BackgroundDifferentiating between adrenal Cushing syndrome (adrenal CS) and Cushing disease (CD) can be challenging if there are equivocal or falsely elevated adrenocorticotropic hormone (ACTH) values. We aim to investigate the diagnostic value of serum steroid profiles in differentiating adrenal CS from CD.MethodA total of 11 serum steroids in adrenal CS (n= 13) and CD (n= 15) were analyzed by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Age- and gender-specific steroid ratios were generated by dividing the actual steroid concentration by the upper limit of the relevant reference range. A principal component analysis (PCA) and an orthogonal partial least squares discriminant analysis (OPLS-DA) were performed.ResultsThe PCA and OPLS-DA analyses showed distinct serum steroid profiles between adrenal CS and CD. Dehydroepiandrosterone sulfate (DHEA-S), dehydroepiandrosterone (DHEA), and androstenedione ratios were identified as biomarkers for discrimination by variable importance in projection (VIP) in combination witht-tests. The sensitivity and specificity of DHEA-S ratios <0.40 were 92.31% (95% CI 64.0%–99.8%) and 93.33% (95% CI 68.1%–99.8%), respectively, in identifying adrenal CS. The sensitivity and specificity of DHEA ratios <0.18 were 100% (95% CI 75.3%–100.0%) and 100% (95% CI 78.2%–100.0%), respectively, in identifying adrenal CS.ConclusionOur data support the clinical use of the DHEA-S and DHEA ratios in the differential diagnosis of adrenal CS and CD, especially when falsely elevated ACTH is suspected.