Simultaneous measurement of cortisol and cortisone in saliva by LC-MS/MS: Application in basal and stimulated conditions

Salivary cortisol is an increasingly popular tool in endocrine, psychological and sports studies. Immunoassays used for its measurement are limited by cross-reactivity from related steroids, mainly cortisone, which is abundant in saliva. A method was developed for the simultaneous measurement of cortisol and cortisone (SalF and SalE respectively) in saliva using LC-MS/MS. 40 μl of extract was injected onto a C8 4×2 mm guard cartridge attached to a C18 3.5 μm 2.1×50 mm analytical column. The eluant was then injected into a tandem mass spectrometer. The method was linear up to 100 nmol/l for SalF and 200 nmol/l for SalE and the lower limits of quantitation were 0.5 nmol/l (SalF) and 2.5 nmol/l (SalE). No evidence of ion suppression was found and precision, accuracy and recovery were within internationally accepted limits for both compounds. Samples were stable at room temperature for at least 2 weeks. The method was applied in 24 healthy volunteer morning and bed-time saliva samples and 96 saliva samples from 32 normal SSTs (peak serum cortisol >500 nmol/l). Results are reported in the table below as median (range). SalF, SalE and the SalF/SalE ratio were significantly greater in the morning (am versuss pm, P