Development and validation of a candidate reference method for serum cortisol by isotope dilution liquid...

Accurate and precise cortisol measurements are requisite for ensuring appropriate diagnosis and management of diseases related with adrenal or pituitary gland disorders. Prompted by the needs in characterization of certified reference materials and quality assurance for serum cortisol measurements, we developed and evaluated a highly reliable measurement procedure based on isotope dilution liquid chromatography-tandem mass spectrometry (ID LC-MS/MS) combined with dextran sulfate-Mg²⁺ precipitation as the sample pretreatment. An appropriate amount of serum was accurately weighed and spiked with the isotope labelled internal standard. After precipitation, massive lipids and lipoproteins were removed from serum matrix. The clear supernatant was transferred and extracted with ethyl acetate-hexane solution. The cortisol was analyzed with LC-MS/MS in positive electrospray ionization mode. The within-run and total coefficient of variations (CVs) ranged from 0.3 to 0.6% and 0.7 to 1.2%, respectively, for a concentration range of 76.30 to 768.04 nmol/L. A regression comparison of the results obtained by the present method and the certified values of ERM-DA451 showed agreement with no statistical difference (Y = 1.0092 X-0.7455; 95% CI for the slope, 0.9940 to 1.0212; 95% CI for the intercept, - 3.6575 to 2.6390, r² = 0.999). All structural analogs of cortisol tested were well resolved from cortisol in 12 min on a phenyl ligand column under an isocratic elution. The limit of quantification was estimated to 5 pg cortisol in absolute amount. This method is accurate and simple and can be served as a candidate reference measurement procedure in establishment of serum cortisol reference system.