Determination of serum cortisol using isotope dilution-liquid chromatography-mass spectrometry as a candidate...

We propose isotope-dilution mass spectrometry as a candidate reference method for determination of serum cortisol. The method uses liquid chromatography-mass spectrometry (LC-MS), interfaced with electrospray ionization, and selective monitoring of the [M + H]+ ions of cortisol and isotopically labeled cortisol. The isotope-dilution-liquid chromatography-mass spectrometry (ID-LC-MS) method simplifies sample-preparation, because samples are processed by simple solvent extraction without further clean-up and derivatization. We studied the time required for complete equilibration of endogenous cortisol and labeled cortisol spiked into serum and found it to be less than 1 h. The repeatability and the reproducibility of the method were evaluated and found to be 0.55% of the measurement value. CRM 192 and 193 from the Bureau Communautaire de Reference were analyzed for verification of the method. The results obtained from the ID-LC-MS method agreed with the certified values. The relative uncertainty of measurement results for samples in the range of a few tens of micrograms per kilogram to several hundred micrograms per kilogram was evaluated and found to be 0.56%. Immunoassay carried out by three independent clinical laboratories produced results more than 15% higher than this ID-LC-MS method, suggesting the presence of bias in the immunoassay methods.