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Steroid profile in dried blood spots by liquid chromatography tandem mass spectrometry: Application to newborn screening for congenital adrenal hyperplasia in China.

X. Zhan,L. Han,W. Qiu,Xuefan Gu,Jun Guo,Siyu Chang,Yu Wang,Huiwen Zhang

Published 2022 in Steroids

ABSTRACT

BACKGROUND Newborn screening for congenital adrenal hyperplasia (CAH) using 17-hydroxyprogesterone dissociation-enhanced, lanthanide fluorescence immunoassay (DELFIA) generates a large number of false-positive results. The present study aimed to improve the sensitivity of the CAH neonatal screening by including second-tier steroid profiling on a dried blood spots (DBS) using liquid chromatography tandem mass spectrometry (LC-MS/MS). METHODS We developed and validated a LC-MS/MS method for simultaneous determination of six steroids in DBS, including androstenedione, testosterone, 17-hydroxyprogesterone, 11-deoxycortisol, 21-deoxycortisol and cortisol. Two 5 mm blood spots were eluted by internal standard working solution. We analyzed 1170 DBS samples from neonates to determine gestational age-specific reference intervals. In order to test the specificity of the second-tier method, we analyzed 707 cards with a positive screening by DELFIA. RESULTS Values of intra- and inter-day precision coefficients of variance, and accuracy were 2.0%∼13.3% and 85.8%∼114.5%, respectively. Recovery ranged from 85.0% to 106.9%. The lower limit of quantification was 0.5 ng/mL for 21-deoxycortisol, 0.25 ng/mL for 17-hydroxyprogesterone and cortisol, 0.1 ng/mL for testosterone, androstenedione and 11-deoxycortisol, respectively. And the linearity range was 0.25-50 ng/mL (R2>0.99). According to the 17-hydroxyprogesterone levels and ratios of (androstenedione +17-hydroxyprogesterone)/cortisol in the 707 positive screening samples, 77 neonates should receive recall visit. The number of false-positive results reduced by 89.1%. Totally, 18 newborns were diagnosed with 21-hydroxylase deficiency, one with P450 oxidoreductase deficiency and one with 11β-hydroxylase deficiency. With two-tier screening, the positive predictive value increased to 26.0%. CONCLUSIONS The second-tier steroid profiling by LC-MS/MS reduce the false-positive rate and improve the positive predictive value of CAH screening. We suggest applying this steroid profiling assay as a second-tier test for CAH screening in China.

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