Introduction: There has been much interest recently in measuring both salivary cortisol and cortisone due to the presence of 11β-hydroxysteroid dehydrogenase type 2 enzyme in the salivary glands. This enzyme facilitates the conversion of cortisol to cortisone; hence the concentration of cortisone in saliva may also be of interest. Studies have shown recently that salivary cortisone and cortisol are good markers of serum free cortisol status.
Methods: Calibrator, quality control or sample (50 μl), 10 μl internal standard (D4 cortisol) and 150 μl water were added to wells of 96 deep-well plate. Online solid phase extraction (SPE) of 150 μl of this was performed using the Spark Holland Symbiosis in XLC mode (eXtraction Liquid Chromatography) with HySphere C18 HD 7 μm cartridges. Chromatography was performed using a Phenomenex Onyx Monolithic C18 column (25×4.6 mm).
Results: Retention times using the chromatography conditions was 2.22 min for cortisol and 1.98 min for cortisone with resolution of the two compounds. The limits of detection were 0.75 nmol/l for cortisol and 0.5 nmol/l minutes for cortisone. No ion suppression was observed 10 patient samples tested using Sarstedt Salivettes for cortisol (blue top), however green top Salivettes were found to be unsuitable due to a large background signal. Extraction cartridges were reliable for up to 15 uses.
Discussion: We have developed a novel assay for measuring salivary cortisol and cortisone in saliva utilising semi-automated solid phase extraction. This allows a sample clean-up step, which improves upon a previously published assay without adding to the length of time required to prepare an assay. We have found that the blue top Salivettes for cortisol are the preferred device for collecting samples for this assay.