title: Update on newborn dried bloodspot testing for cerebrotendinous xanthomatosis: An available high-throughput liquid-chromatography tandem mass spectrometry method.
abstract: BACKGROUND
  Cerebrotendinous xanthomatosis (CTX) is a rare genetic disorder of bile acid synthesis that can cause progressive neurological damage and premature death. Detection of CTX in the newborn period would be beneficial since an effective treatment is available. We previously described a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) test with potential to screen newborn dried bloodspots (DBS) for CTX. We report here modifications to the methodology and application of the modified test to analysis of DBS from a CTX-affected and unaffected newborns.
  
  
  METHODS
  The testing methodology utilizes keto derivatization to enable sensitive LC-ESI-MS/MS measurement of elevated 7α,12α-dihydroxy-4-cholesten-3-one (7α12αC4) in CTX newborn DBS. We report here method modifications, including use of a DBS extraction procedure used in newborn screening laboratories and a reduced analysis time of 2 min per sample.
  
  
  RESULTS
  Rapid isotope-dilution LC-ESI/MS/MS quantification of the ketosterol bile acid precursor 7α12αC4 provides a test that could readily discriminate a CTX positive newborn DBS sample (with a concentration of 104.4 ng/ml) from unaffected newborn samples (with a mean concentration of 4.1 ± 3.4 ng/ml; range 0.2-15.6 ng/ml, n = 39) analyzed in a blinded manner.
  
  
  CONCLUSIONS
  We provide additional evidence suggesting 7α12αC4 may be a promising test marker to screen newborn DBS for CTX. Early detection and intervention through newborn screening would greatly benefit those affected with CTX, preventing morbidity and mortality.
authors.affiliation: BioAnalytical Shared Resource Facility, Department of Physiology & Pharmacology, Oregon Health & Science University (OHSU), Portland, OR, United States.
authors.forename: Lisa
authors.initials: L
authors.lastname: Bleyle
authors.affiliation: Department of Pediatrics, Center for Lysosomal and Metabolic Diseases, Erasmus Medical Center, Rotterdam, The Netherlands; Department of Pediatrics, Academic Medical Center, Amsterdam, The Netherlands.
authors.forename: Hidde H
authors.initials: HH
authors.lastname: Huidekoper
authors.affiliation: Laboratory Genetic Metabolic Diseases, Academic Medical Center, Amsterdam, The Netherlands.
authors.forename: Frederic M
authors.initials: FM
authors.lastname: Vaz
authors.affiliation: BioAnalytical Shared Resource Facility, Department of Physiology & Pharmacology, Oregon Health & Science University (OHSU), Portland, OR, United States.
authors.forename: Renu
authors.initials: R
authors.lastname: Singh
authors.affiliation: University of Wisconsin, Madison, WI, United States.
authors.forename: Robert D
authors.initials: RD
authors.lastname: Steiner
authors.affiliation: BioAnalytical Shared Resource Facility, Department of Physiology & Pharmacology, Oregon Health & Science University (OHSU), Portland, OR, United States.
authors.forename: Andrea E
authors.initials: AE
authors.lastname: DeBarber
keywords: 7α12αC4, 7α,12α-dihydroxy-4-cholesten-3-one; Bile acid synthesis; CDCA, chenodeoxycholic acid; CTX, cerebrotendinous xanthomatosis; CYP27A1; DBS, dried bloodspots; ESI-MS/MS, liquid chromatography-electrospray ionization-tandem MS; GC–MS, gas chromatography–mass spectrometry; IRB, institutional Review Board; Ketosterols; LC-ESI-MS/MS; LLOQ, lower limit of quantification; Leukodystrophy; MRM, multiple reaction monitoring; Newborn screening; QAO, quaternary amonoxy; QCs, quality control samples; RSD, relative standard deviation; S/N, signal-to-noise