Confirmation of congenital adrenal hyperplasia by adrenal steroid profiling of filter paper dried blood samples using ultra-performance liquid chromatography-tandem mass spectrometry

Claudia Rossi, Lisa Calton, Heather A. Brown, Scott Gillingwater, A. Michael Wallace, Francesca Petrucci, Domenico Ciavardelli, Andrea Urbani, Paolo Sacchetta, Michael Morris

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Pages (from-to)677-684
Number of pages8
JournalClinical Chemistry and Laboratory Medicine
Volume49
Issue number4
DOIs
Publication statusPublished - Apr 1 2011

Keywords

  • congenital adrenal hyperplasia
  • dried blood spot
  • liquid chromatography- tandem mass spectrometry
  • steroid profiling

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Biochemistry, medical

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