Candidate Reference Method for Total Thyroxine in Human Serum

Abstract

Background: There is a need for a critically evaluated reference method for thyroxine to provide an accuracy base to which routine methods can be traceable. We describe a candidate reference method involving isotope-dilution coupled with liquid chromatography/mass spectrometry. Methods: An isotopically labeled internal standard, thyroxine-d5, was added to serum, followed by equilibration, protein precipitation, and ethyl acetate and solid-phase extractions to prepare samples for liquid chromatography-mass spectrometry electrospray ionization (LC/MS-ESI) analysis. For separation, a Zorbax Eclipse XDB-C18 column was used with a mobile phase consisting of 1 mL/L acetic acid in acetonitrile-water (32:68 by volume) for positive ions and a Zorbax Extend-C18 column with a mobile phase consisting of 2 mL/L ammonium hydroxide in methanol-water (32:68 by volume) for negative ions. [M + H]+ ions at m/z 778 and 783 for thyroxine and its labeled internal standard were monitored for positive ions and [M − H]− ions at m/z 776 and 781 for negative ions. Samples of frozen serum pools were prepared and measured in three separate sets. Results: Within-set CVs were 0.2–1.0%. The correlation coefficients of all linear regression lines (measured intensity ratios vs mass ratios) were 0.999–1.000. Positive- and negative-ion measurements agreed with a mean difference of 0.45% at three concentrations (50, 110, and 168 μg/L). The detection limits (at a signal-to-noise ratio of ∼3 to 5) were 30 and 20 pg for positive and negative ions, respectively. The results from the LC/MS-ESI method were within 1 SD of the composite means from many routine clinical methods, although it appears that the clinical method means may be biased high by 4–5 μg/L across the concentrations. Some routine clinical methods may be biased by up to 20% at low concentrations. Conclusions: This well-characterized LC/MS-ESI method for total serum thyroxine with a theoretically sound approach, demonstrated good accuracy and precision, and low susceptibility to interferences qualifies as a candidate reference method. Use of this reference method as an accuracy base may reduce the apparent biases in routine methods along with the high interlaboratory imprecision.