title: A validated method for measurement of serum total, serum free, and salivary cortisol, using high-performance liquid chromatography coupled with high-resolution ESI-TOF mass spectrometry.
abstract: 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 <2.5 ppm. Serum total cortisol levels were in the range 35.6-1088 nmol L(-1), and serum free cortisol levels were in the range 0.5-12.4 nmol L(-1). Salivary cortisol levels were in the range 0.7-10.4 nmol L(-1). Mass accuracy was equal to or below 2.5 ppm, resulting in a mass error less than 1 mDa and thus providing high specificity. We did not observe any interference with routinely used steroidal drugs. The method is capable of specific cortisol quantification in different matrices on the basis of accurate mass identification.
authors.affiliation: Department of Laboratory Medicine, Faculty of Medicine, University of Pécs, 13 Ifjúság str., 7624, Pécs, Hungary.
authors.forename: Gergely
authors.initials: G
authors.lastname: Montskó
authors.forename: Zita
authors.initials: Z
authors.lastname: Tarjányi
authors.forename: Emese
authors.initials: E
authors.lastname: Mezősi
authors.forename: Gábor L
authors.initials: GL
authors.lastname: Kovács
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D006854:Hydrocortisone / Q000032:analysis* / Q000097:blood; D012463:Saliva / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D006854:Hydrocortisone