3. Isotope Dilution Analysis of Cortisol, Androstenedione and 17-alpha-Hydroxyprogesterone on the Varian 320 LC/MS/MS to Screen for Congenital Adrenal Hyperplasia (CAH) in Newborns

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Brian Nies Varian

Brian Nies, Varian Inc. Lisa Bates-Dubrow, Metabolics Genetics Laboratory, Arkansas Children's Hospital Carolyn Sparks, Metabolics Genetics Laboratory, Arkansas Children's Hospital Patricia Whittecar, Metabolics Genetics Laboratory, Arkansas Children's Hospital Kurt Thaxton, Varian Inc.

Congenital adrenal hyperplasia (CAH) consists of a family of disorders resulting from enzymatic defects in the metabolic pathway to make steroids in the body (steroidogenesis) and inherited as autosomal (male or female) recessive traits. Each enzyme deficiency produces characteristic hormonal abnormalities and associated clinical syndromes. Among these syndromes are steroid 21-hydroxylase deficiency and nonclassic forms of steroidogenic defects due to genetic allelism (substitution of an alternative form of a gene in a chromosome). Since the nonclassic 21-hydroxylase deficiency is the most common autosomal recessive defect in humans and, unlike other androgen-excess syndromes, responds readily to treatment, correct diagnosis is important. Androstenedione, Cortisol and 17á-Hydroxyprogesterone (17á-OHP) steroids are used as biochemical markers for the detection of Congenital Adrenal Hyperplasia in newborns using isotope dilution/ high performance liquid chromatography/tandem Mass Spectrometry (ID/LC/MS/MS). These markers are extracted from dried blood spot samples that have been applied to a filter paper and analyzed by LC-MS/MS. A positive result for CAH is indicated by decreased cortisol levels and increased levels of both 17á-OHP and androstenedione. Analysis is rapid, requiring less than ten minutes per sample. The analysis is linear over approximately three orders of magnitude with limits of detection less than 1 ng/mL for each analyte.

Email: brian.nies@varianinc.com

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