio respectively and the response was linear over the range 0-100nmol/l of blood. The within- and between-day imprecision was <6.4 and 10.8% respectively. Reference ranges for newborns, infants and children/adult were established. The method was tested in 11 patients affected by BH4 defects.
  
  
  CONCLUSIONS
  The assessment of Neo and Bio in DBS is reliable and sensitive and may be proposed as a second tier test for the newborns with hyperphenylalaninemia (HPA) as well as a new potential diagnostic tool for symptomatic subjects with BH4 deficiencies.
authors.affiliation: Department of Experimental Medicine, Sapienza University of Rome, Viale del Policlinico 155, 00161 Rome, Italy. Electronic address: silviasantagata1@gmail.com.
authors.forename: Silvia
authors.initials: S
authors.lastname: Santagata
authors.affiliation: Department of Molecular Medicine, Sapienza University of Rome, Viale del Policlinico 155, 00161 Rome, Italy. Electronic address: emanuele_dicarlo@yahoo.com.
authors.forename: Emanuele
authors.initials: E
authors.lastname: Di Carlo
authors.affiliation: Department of Experimental Medicine, Sapienza University of Rome, Viale del Policlinico 155, 00161 Rome, Italy. Electronic address: carla.carducci@uniroma1.it.
authors.forename: Carla
authors.initials: C
authors.lastname: Carducci
authors.affiliation: Department of Pediatrics, Child Neurology and Psychiatry, Sapienza University of Rome, Via dei Sabelli 108, 00185 Rome, Italy. Electronic address: vincenzo.leuzzi@uniroma1.it.
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Leuzzi
authors.affiliation: Department of Molecular Medicine, Sapienza University of Rome, Viale del Policlinico 155, 00161 Rome, Italy. Electronic address: antonio.angeloni@uniroma1.it.
authors.forename: Antonio
authors.initials: A
authors.lastname: Angeloni
authors.affiliation: Department of Experimental Medicine, Sapienza University of Rome, Viale del Policlinico 155, 00161 Rome, Italy. Electronic address: claudia.carducci@uniroma1.it.
authors.forename: Claudia
authors.initials: C
authors.lastname: Carducci
mesh_terms: D000293:Adolescent; D001708:Biopterins / Q000097:blood*; D002648:Child; D002675:Child, Preschool; D002853:Chromatography, Liquid / Q000379:methods; D059788:Dried Blood Spot Testing / Q000379:methods* / Q000592:standards; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D015997:Neonatal Screening / Q000379:methods; D019798:Neopterin / Q000097:blood*; D010661:Phenylketonurias / Q000097:blood / Q000175:diagnosis*; D012016:Reference Values; D053719:Tandem Mass Spectrometry / Q000379:methods / Q000592:standards; D055815:Young Adult
chemical_list: D001708:Biopterins; D019798:Neopterin
keywords: BH4 deficiencies; Biopterin; Dried blood spot; Neopterin; Tandem mass spectrometry