title: Rapid steroid hormone quantification for congenital adrenal hyperplasia (CAH) in dried blood spots using UPLC liquid chromatography-tandem mass spectrometry.
abstract: Newborn screening for congenital adrenal hyperplasia (CAH) is usually done by quantifying 17α-hydroxyprogesterone using immunoassay. However, this test produces high rates of false positive results caused by cross reacting steroids. Therefore we have developed a selective and specific method with a short run time (1.25 min) for quantification of 17α-hydroxyprogesterone, 21-deoxycortisol, 11-deoxycortisol, 11-deoxycorticosterone and cortisol from dried blood spots. The extraction procedure is very simple and steroid separation is ensured on a BEH C18 column and an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Analysis was done in positive ionization mode (ESI+) and recorded in multiple reaction monitoring mode (MRM). The method gave linear results for all steroids over a range of 5-200 (cortisol: 12.5-500)nmol/L with coefficients of regression >0.992. Absolute recovery was >64.1%. Across the analytical range the inter-assay coefficient of variation (CV) was <3%. Newborn blood samples of patients with confirmed 21-CAH and 11-CAH could clearly be distinguished from samples of unaffected newborns falsely positive on immunoassay. The method is not influenced by cross reactions as found on immunoassay. Analysis of dried blood spots shows that this method is sensitive and fast enough to allow rapid analysis and can therefore improve the newborn screening program.
authors.affiliation: Clinic for Pediatric Kidney-, Liver- and Metabolic Diseases, Hannover Medical School, Hannover, Germany. Janzen.Nils@mh-hannover.de
authors.forename: Nils
authors.initials: N
authors.lastname: Janzen
authors.forename: Stefanie
authors.initials: S
authors.lastname: Sander
authors.forename: Michael
authors.initials: M
authors.lastname: Terhardt
authors.forename: Ulrike
authors.initials: U
authors.lastname: Steuerwald
authors.forename: Michael
authors.initials: M
authors.lastname: Peter
authors.forename: Anibh M
authors.initials: AM
authors.lastname: Das
authors.forename: Johannes
authors.initials: J
authors.lastname: Sander
mesh_terms: D000312:Adrenal Hyperplasia, Congenital / Q000097:blood* / Q000175:diagnosis; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D059788:Dried Blood Spot Testing / Q000379:methods*; D006728:Hormones / Q000097:blood* / Q000302:isolation & purification; D006801:Humans; D007231:Infant, Newborn; D057230:Limit of Detection; D016014:Linear Models; D013256:Steroids / Q000097:blood* / Q000302:isolation & purification; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013997:Time Factors
chemical_list: D006728:Hormones; D013256:Steroids