Dried blood spot multiplexed steroid profiling using liquid chromatography tandem mass spectrometry in Korean neonates

Background: Screening for congenital adrenal hyperplasia (CAH) using immunoassays for 17α-hydroxyprogesterone generates many false-positive results. We developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for simultaneous quantification of nine steroid hormones in dried blood spot (DBS) samples, and established reference intervals for these hormones. Methods: We examined our method for linearity, precision, accuracy, extraction recovery, and matrix effects and determined the reference intervals of cortisol, 17α-hydroxyproges-terone, 11-deoxycortisol, 21-deoxycortisol, androstenedione, corticosterone, 11-deoxycorticosterone, testosterone, and progesterone in 1,146 DBS samples (from 272 preterm and 874 full-term neonates). Immunoassay and LC-MS/MS methods were compared for 17α-hydroxyprogesterone. Fourteen additional samples were tested to validate the clinical applicability of the LC-MS/MS method. Results: The linearity range was 2.8-828.0 nmol/L for cortisol and 0.9-40.0 nmol/L for the other steroids (R2>0.99). Intra-day and inter-day precision CVs were 2.52-12.26% and 3.53-17.12%, respectively. Accuracy was 80.81-99.94%, and extraction recovery and matrix effects were 88.0-125.4% and 61.7-74.2%, respectively. There was a negative bias, with higher values measured by immunoassay compared with LC-MS/MS (r=0.8104, P< 0.0001). The LC-MS/MS method was successfully applied to the analysis of nine steroids in DBS for screening and diagnosis of CAH using the 14 additional samples. Conclusions: Our method enables highly sensitive and specific assessment of nine steroids from DBS and is a promising tool for clinical analysis of CAH.