Serum and urine steroid metabolites
Prednisolone, prednisone, cortisol and cortisone were extracted from participants’ serum, calibration standards and quality control by liquid:liquid extraction using diethyl ether. The upper solvent layer containing the steroids was separated from the bottom aqueous layer using an ice-bath (Thermofisher) filled with ethanol. A rotary evaporator was used to dry down extracts and they were then reconstituted in 50:50 methanol-water before analysis on the mass spectrometer.Total urinary cortisol, cortisone, THF, alloTHF and THE were measured by using a validated high‐performance liquid chromatography tandem‐mass spectrometry assay