Measurement of twenty-one serum steroid profiles by UPLC-MS/MS for the diagnosis and monitoring of congenital adrenal hyperplasia.

Background: Congenital adrenal hyperplasia (CAH) represents a group of inherited disorders affecting steroidogenesis. Early and accurate diagnosis is crucial for effective treatment, particularly for preventing adrenal insufficiency and minimizing androgen excess. This study aims to develop and validate an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous quantification of 21 steroid hormones, including 11-oxygenated androgens, which are critical for diagnosing and monitoring various forms of CAH. Methods: We utilized a microbore column UPLC combined with hydroxylamine derivatization, which enabled excellent chromatographic separation and enhanced sensitivity of all target compounds. The method was evaluated for precision, linearity, recovery, ion suppression, and carryover according to FDA and CLSI guidelines. Steroid profiles from healthy controls and CAH patients were compared using Mann-Whitney tests. Results: The UPLC-MS/MS method demonstrated excellent precision (<20 % except for 11-ketoandrostenedione), linearity (R 2 > 0.99), low limits of detection and quantification, and satisfactory recovery (57-86 % absolute, 99-111 % relative). Our method showed good correlation with proficiency testing group means, although significant negative biases were noted for androstenedione, progesterone, and 11-deoxycortisol. In a clinical setting, significant increases in pregnenolone, progesterone, 17-hydroxyprogesterone, dehydroepiandrosterone, and other key steroids were observed in patients with 21-hydroxylase deficiency, while distinct profiles were identified for patients with 17-hydroxylase deficiency, cytochrome P450 oxidoreductase deficiency, and lipoid CAH. Conclusions: Our UPLC-MS/MS method provides a sensitive and specific tool for the comprehensive profiling of adrenal steroids, offering improved diagnostic accuracy for CAH. Its ability to differentiate between various CAH subtypes highlights its potential clinical utility in both diagnosis and monitoring.