Ann Lab Med.  2019 May;39(3):263-270. 10.3343/alm.2019.39.3.263.

Dried Blood Spot Multiplexed Steroid Profiling Using Liquid Chromatography Tandem Mass Spectrometry in Korean Neonates

• Choi R
• Park HD
• Oh HJ
• Lee K
• Song J
• Lee SY

• KMID: 2431603
• DOI: http://doi.org/10.3343/alm.2019.39.3.263

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-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.

Keyword

17α-hydroxyprogesterone; Steroid hormones; Congenital adrenal hyperplasia; reference intervals; LC-MS/MS; Dried blood spot

MeSH Terms

Adrenal Hyperplasia, Congenital
Androstenedione
Bias (Epidemiology)
Chromatography, Liquid*
Corticosterone
Cortodoxone
Diagnosis
Humans
Hydrocortisone
Immunoassay
Infant, Newborn*
Mass Screening
Mass Spectrometry
Methods
Progesterone
Steroids
Tandem Mass Spectrometry*
Testosterone
Androstenedione
Corticosterone
Cortodoxone
Hydrocortisone
Progesterone
Steroids
Testosterone

Figure

• 

  Fig. 1 Comparison of LC-MS/MS and immunoassay. Correlation (A), absolute difference (B), and percent bias (C) between LC-MS/MS and immunoassay results for 17α-hydroxyprogesterone in 1,146 dried blood spots from Korean neonates. The solid line in (A) is the best-fit regression line and dashed lines in (A) represent 95% confidence interval. Solid lines in (B) and (C) represent average differences, and dashed lines in (B) and (C) represent limit of agreement.Abbreviation: LC-MS/MS, liquid chromatography-tandem mass spectrometry.

• 

  Fig. 2 Steroid profiles for 14 additional samples from neonates and children used to evaluate the clinical applicability of the LC-MS/MS method. Full-term neonates with 21-CAH (Δ) had distinctively low concentrations of cortisol, high concentrations of 21-deoxycortisol, 17α-hydroxyprogesterone, 11-deoxycorticosterone, and high values of (androstenedione+17α-hydroxyprogesterone)/cortisol ratio (A & B). Other steroid hormone concentrations in full-term neonates with 21-CAH were variable (C). Two children with 21-CAH had high concentrations of cortisol and 17α-hydroxyprogesterone, progesterone, and androstenedione. Concentrations of other steroids were not distinctive. One child with CALH had high concentration of cortisol and low concentrations of other steroids.Abbreviations: CAH, congenital adrenal hyperplasia; 21-CAH, congenital adrenal hyperplasia caused by 21-hydroxylase deficiency; CALH, congenital adrenal lipoid hyperplasia due to the StAR mutation; LC-MS/MS, liquid chromatography-tandem mass spectrometry.

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