박소연 (한국과학기술연구원)

최만호 (한국과학기술연구원)

박소연 (한국과학기술연구원)

이준석 (한양대학교)

최만호 (한국과학기술연구원)

Salivary steroids have emerged as an alternative to serum steroids due to their correlation with serum levels and the stress-free nature of sampling. Although mass spectrometry has been the gold standard for steroid profiling in endocrine tests, it requires complex sample preparation to analyze trace amounts of steroids. In this study, a liquid chromatography-mass spectrometry (LC-MS)-based reliable sample preparation method was developed to measure five adrenal steroids (cortisol, cortisone, DHEA sulfate, 11-dehydrocorticosterone, and 11-ketoandrostenedione) and four synthetic steroids (dexamethasone, fludrocortisone, prednisolone, and prednisone) in human saliva. Saliva (100 μL) was diluted with methanol, containing deuterated steroids, followed by the addition of water. After removing precipitates via centrifugal filtration, the sample was injected into the LC-MS. The lower limit of quantification for all analytes was 0.2 to 2.0 ng/mL with good linearity (r² > 0.97) over the calibration range of 0.1 to 100 ng/mL. Precision and accuracy ranged from 1.5 to 24.7% and 83.1 to 114.7% for intra-day repeatability, respectively, and from 2.8 to 19.1% and 81.1 to 109.1% for inter-day reproducibility, respectively. Recovery rates ranged from 88% to 128%. This novel method is simple and less dependent on experimental expertise, making it widely applicable in clinical settings where steroid levels serve as biomarkers for diagnostic purposes or to reflect steroid exposure under physiological conditions.