A simplified liquid chromatography tandem mass spectrometry assay, using on-line solid-phase extraction, for the quantitation of cortisol in saliva and comparison with a routine DELFIA method

BACKGROUND: We have developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for measuring salivary cortisol, which requires only 200 microL sample and no extraction. METHODS: Sample (200 microL) and 25 microL internal standard were added directly to a 96-deep-well plate. Of this, 50 microL was loaded onto a guard cartridge, the cartridge was then washed and the eluate was diverted to waste. The compounds were eluted from the guard cartridge onto the C18 analytical column. Cortisol and deuterated cortisol were monitored using transitions m/z 363.2 > 121.1 and 365.1 > 122.2, respectively. RESULTS: The method had a lower limit of quantitation of 2 nmol/L. Intra-assay and inter-assay imprecision were better than 9.5%. Comparison with an established dissociation enhanced lanthanide fluorescent immunoassay (DELFIA) gave good agreement for the majority of samples; LC-MS/MS = 1.0065 x DELFIA - 3.7 (n = 130). The reference range was determined to be 5.8-45.7 nmol/L at 08:00 h and <6.4 nmol/L at 23:00 h (n = 44). CONCLUSIONS: We have developed a simple, robust assay to measure salivary cortisol using on-line solid-phase extraction to reduce sample clean-up requirements.