MSACL 2022 Abstract

Urine Free Cortisol by LC-MS/MS: Monitoring Known Interferences as Potential Culprits of Iatrogenic Cushing’s Syndrome

Julie A. Ray1, Amol O. Bajaj1, Erik Kish-Trier1, Lisa M. Johnson1,2
1 ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, 84108, United States 2 Department of Pathology, University of Utah, Salt Lake City, UT, 84108, United States

Julie Ray, Ph.D (Presenter) ARUP Laboratories

Presenter Bio: Julie A. Ray is the Lead Scientist in the R&D Mass Spectrometry department at ARUP Laboratories in Salt Lake City, Utah. She specializes in developing high-sensitivity LC-MS/MS assays for small molecules, with a primary focus on steroids. Her work involves the extensive validation of quantitative methods and optimizing sample preparation to streamline high-throughput production.

Relevant Financial Disclosures (within past 24 months, reported on Apr 22, 2026) No relevant financial relationship(s) to disclose. Discussing Products? NO, presenter will NOT present educational content related to products/services of the ineligible company(ies) listed above. Conflict mitigation NOT REQUIRED.

Background: Cushing’s syndrome is caused by an excess of glucocorticoids that results in a variety of symptoms such as central obesity, moon faces, hirsutism, and reddish-purple stretch marks. Cortisol is the most potent endogenous glucocorticoid, and measuring the total amount excreted in the urine over a 24-hour period is useful to screen for Cushing’s syndrome. However, exogenous administration of glucocorticoids is thought to cause a majority of Cushing’s syndrome cases. This is often referred to as iatrogenic (drug-related or exogenous) Cushing’s syndrome. The structurally similar synthetic glucocorticoids prednisone and prednisolone are potential culprits and, importantly, potential LC-MS/MS interferences. Therefore, it is important to ensure that an LC-MS/MS method that monitors cortisol and cortisone can resolve prednisone and prednisolone.

Objectives: The primary objective of this study was to improve our current analytical LC-MS/MS method for urinary cortisol and cortisone, in order to screen for the presence of prednisone and prednisolone in patient samples. We used the new method to investigate the prevalence of prednisone and prednisolone in samples sent to ARUP Laboratories.

Methods: 250 µL aliquots of urine were mixed with 13C3 internal standards of cortisol and cortisone and extracted with 3:1 ethyl acetate: hexanes and dried down. Reconstituted extracts were analyzed using a Shimadzu LC20A LC with Restek Aromax (100 mm x 3 mm, 3 µm) column coupled to an AB SciexTM Triple Quad 5500 mass spectrometer setup for atmospheric pressure chemical ionization (APCI) in positive mode and Multiple Reaction Monitoring (MRM) acquisition. Calibrators were prepared in synthetic urine over a concentration range of 1-200 ng/mL. MRM transitions were added to monitor for the presence of prednisone and prednisolone using purchased chemicals (Cerilliant, Round Rock, TX). A threshold for interference was established and samples from nine deidentified patients known to be on prednisone and prednisolone were evaluated for specificity. Clinical data was then retrospectively analyzed over five months to investigate the presence of prednisone and prednisolone >5 ng/mL.

Results: The improved LC-MS/MS method was able to separate prednisone and prednisolone from cortisone and cortisol. During the validation, we determined that up to 5000 ng/mL prednisone and 1000 ng/mL of prednisolone would not interfere with results for cortisone and cortisol, respectively. Retrospective analyses of clinical data showed that approximately 0.9% of our samples had prednisone or prednisolone present >5 ng/mL. Thirty-three samples with cortisol concentrations <1 ng/mL, had prednisone concentrations ranging between 6.0-209 ng/mL and prednisolone concentrations ranging between 9.6-783 ng/mL. The presence of these exogenous glucocorticoids in low urine free cortisol samples is the expected pattern for potential iatrogenic Cushing’s syndrome.

Discussion and Conclusion: We developed and validated a robust, sensitive, and specific LC-MS/MS method for the measurement of free cortisol and cortisol in urine. Considering the metabolism of inactive prednisone to active prednisolone in the body, monitoring the presence of both exogenous drugs is of significance. Our improved LC-MS/MS method screens for prednisone and prednisolone use, which may assist in the diagnosis of iatrogenic Cushing’s syndrome.