Serum steroid assays play an important role in the clinical evaluation of many common endocrine disorders [1]. Congenital adrenal hyperplasia (CAH), the most common adrenal gland disorder in infants and children, is a group of autosomal recessive disorders [2]. The 21-hydroxylase catalyzes hydroxylation of 17α-hydroxyprogesterone to 11-deoxycortisol in the glucocorticoid pathway [1]. In particular, 21-hydroxylase deficiency is the cause of approximately 95% of CAH cases [2]. Most neonatal screening programs worldwide include the detection of CAH, and the standard test parameter is 17α-hydroxyprogesterone [3^,4].

Although immunoassays to evaluate 17α-hydroxyprogesterone are easy to handle, rapid, and highly sensitive, they have questionable reliability because of a lack of specificity to CAH and matrix effects [5]. Conventional screening for CAH using immunoassays for 17α-hydroxyprogesterone generates many false-positive results [3^,5], which not only upset parents and medical staff but also necessitate subsequent costly clinical and laboratory analyses [3].

While the inclusion of extraction steps improves immunoassay specificity, all interfering molecules cannot be completely eliminated [5]. Several studies attribute the low specificity to cross-reactivity of antibodies with steroids other than 17α-hydroxyprogesterone, such as steroid sulfates, 17α-hydroxypregnenolone, and 17α-hydroxypregnenolone sulfate [1^,3^,5]. Poor immunoassay specificity is an issue, especially in preterm or stressed infants, who have high concentrations of delta-5 cross-reacting steroids [2]. Preterm neonates often have high concentrations of 17α-hydroxyprogesterone because of stress or delayed maturation of 11-hydroxylase [1], which can reduce diagnostic specificity in screening for CAH by antibody-based methods, potentially resulting in false-positive diagnosis and generating demand for a second-tier confirmation test [2]. Using steroid profiling by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in neonate screening for CAH yields fewer false-positive results because of its high analytical specificity and potential to quantify several compounds in a single run [2].

LC-MS/MS is the most accurate method currently available for measuring small molecules, and it can measure multiple steroid hormones simultaneously [6]. Steroid profiling with LC-MS/MS allows for evaluation of the status of enzymes involved in adrenal steroid biosynthetic pathways; thus, it is a better diagnostic tool than the evaluation of a single steroid [6]. A specific LC-MS/MS method with simultaneous detection of multiple steroids has been introduced to minimize unnecessary follow-up analyses [3^,6]. Although previous studies examined multiple steroid hormonal analyses using LC-MS/MS in Korea, they included limited numbers of steroids [6^,7] or did not evaluate clinical screening for CAH using patient samples [6]. Testosterone and progesterone, steroid hormones useful for the screening and diagnosis of CAH, have not been studied using dried blood spots (DBS) in Korean neonates [6^,7].

We developed and validated an LC-MS/MS method that accurately detects cortisol, 17α-hydroxyprogesterone, 11-deoxycortisol, 21-deoxycortisol, androstenedione, corticosterone, 11-deoxycorticosterone, testosterone, and progesterone in DBS, so as to be suitable for the screening and diagnosis of CAH in neonates. As the reference interval is not only method-specific [8] but also age-specific [9], we also determined age-specific reference intervals for the nine aforementioned steroids for healthy Korean neonates, including preterm neonates.

We developed and validated an LC-MS/MS method for the simultaneous quantification of nine steroids in DBS. In addition, we determined age-specific reference intervals of the nine steroids for Korean neonates (preterm and full-term). To the best of our knowledge, this is the first attempt to determine reference intervals for testosterone and progesterone concentrations in DBS samples from Korean neonates.

Intra-day precision and inter-day precision CVs were comparable with results in the literature and guidelines [2^,12^,13^,16^,22]. Accuracy was within the acceptable range for neonatal screening using MS/MS [2^,4^,12^,13^,16^,17^,22]. Extraction recovery and the matrix effect were comparable to the previous findings and existing guidelines for neonatal screening by MS/MS [2^,11^,12^,13^,16^,17^,20^,21^,22^,23]. In our study, the bias between LC-MS/MS and immunoassay findings changed over the measurement interval in a linear fashion, and there was a negative bias, with higher values measured by immunoassay than LC-MS/MS over the entire range.

To avoid unnecessary tests, a sensitive and specific test, such as the LC-MS/MS method, is advantageous [1^,7]. Our method allows for simultaneous and rapid quantitation of nine steroids related to CAH with high specificity and without compromising chromatographic resolution.

Differential diagnosis of CAH subtypes is possible by steroid profiling using the LC-MS/MS method [1]. In this method, diagnosis of CAH depends on not only the absolute quantification of 17α-hydroxyprogesterone but also the ratio of multiple steroids to determine a positive result: 17α-hydroxyprogesterone (a direct substrate for 21-hydroxylase), cortisol (the final product of the adrenal enzyme's action), and androstenedione (which secondarily accumulates in 21-hydroxylase deficiency) [2]. Although the prevalence of CAH other than 21-hydroxylase deficiency is very low, and samples from patients with those diseases were not tested in this study, steroid profiling on the basis of ratios of multiple steroids, including precursor hormone and product hormones, used in this method has diagnostic strengths.

According to the CLSI guidelines [9], at least 120 samples for analysis are required to establish reference intervals. Some previous studies on the development and application of mass spectrometry for steroid profiling included less than 120 samples for establishing reference intervals [6^,8^,23^,25^,27]. Although the various reference intervals presented in these studies corresponded to different analytical conditions, they were quite similar to the present ones, as shown in Tables 3 and 4 [6^,8^,23^,24^,25^,27]. We included more than 120 samples for all nine steroids, especially testosterone and progesterone, which previous studies did not do [8^,25^,27].

Furthermore, we successfully demonstrated the clinical applicability of steroid profiling to differentiate between neonates with 21-hydroxylase deficiency and normal neonates. However, the reference interval was not established using samples from children to appropriately differentiate children with CAH from the normal ones. Considering that there was a 3-year-old girl with congenital adrenal lipoid hyperplasia due to StAR mutation whose cortisol concentration was higher than the upper limit of the reference interval for full-term neonates, whereas the other steroids were within the reference interval for full-term neonates, the reference interval of hormone analyses should be established separately for different age groups [6^,9].

The limitation of this study was the limited numbers of samples for validation, because the prevalence of CAH is low [25]. Previous studies also included limited numbers of CAH patients for clinical application due to the low prevalence of CAH, but they reported successful clinical application of the methods in a manner similar to this study [2^,5^,7].

In conclusion, our method produced lower values for 17α-hydroxyprogesterone than an immunoassay, consistent with the relatively low analytical specificity of immunoassays compared with LC-MS/MS. We also established reference intervals for nine steroid hormones in Korean neonates, which were comparable with those in other populations. Steroid profiling by LC-MS/MS highlights the utility of secondary diagnostic markers as a means of reducing false-positive results due to stress- or illness-induced transient elevation of 17α-hydroxyprogesterone. Thus, our sensitive, specific, and accurate steroid profiling method is suitable for routine neonatal screening for CAH.

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.

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.