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

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 (R 2 >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.

Evaluation of a Two-Tier Screening Pathway for Congenital Adrenal Hyperplasia in the New South Wales Newborn Screening Programme

“…There have been various studies investigating the feasibility of increasing the number of analytes in the steroid panel to increase specificity and sensitivity [ 44 , 45 , 46 , 47 , 48 ]. There are studies that show that the inclusion of 21-deoxycortisol and 11-deoxycortisol is more specific for detecting SWCAH by excluding β-hydroxylase deficiency [ 47 , 48 , 49 , 50 ].…”

The Potential of Steroid Profiling by Mass Spectrometry in the Management of Adrenocortical Carcinoma

“…In fact, the diagnosis, prognosis, and management of such diseases strongly depend on accurate quantification of steroid hormones in biological fluids. Imbalances in production or metabolism of steroids lead to a wide range of steroid related disorders, sometimes complex adrenal diseases, including congenital adrenal hyperplasia (CAH) [71][72][73][74]82,83], adrenal cancer [4,6,7,45,75,76], hyperaldosteronism [45,[84][85][86][87], Cushing's syndrome [68], disorder of sexual development (DSD) [44], and polycystic ovary syndrome (PCOS) [44,69,88]. For some of these disorders, it is still open to debate on which steroids are most appropriate to be quantified for the related diagnosis.…”

Adrenal Cushing Syndrome Diagnosed During Pregnancy: Successful Medical Management With Metyrapone

“…Therefore, these results were interpreted as appropriate for the child’s gestational age. Defining normal cortisol values is difficult in the neonatal period as studies have shown a large range of reference values [ 10 ]. There was no evidence of hypoglycemia, hyponatremia, hyperkalemia, lethargy, or vomiting.…”