A validated method for measurement of serum total, serum free, and salivary cortisol, using high-performance liquid chromatography coupled with high-resolutionESI-TOF mass spectrometry

Blood cortisol level is routinely analysed in laboratory medicine, but the immunoassays in widespread use have the disadvantage of cross-reactivity with some commonly used steroid drugs. Mass spectrometry has become a method of increasing importance for cortisol estimation. However, current methods do not offer the option of accurate mass identification. Our objective was to develop a mass spectrometry method to analyse salivary, serum total, and serum free cortisol via accurate mass identification. The analysis was performed on a Bruker micrOTOF high-resolution mass spectrometer. Sample preparation involved protein precipitation, serum ultrafiltration, and solid-phase extraction. Limit of quantification was 12.5 nmol L −1 for total cortisol, 440 pmol L −1 for serum ultrafiltrate, and 600 pmol L −1 for saliva. Average intra-assay variation was 4.7 %, and inter-assay variation was 6.6 %. Mass accuracy was