title: Candidate reference method for total thyroxine in human serum: use of isotope-dilution liquid chromatography-mass spectrometry with electrospray ionization.
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-d(5), 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-C(18) 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-C(18) 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 microg/L). The detection limits (at a signal-to-noise ratio of approximately 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 microg/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.
authors.affiliation: Analytical Chemistry Division, National Institute of Standards and Technology, Gaithersburg, MD 20899-8392, USA. susan.tai@nist.gov
authors.forename: Susan S-C
authors.initials: SS
authors.lastname: Tai
authors.forename: Lorna T
authors.initials: LT
authors.lastname: Sniegoski
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Welch
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods* / Q000706:statistics & numerical data; D006801:Humans; D016014:Linear Models; D011786:Quality Control; D012015:Reference Standards; D012016:Reference Values; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods* / Q000706:statistics & numerical data; D013974:Thyroxine / Q000097:blood*
chemical_list: D013974:Thyroxine