Development and Evaluation of a Candidate Reference Method for Cortisol in Human Serum by Isotope Dilution Liquid Chromatography-Tandem Mass Spectrometry
Objective To develop a candidate reference method for cortisol in human serum by isotope dilution liquid chromatography-tandem mass spectrometry(ID-LC/MS/MS).Methods An isotope-labeled internal standard was added to samples,followed by alkalizing with sodium carbonate solution and liquid-liquid extraction with n-hexane/ethyl acetate.Isoelution with a methanol aqueous solution was employed for liquid chromatographic separation,while ESI in the positive ion and multiple reaction monitoring mode were used for mass spectrometry.The volume of sample,standard,and internal standard solutions were all controlled by weighing,and the results were calculated by bracketing method.The accuracy,precision,specificity,linearity,LOD and LOQ of this method were evaluated referring to the CLSI C62-A and C50-A guidelines and the domestic expert consensus documents.Results The method demonstrated excellent accuracy and precision with the bias of ERM-DA192,ERM-DA193 and RELA 2021-2023 all being less than 2％.The recovery of added cortisol ranged from 98.2％to 101.1％.Both intra-and inter-assay imprecisions was＜2％.The method was free from interference by structural analogue and showed a good linearity in the range of 25-1 600 nmol/L.The LOD and LOQ were 0.5 nmol/L and 1.0 nmol/L,respectively.A cortisol assay kit(chemiluminescence immunoassay)traced to this candidate reference method was used to determine 46 clinical serum samples concurrently,and the two methods exhibited good correlation.Conclusions A candidate reference method for the determination of cortisol in serum was established,demonstrating high sensitivity,good repeatability and accuracy.This method can serve as a reference for the measurement traceability and accuracy evaluation of routine methods.
HydrocortisoneIsotope dilution liquid chromatography-tandem mass spectrometryCandidate reference method
万方数据
维普
