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 ( 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.