Newborn screening for congenital adrenal hyperplasia: Additional steroid profile using liquid chromatography-tandem mass spectrometry

Background: Neonatal screening programs for congenital adrenal hyperplasia (21-CAH) using an immunoassay for 17 alpha-hydroxyprogesterone (17-OHP) generate a high rate of positive results attributable to physiological reasons and to cross-reactions with steroids other than 17 alpha-OHP, especially in preterm neonates and in critically ill newborns. Methods: To increase the specificity of the screening process, we applied a liquid chromatography-tandem mass spectrometry method quantifying 17 alpha-OHP, 11-deoxycortisol, 21-deoxycortisol, cortisol, and androstenedione. The steroids were eluted in aqueous solution containing d(8)-17 alpha-OHP and d(2)-cortisol and quantified in multiple reaction mode. Results: Detection limit was below 1 nmol/liter, and recovery ranged from 64% (androstenedione) to 83% (cortisol). Linearity was proven within a range of 5-100 nmol/liter (cortisol, 12.5-200 nmol/liter), and total run time was 6 min. Retrospective analysis of 6151 blood samples and 50 blood samples from newborns with clinically confirmed 21-CAH, as well as prospective analysis of 1609 samples of a total of 242,500 testing positive in our routine 17-OHP immunoassay, allowed clear distinction of affected and nonaffected newborns. High levels of 21-deoxycortisol were only found in children with 21-hydroxylase deficiency. Calculating the ratio of 17 alpha-OHP to 21-deoxycortisol divided by cortisol further increased the sensitivity of the method. Conclusion: Our liquid chromatography-tandem mass spectrometry procedure as a second-tier test can be used to reduce false-positive results of standard 21-CAH screening. The short total run time of 6 min allows for immediate reanalysis of all immunoassay results above the cutoff.