title: Confirmation of congenital adrenal hyperplasia by adrenal steroid profiling of filter paper dried blood samples using ultra-performance liquid chromatography-tandem mass spectrometry.
abstract: BACKGROUND
  The specificity of screening for congenital adrenal hyperplasia by direct measurement of 17-hydroxyprogesterone in filter paper dried blood spot samples by immunoassay is low and has a high false-positive rate. In order to reduce the false-positive rate of this test, we developed a rapid, robust, specific confirmatory procedure in which cortisol, 4-androstene-3,17-dione and 17-hydroxyprogesterone were measured simultaneously by ultra-performance liquid chromatography-tandem mass spectrometry.
  
  
  METHODS
  After extraction, samples were analysed by ultra-performance liquid chromatography-tandem mass spectrometry and 17-hydroxyprogesterone was quantified accurately. Other steroids were determined using stable deuterated internal standards. In total, 25 patient blood spot samples and 92 control samples were analysed.
  
  
  RESULTS
  The assay was linear for 17-hydroxyprogesterone, with a coefficient of determination >0.997 and imprecision ≤ 6.5%. An upper limit of normal for 17-hydroxyprogester-one of 4.45 nmol/L was established by analysing a cohort of samples from unaffected newborns. In addition, a cut-off of 3.5 for the peak areas ratio (17-hydroxyprogesterone+4-androstene-3,17-dione)/cortisol, allows confirmation of the affected steroidogenic enzyme.
  
  
  CONCLUSIONS
  A high throughput method for the detection of steroids related to congenital adrenal hyperplasia has been developed, allowing the false-positive rate associated with screening for 17-hydroxyprogesterone by immunoassay to be determined.
authors.affiliation: Centre of Study on Aging (CeSI), G. d'Annunzio University Foundation, Chieti, Italy. claudia.rossi@unich.it
authors.forename: Claudia
authors.initials: C
authors.lastname: Rossi
authors.forename: Lisa
authors.initials: L
authors.lastname: Calton
authors.forename: Heather A
authors.initials: HA
authors.lastname: Brown
authors.forename: Scott
authors.initials: S
authors.lastname: Gillingwater
authors.forename: A Michael
authors.initials: AM
authors.lastname: Wallace
authors.forename: Francesca
authors.initials: F
authors.lastname: Petrucci
authors.forename: Domenico
authors.initials: D
authors.lastname: Ciavardelli
authors.forename: Andrea
authors.initials: A
authors.lastname: Urbani
authors.forename: Paolo
authors.initials: P
authors.lastname: Sacchetta
authors.forename: Michael
authors.initials: M
authors.lastname: Morris
mesh_terms: D000311:Adrenal Glands / Q000378:metabolism*; D000312:Adrenal Hyperplasia, Congenital / Q000097:blood* / Q000175:diagnosis; D001774:Blood Chemical Analysis / Q000379:methods*; D001800:Blood Specimen Collection / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D005374:Filtration / Q000295:instrumentation*; D006801:Humans; D010209:Paper; D011786:Quality Control; D013256:Steroids / Q000097:blood* / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D013256:Steroids