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Congenital adrenal hyperplasia (CAH) is a severe inherited disorder of cortisol biosynthesis that is potentially lethal or can seriously affect quality of life. For the first time, we aimed to assess the stability of 21-deoxycortisol (21Deox), 11-deoxycortisol (11Deox), 4-androstenedione (4AD), 17-hydroxyprogesterone (17OHP) and cortisol (Cort), diagnostic for CAH, in dried blood spots (DBSs) during a 1 year storage at different temperatures. Spiked DBS samples were stored at room temperature, 4 °C, -20 °C or -70 °C, respectively and ana