SUN-065 Clinical Utility of a Multiplexed LC-MS/MS Cortisol and Cortisone Assay

Abstract Disclosure: T.I. Muamar: None. B. Holmquist: None. Introduction: The Labcorp Endocrine Sciences / Esoterix laboratory has offered saliva cortisol testing for several decades to assist with Late Night Saliva Cortisol (LNSC) testing in accordance with the Endocrine Society guideline on the diagnosis of Cushing’s syndrome. In 2008 the method was converted from RIA to LC-MS/MS and in 2018 the assay was modified to include cortisone as an internal tool to detect exogenous interference of hydrocortisone cream (cortisol) from the patient’s hands during collection. Methods An analytical method was developed using a TX-4 HPLC system (Thermo-Fisher) with Agilent® 1200 pumps (Agilent Technologies, Inc.) and a Sciex® 5000 (Danaher) triple quadrupole mass spectrometer. Calibration curve based on powdered Cortisol and Cortisone (Sigma) was prepared in protein-enriched phosphate buffered saline. Metrology was verified by comparison to traceable reference materials (ChromSystems). Sample preparation consisted of isotope dilution using deuterated heavy isotope internal standard (ASI, Inc) followed by liquid-liquid extraction, evaporation and reconstitution. A reversed phase 3mm x 50mm C18 fused core analytical column (Phenomenex) was used with an acidified water/acetonitrile/methanol solvent gradient to achieve chromatographic separation of both analytes. Positive mode electrospray ionization (ESI) was used for detection in Multiple Reaction Monitoring (MRM) mode. Results Analytical range was 0.01 - 1 μg/dL. Inter-assay precision ranged from 9.8-12.4% and inter-assay accuracy ranged from 90.2 - 101.4%. Specimen stability was interrogated at ambient, refrigerated and frozen conditions. Multiple saliva collection devices and/or containers from different vendors were evaluated for suitability. Reference interval for adults were developed using residual specimen collected from employees at specific times. Clinical Utility Using historical data, thresholds for suspected interference were developed by monitoring the ratio of the two glucocorticoids. Retrospective analysis of our data (17 years for cortisol, five years for cortisone) has allowed us to evaluate contamination rate for specimens as well as perform indirect reference range analyses for both analytes at multiple collection times within the circadian cycle. Presentation: Sunday, July 13, 2025