Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
1967184 | Clinica Chimica Acta | 2010 | 7 Pages |
BackgroundDiagnosis of Congenital Adrenal Hyperplasia (CAH) is based on the quantification of 17-hydroxyprogesterone (17-OHP), usually by immunoassay. During the neonatal period the specificity of screening for CAH by blood spot 17-OHP immunoassay is low. High false-positive rates result in a relatively high demand for a second-tier serum confirmation test. A robust, specific and selective method for measurement of cortisol, 21-deoxycortisol, 11-deoxycortisol, 4-androstene-3,17-dione (A4) and 17-OHP in serum has been developed. The method involves a simple extraction procedure and a fast analysis using ultra-performance liquid chromatography–tandem mass spectrometry (UPLC/MS/MS).MethodsThe steroids were extracted from 50 µl of serum using methyl-tert-butyl-ether. Analysis was performed on a UPLC tandem quadrupole mass spectrometer system in positive mode electrospray ionization and multiple reaction monitoring acquisition.ResultsThe assay was linear over each analyte concentration range with all correlation coefficients (r2) > 0.996. Inter- and intra-day CVs were ≤ 10% across the analytical range. In addition simultaneous measurement of the full range of steroids on the pathway to cortisol allows confirmation of the affected steroidogenic enzyme.ConclusionsA second-tier test for the confirmation of CAH has been developed. The method allows for detection and quantification of 5 steroids related to CAH over the range of the clinical assay with good linearity, sensitivity and precision.