Endocrine Abstracts

Cortisol is a glucocorticoid hormone with low molecular weight (362 Da) synthesized from cholesterol in the adrenal gland. The release is regulated by the hypothalamic–pituitary-adrenal (HPA) axis. Approximately 95% of the circulating amount is bound to proteins (CBG and albumin), but only the remaining free fraction is biologically active. Serum cortisol level is routinely analysed in laboratory medicine, though the widespread immunoassays (RIA and ECLIA) have the disadvantage of cross-reactivity with some commonly used steroid drugs, which can be eliminated by mass spectrometry. On the other hand in routine diagnostics we only have the opportunity to measure total cortisol concentration, but the identification of free cortisol provides more informative results. MS has become a method of increasing importance for cortisol estimation because after suitable sample preparation it provides the measurement of free fraction as well. Our aim was to develop a mass spectrometric method to analyze serum total, serum free, and salivary cortisol based on accurate mass identification, which can be reliably used in diagnostics and in therapy follow-up.

The analysis was carried out on a Bruker micrOTOF mass spectrometer using deuterated (9,12,12-D3) cortisol as internal standard. Sample preparation involved protein precipitation, serum ultrafiltration, and solid phase extraction. The limit of detection (LOD) for total cortisol measurements was 9 nmol/l and the limit of quantification (LOQ) was 15 nmol/l. The calibration curve was linear from 25 fmol to 1046 pmol (on column). Average intra-assay variation was 3.1%, while the inter-assay variation was 6.3%. Results were compared with the data of the Roche Modular Analytics E 170 ECLIA assay. The comparison resulted in eligible correlation (R²=0.96, slope=0.9725, CI 0.971–0.991 at 95%) in every (low, medium, and high) concentration range.

We can conclude that MS coupled with HPLC has higher specificity compared to immunoassays as identification is based on compound mass, instead of structural characteristics. We did not observe any interference with the therapeutically used steroid drugs, which is a common limitation of the immunoassays. Our method is capable of specific cortisol quantification in different matrices based on accurate mass identification.