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

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.

Free and total cortisol levels are useful prognostic markers in critically ill patients: a prospective observational study

“…Commonly, targeted mass-spectrometry analysis is carried out using triplequadrupole mass spectrometers. The method and the way of analysis are different compared with our measurement, but both assessments give similar results without the disadvantages of immunoassays (28). We believe that our results are fully transferable to cortisol levels determined by triple-quadrupole MS.…”

“…Measurement of cortisol levels using immunoassays is disturbed by many analytical errors, especially in critically ill patients. Our method of measuring cortisol levels using LC-MS was previously validated in normal subjects (28). The method is capable of specific cortisol quantification in different matrices on the basis of accurate mass identification.…”

“…The sample preparation and the measurements for TC and FC analysis were carried out using HPLC-coupled high-resolution ESI-TOF MS according to the validated method described by Montskó et al (28). Briefly, during TC analysis, 20 ml of internal standard solution (1.1 mmol/l) was added to 100 ml serum, protein was precipitated by 300 ml acetonitrile, centrifuged at 14 000 g for 10 min.…”

“…Recently, a major development has been achieved in the measurement of cortisol levels by the technique of liquid chromatography coupled mass spectrometry (LC-MS) (22,28). LC-MS has a greater analytical specificity in the detection of total and free serum cortisol than immunoassays and is able to separate and quantify serum TC in the presence of other steroids and metabolites (25,26,27).…”

Use of Objective Neurocognitive Measures to Assess the Psychological States that Influence Return to Sport Following Injury

“…The above techniques could further be coupled with real-time measures of stress such as electrodermal activity (EDA; a measure of the sympathetic nervous system) [33,34] or serum/salivary cortisol levels [35] before, during, and after an anticipated kinesiophobic event. This correlated data would further identify when an individual is truly experiencing kinesiophobia or reinjury anxiety.…”