Serum total thyroxine measured by isotope dilution liquid chromatography tandem mass spectrometry

Objective An isotope dilution liquid chromatography tandem mass spectrometric (ID-LC/MS/MS) method was developed for serum total thyroxine. Methods Serum samples were sampled by weight and the 13C6-thyroxine internal standards were added volumetrically using automated dilutors, followed by equilibration, protein precipitation, and cation exchang solid-phase extractions(SPE). After SPE, the eluates were evaporated to dryness under nitrogen and then the evaporated residues were reconstituted to prepare samples for liquid chromatography-mass spectrometry electrospray ionization (LC/MS-ESI) analysis.Seven kinds of serum pools were used to analyze the precision of the method.The analytical recovery, the limit of detection(signal to noise ratio, S/N ratio 3∶1) and the limit of quantification (S/N ratio 10∶1)for thyroxine were calculated.Also, in order to evaluate the accuracy of this method, the candidate reference materials from German Societies for Clinical Chemistry were analyzed. Results The within-run and total coefficients of variation were: 0.64%(0.35%-0.89 %)and 0.83%(0.57%-1.37 %), respectively.The analytical recoveries ranged from 99.6% to 100.7%.The limit of detection was 0.12 nmol/L and the limit of quantification was 0.41 nmol/L for thyroxine in human serum.The results of analyzing the reference material of CRM21201 and CRM21202 showed biases of -0.30% (ranged from -0.13% to 0.73%). Conclusions A highly precise and accurate method for measuring serum total thyroxine has been developed by ID-LC/MS/MS. The method is less sample volume demand and less time-consuming and may be used as a candidate reference method. Key words: Thyroxine; Isotopes; Indicators and reagents; Chromatography, liquid; Tandem mass spectrometry