title: An improved micro-method for the measurement of steroid profiles by APPI-LC-MS/MS and its use in assessing diurnal effects on steroid concentrations and optimizing the diagnosis and treatment of adrenal insufficiency and CAH.
abstract: Our goals were to (1) develop an improved micro-method usable for neonates for steroid profile measurements and a method to measure androsterone, a key steroid in the recently described androgen backdoor pathway together, with dehydroepiandrosterone and (2) to assess if dehydroepiandrosterone diurnal concentration fluctuations exist potentially necessitating strict adherence to time of blood sample draw and requirement of separate time-dependent reference intervals. Liquid chromatography-tandem mass spectrometry was performed with an atmospheric pressure photoionization source [1]. For each sample 50μL (100μL for the backdoor pathway) of serum was deproteinized by adding 75μL (150μL for the backdoor pathway) of acetonitrile containing the internal standards. After centrifugation, 75μL (150μL for the backdoor pathway) of supernatant was diluted with 250μL of water and injected onto a Poroshell 120 EC-C8 column (SB-C8 column for the backdoor pathway). Within-run coefficients of variation ranged from 2.4 to 10.4% and between-day coefficients of variation from 2.9 to 11.2%. Comparison studies yielded correlation coefficient between 0.97 and 1.00 with recoveries of 90% or greater. Our methods analyze a 9 steroid profile and an additional 2 steroid profile (backdoor pathway) with minimal sample volume (usable in neonates optimizing early diagnosis of endocrinopathies and genetic diseases). Low limits of quantitation make these methods ideal for steroid measurement in women and prepubertal children. As diurnal variations of dehydroepiandrosterone and other steroids [2] concentrations are clinically significant we recommend that separate reference intervals be developed for 8 am, 8 pm, and midnight sample draws. The use of this approach in improving the diagnosis of patients with adrenal insufficiency and congenital adrenal hyperplasia is discussed.
authors.affiliation: Department of Laboratory Medicine, National Institutes of Health, 10 Center Drive, Building 10, Room 2C-306, Bethesda, MD 20814, USA.
authors.forename: Brian R
authors.initials: BR
authors.lastname: Stolze
authors.affiliation: Department of Laboratory Medicine, National Institutes of Health, 10 Center Drive, Building 10, Room 2C-306, Bethesda, MD 20814, USA.
authors.forename: Verena
authors.initials: V
authors.lastname: Gounden
authors.affiliation: Department of Laboratory Medicine, National Institutes of Health, 10 Center Drive, Building 10, Room 2C-306, Bethesda, MD 20814, USA.
authors.forename: Jianghong
authors.initials: J
authors.lastname: Gu
authors.affiliation: Department of Laboratory Medicine, National Institutes of Health, 10 Center Drive, Building 10, Room 2C-306, Bethesda, MD 20814, USA.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Elliott
authors.affiliation: Department of Laboratory Medicine, National Institutes of Health, 10 Center Drive, Building 10, Room 2C-306, Bethesda, MD 20814, USA.
authors.forename: Likhona S
authors.initials: LS
authors.lastname: Masika
authors.affiliation: National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health, 10Center Drive, Building 10, Room 6-3940, Bethesda, MD, 20814, USA.
authors.forename: Brent S
authors.initials: BS
authors.lastname: Abel
authors.affiliation: Clinical Center and Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health, 10 Center Drive, Building 10, Room 1-2740, Bethesda, MD, 20814, USA.
authors.forename: Deborah P
authors.initials: DP
authors.lastname: Merke
authors.affiliation: National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health, 10Center Drive, Building 10, Room 6-3940, Bethesda, MD, 20814, USA.
authors.forename: Monica C
authors.initials: MC
authors.lastname: Skarulis
authors.affiliation: Department of Laboratory Medicine, National Institutes of Health, 10 Center Drive, Building 10, Room 2C-306, Bethesda, MD 20814, USA; Department of Medicine, Division of Endocrinology and Metabolism, Georgetown University, 3700 O St NW, Washington, D.C. 20057, USA. Electronic address: steven.soldin@nih.gov.
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Soldin
mesh_terms: D000312:Adrenal Hyperplasia, Congenital / Q000097:blood* / Q000175:diagnosis; D000309:Adrenal Insufficiency / Q000097:blood* / Q000175:diagnosis; D000728:Androgens / Q000097:blood; D000738:Androsterone / Q000097:blood; D002853:Chromatography, Liquid / Q000379:methods*; D003687:Dehydroepiandrosterone / Q000097:blood; D006801:Humans; D012680:Sensitivity and Specificity; D013256:Steroids / Q000097:blood*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000728:Androgens; D013256:Steroids; D003687:Dehydroepiandrosterone; D000738:Androsterone
keywords: Dehydroepiandrosterone; Diurnal variation; Micro-method atmospheric pressure photoionization LC–MS/MS; Steroids