Measuring steroids from dried blood spots using tandem mass spectrometry to diagnose congenital adrenal hyperplasia

Background: Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders that occur due to defects in the steroidogenesis pathway. Approximately 90% of CAH cases can be diagnosed by the measurement of serum 17-hydroxyprogesterone alone. However, the quantification of six additional steroids could significantly improve CAH laboratory diagnosis. Using dried blood spot (DBS) as specimen of choice can further improve patient care due to the small sample volume required for CAH diagnosis in neonates. Methods: An optimized DBS sample preparation method was employed for steroids quantification without the need of derivatization. A LC-MS/MS assay was developed and optimized using a reverse phase-ultra highpressure liquid chromatography (RP-UHPLC) system combined with triple quadrupole mass spectrometry using positive electrospray ionization mode. Results: The assay was validated according to CLSI analytical guidelines, including lower limit of quantification (LLOQ), linearity, precision, accuracy, carryover, and method comparison. The analytical measuring range of the method for all steroids was 2.5, 5, or 10 ng/ml to 250 ng/ml in DBS, r2 >= 0.995. The LLOQ, intra-day and interday precision were 0.11-1.8 ng/ml, 1.2-6.4 ng/ml, 1.8-11.5%, and 5.3-13.8%, respectively. Conclusions: Our LC-MS/MS assay simultaneously detects 7 steroids for the diagnosis of CAH and can be readily implemented in clinical laboratories to provide superior analytical performance over traditional immunoassays.

Automated summary

CAH is a group of autosomal recessive disorders caused by defects in the steroidogenesis pathway. The measurement of serum 17-hydroxyprogesterone can diagnose approximately 90% of cases, but quantifying six additional steroids can significantly improve laboratory diagnosis accuracy. Using DBS as specimen can further improve patient care and diagnosis accuracy in neonates.