SUN-452 - Isotope Dilution LC-MS Assay for Salivary Steroid Profiling in the Assessment of Adrenal Insufficiency

Although mass spectrometry has been the promising tool for steroid profiling in endocrine tests, the complex sample preparation limits its clinical feasibility. An isotope dilution mass spectrometry coupled to liquid chromatography (LC-IDMS) was developed for quantification of cortisol, cortisone and DHEA sulfate (DHEA-S) in human saliva. The assay was fully validated and assessed with saliva samples collected from 11 patients with adrenal insufficiency (AI) and 22 control subjects without adrenal disorders. Levels of salivary cortisol, cortisone and DHEA-S at baseline, and 30 min and 60 min after Synacthen test were investigated. Saliva (100 μL) diluted with methanol, containing deuterated adrenal steroids, and water was processed via centrifugal filtration, and directly injected into the LC-MS. The lower limit of quantification for all analytes was 0.5 to 1.0 ng/mL with good linearity (r2 > 0.97) over the calibration range of 0.1 to 100 ng/mL. Precision and accuracy ranged from 2.1 to 12.1% and 97.2 to 110.0% for intra-day repeatability, respectively, and from 2.8 to 19.1% and 81.9 to 106.4% for inter-day reproducibility, respectively. Recovery rates ranged from 95.8% to 103.9%. In addition to baseline cortisol and cortisone (AUROC 0.57, 95% CI 0.35~0.78; cut-off 0.54 ng/mL vs. AUROC 0.72, 95% CI 0.52~0.91; cut-off 5.98 ng/mL), both cortisol and cortisone at 30 min and 60 min after Synacthen stimulation test had good diagnostic performance with AUROC 0.84~0.86 (accuracy 85% and 82% for cortisol; 73% and 70% for cortisone). The peak concentrations of cortisol and cortisone provided the AUROC of 0.86 (95% CI 0.72~1.00; cut-off 3.06 ng/mL) and 0.83 (95% CI 0.68~0.98; cut-off 16.21 ng/mL), respectively. The baseline salivary cortisone and both salivary cortisol and cortisone combined with Synacthen test had good diagnostic performance in assessing AI. This novel LC-IDMS method provides a straightforward analytical assay with less dependent on experimental expertise, enhancing its feasibility in clinical practice.

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