Determination of serum cortisol using isotope dilution?liquid chromatography?mass spectrometry as a candidate reference method

Abstract

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.

Development of a Candidate Reference Measurement Procedure for the Analysis of Cortisol in Human Serum Samples by Isotope Dilution-Gas Chromatography-Mass Spectrometry

“…This density value was referred the previous paper. 10 b. The numbers after "±" are the expanded uncertainties, with a level of confidence of 95%, of the proceeding IDMS results.…”

“…The optimal equilibration time of cortisol and labeled compound in human serum samples has already been reported. 9,10 When the equilibration time was set to 2 h by referring to a previous paper, a high reliability measurement was possible. Therefore, the equilibration time was set to 2 h. Extraction efficiency of cortisol from a serum sample.…”

“…[5][6][7][8] On the other hand, highly reproducible, selective, and sensitive ID-liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS-MS) with electrospray ionization (ESI) were developed. 9,10 Two lyophilized human serum reference materials for cortisol 11,12 as Certified Reference Materials (CRMs), European Reference Material (ERM)-DA 192 and 193 (from European Commission Institute of Reference Materials and Measurements (IRMM)) and a reference panel of fresh frozen single-donation sera for cortisol 13 were certified using GC-MS reference methods for traceability assessment.…”

“…5,9,10 In previous methods, LLE using the dichloromethane method, 10 LLE using dichloromethane followed by a gel filtration chromatography method, 5 and SPE using C18 followed by LLE using an ethyl acetate method 9 were reported. In this work, we have developed a candidate reference measurement procedure for total cortisol in human serum involving ID-GC-MS with HFBA derivatization.…”

Development of Isotope Dilution-Liquid Chromatography/Tandem Mass Spectrometry as a Candidate Reference Method for the Determination of Folic Acid in Infant Milk Formula

“…Most of the information required to evaluate uncertainty were already obtained during the repeatability and reproducibility test. The standard deviation of the measurement results of multiple subsamples within a period is the combination of random uncertainties 36,37 due to the uncertainties in weighing sample taken for analysis, weighing the 13 C 5 -folic acid solution spiked to the sample, and the LC/MS/MS measurements of calibration mixtures and sample extracts. The uncertainties in the standard solution and the calibration mixtures give systematic effects on the results and are not included in the repeatability.…”