Endocrine Abstracts

Steroids play key roles in numerous biological processes, including blood pressure regulation, glucose metabolism, stress response, muscle development, and reproduction. Accurate, precise quantification in biological fluids is achieved using mass spectrometry. To date, most research has focused on serum or plasma requiring invasive sampling or on 24-hour urine, which is an inconvenient matrix to collect. Saliva provides a simple, non-invasive alternative, however current applications have been limited to single analytes or small steroid panels for targeted clinical use, such as diagnosing Cushing’s syndrome. The broader salivary steroid profile remains less characterised, and its utility as a biofluid for steroid research is not well established. We therefore aimed to firstly comprehensively investigate the salivary steroid profile and secondly to develop a single liquid chromatography–triple quadrupole mass spectrometry (LC-MS/MS) method for their quantification. Using a top-down approach, saliva from six healthy volunteers (three males, three females) was screened across multiple LC-MS/MS and gas chromatography–mass spectrometry (GC–MS) assays for 68 candidate steroids. Of these, 44 were detectable, spanning androgens, 11-oxygenated androgens, glucocorticoids, mineralocorticoids, precursor steroids, and compounds usually observed in urine (e.g., THE, 20α/βDHE). A single LC-MS/MS extraction and analysis method for these steroids was then developed and validated according to EMA bioanalytical guidelines (2023). Its applicability was demonstrated in a cohort of healthy participants. These findings highlight saliva’s potential as a practical, non-invasive bio-fluid with an extensive steroid profile. Future work will compare salivary, plasma, and urinary profiles, supporting saliva’s use as a primary biofluid for endocrine research.