Endocrine Abstracts

Steroid profiling of biological fluids has been used for clinical and diagnostic research since the 1950s. Most methods focus on blood and urine, however, more recently saliva analysis has been employed as a non-invasive, simple to collect sample for the diagnosis of conditions such as Cushing’s and androgen excess. Despite its ease of collection, saliva remains an underutilised biofluid. To investigate the use of salivary steroid profiling for endocrine research, we aimed to develop and validate a liquid chromatography-tandem mass spectrometry method to quantify salivary androgens, glucocorticoids, and mineralocorticoids. Furthermore, we aimed to investigate the correlations between urine, serum, and salivary steroids. Twenty steroids were included in the assay. Calibrants and samples were spiked with isotopically labelled internal standards and extracted via supported liquid extraction (SLE) using methyl-tertbutyl ether. Steroids were separated on a Phenomenex Luna Omega C18 column (1.6 µm, 100 Æ, 2.1 x 50 mm), using a methanol and water (0.1% formic acid) linear gradient on an Acquity UPLC chromatography system with post-column infusion of ammonium fluoride. Quantification was performed on a Waters TQ-XS mass spectrometer using electrospray ionisation in positive ion mode. The method was clinically validated. The lower limit of quantification of the assay was ≤0.2 ng/ml for all steroids. Assay precision at low, medium, and high (0.2, 0.5, and 1 ng/ml) spiked QCs resulted in a variation of ≤20%. Calibrations were linear from 0.02-10 ng/ml with correlation coefficients (R2) of ≥0.98. Matrix effects, analyte recovery, reproducibility, and carryover demonstrated acceptable validation outcomes. Ten healthy participants provided matched saliva, serum, and urine to investigate correlations between the biofluids. 10 of the 20 steroids were detectable in saliva from healthy volunteers. In the future, this method will be used to obtain a healthy control reference cohort and investigate endocrine conditions.