Ann Lab Med 2019; 39(3): 263-270  https://doi.org/10.3343/alm.2019.39.3.263
Dried Blood Spot Multiplexed Steroid Profiling Using Liquid Chromatography Tandem Mass Spectrometry in Korean Neonates
Rihwa Choi, M.D.1,2,3, Hyung-Doo Park , M.D., Ph.D.2,3, Hyeon Ju Oh, M.S.2, Kyounghoon Lee, M.D.2,3, Junghan Song, M.D., Ph.D.4, and Soo-Youn Lee , M.D., Ph.D.2,3
1Department of Laboratory Medicine, Green Cross Laboratories, Yongin, Korea; 2Department of Laboratory Medicine and Genetics, Samsung Medical Center, Seoul, Korea; 3Department of Laboratory Medicine and Genetics, Sungkyunkwan University School of Medicine, Seoul, Korea; 4Department of Laboratory Medicine, Seoul National University College of Medicine, Seoul National University Bundang Hospital, Seongnam, Korea
Corresponding author: Hyung-Doo Park, M.D. https://orcid.org/0000-0003-1798-773X
Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, 81 Irwon-ro, Gangnam-gu, Seoul 06351, Korea
Tel: +82-2-3410-0290
Fax: +82-2-3410-2719
E-mail: nayadoo@hanmail.net
Soo-Youn Lee, M.D. https://orcid.org/0000-0001-7595-4042
Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, 81 Irwon-ro, Gangnam-gu, Seoul 06351, Korea
Tel: +82-2-3410-1834
Fax: +82-2-3410-2719
E-mail: sy117.lee@samsung.com
Received: June 10, 2018; Revised: September 7, 2018; Accepted: December 17, 2018; Published online: May 1, 2019.
© Korean Society for Laboratory Medicine. All rights reserved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
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-hydroxyprogesterone, 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.
Keywords: 17α-hydroxyprogesterone, Steroid hormones, Congenital adrenal hyperplasia, reference intervals, LC-MS/MS, Dried blood spot



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