title: An improved ultra performance liquid chromatography-tandem mass spectrometry method for the determination of alloisoleucine and branched chain amino acids in dried blood samples.
abstract: BACKGROUND
  Branched chain amino acid (BCAA) analysis is needed for the diagnosis and management of patients with maple syrup urine disease (MSUD). We report an improved ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the determination of BCAAs and Allo-Ile in dried blood spot (DBS) samples.
  
  
  METHODS
  BCAAs were extracted from a 3 mm blood spot into methanol/water containing stable isotope internal standards. Eluents were dried and reconstituted in the mobile phase. Gradient elution was performed on an Acquity™ BEH C(18) (100 × 2.1 mm, 1.7 μm) column. BCAAs were detected and quantified in the multiple reaction monitoring mode in a five-minute analysis.
  
  
  RESULTS
  The assay was calibrated to give best possible alignment with plasma results. Retrospective analysis of newborn DBSs from six classic MSUD patients showed elevated alloisoleucine (Allo-Ile) in all cases. Two of four patients with mild disease had normal values; the other two had significant elevations in Allo-Ile.
  
  
  CONCLUSIONS
  Analysis of BCAA in DBS by UPLC-MS/MS is a useful second tier newborn screening test to identify classical MSUD and for monitoring of remote patients.
authors.affiliation: Discipline of Paediatrics and Child Health, University of Sydney, Sydney, Australia.
authors.forename: A
authors.initials: A
authors.lastname: Alodaib
authors.forename: K
authors.initials: K
authors.lastname: Carpenter
authors.forename: V
authors.initials: V
authors.lastname: Wiley
authors.forename: K
authors.initials: K
authors.lastname: Sim
authors.forename: J
authors.initials: J
authors.lastname: Christodoulou
authors.forename: B
authors.initials: B
authors.lastname: Wilcken
mesh_terms: D000597:Amino Acids, Branched-Chain / Q000097:blood*; D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods* / Q000592:standards; D059788:Dried Blood Spot Testing / Q000379:methods* / Q000592:standards; D006801:Humans; D007231:Infant, Newborn; D007532:Isoleucine / Q000097:blood*; D008375:Maple Syrup Urine Disease / Q000097:blood / Q000175:diagnosis; D015997:Neonatal Screening; D058996:Quality Improvement; D012015:Reference Standards; D053719:Tandem Mass Spectrometry / Q000379:methods* / Q000592:standards
chemical_list: D000597:Amino Acids, Branched-Chain; D007532:Isoleucine