Quantification of free serum cortisol based on equilibrium dialysis and isotope dilution-liquid chromatography–tandem mass spectrometry

ObjectivesOnly unbound serum cortisol is bioactive and protein binding of cortisol is highly variable. Thus, the quantification of free serum cortisol (FSC) is of superior biological relevance compared to total serum cortisol quantification. Consequently, the development of automated routine tests for FSC for endocrine testing is desirable — along the lines of free thyroid hormone measurement. Since the availability of a reliable and matrix-independent method is an important tool for this goal, we have developed a highly standardised mass spectrometric FSC method.Design and methodsWe used equilibrium dialysis (ED) to obtain a protein-free fraction from serum samples. The cortisol content of the dialysate was quantified using isotope-dilution two dimensional liquid chromatography (LCxLC–MS/MS).ResultsComprehensive evaluation characterised the method as reliable and robust; using commercially available dialysis cells, convenient handling was realised.ConclusionsThe method described in this article can be suggested for the implementation of a reference measurement system for FSC.