Analytical validation of a dried blood microsampling method to measure androstenedione, 17α‑hydroxyprogesterone, and 11‑ketotestosterone for congenital adrenal hyperplasia monitoring.

Measurement of androstenedione (4-AD) and 17α-hydroxyprogesterone (17-OHP) in blood is crucial for diagnosing and monitoring endocrine disorders such as congenital adrenal hyperplasia (CAH), which results from enzymatic defects in the steroidogenesis pathway. CAH patients require lifelong hormone replacement therapy to manage adrenal insufficiency and control androgen excess. Microsampling offers a less invasive alternative to frequent venous blood sampling, particularly in paediatric CAH patients, and enables at-home monitoring. Including 11-ketotestosterone (11-KT), an emerging biomarker in CAH, further enhances the clinical relevance of this approach. Liquid chromatography-tandem mass spectrometry-based methods were established and fully validated for the determination of 4-AD, 17-OHP and 11-KT in plasma, blood and 20 μL dried blood microsamples collected by volumetric absorptive microsampling (VAMS). For VAMS samples, we paid particular attention to robustness regarding storage and hematocrit to support future home sampling applications. The method's applicability was evaluated by assessing the agreement with plasma as the reference matrix for steroid hormone determination. A robust extraction procedure was developed, ensuring consistent recoveries across storage conditions and hematocrits (0.20 - 0.60 L/L). The method was successfully validated for 4-AD, 17-OHP and 11-KT in VAMS, blood and plasma samples, achieving lower limits of quantification of 30, 144 and 81 pg/mL in VAMS samples, respectively. Accuracy and precision were within 6.6% and 14.0% for all matrices. VAMS samples remained stable for up to one week at room temperature and after postal sending. A proof-of-concept study showed that plasma concentrations can reliably be derived from VAMS concentrations, when taking into account the hematocrit.