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Clinical Chemistry 54: 642-651, 2008. First published February 7, 2008; 10.1373/clinchem.2007.098293
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Right arrow Endocrinology and Metabolism
(Clinical Chemistry. 2008;54:642-651.)
© 2008 American Association for Clinical Chemistry, Inc.

Endocrinology and Metabolism

Free Thyroid Hormones in Serum by Direct Equilibrium Dialysis and Online Solid-Phase Extraction–Liquid Chromatography/Tandem Mass Spectrometry

Bingfang Yue1,a, Alan L. Rockwood1,2, Tanya Sandrock1, Sonia L. La’ulu1, Mark M. Kushnir1 and A. Wayne Meikle1,2,3

1 ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah; 2 Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah; 3 Department of Medicine, University of Utah, Salt Lake City, Utah.

aAddress correspondence to this author at: ARUP Institute for Clinical and Experimental Pathology, 500 Chipeta Way, Salt Lake City, UT 84108-1221. Fax 801-584-5207; e-mail bingfang.yue{at}aruplab.com.

Background: Measurements of free thyroxine (FT4) and free triiodothyronine (FT3) are important for the diagnosis and monitoring of thyroid diseases. Considerable differences among methods limit their clinical utility, however, and accurate methods are needed for various clinical specimens. We describe a direct equilibrium dialysis (ED)–liquid chromatography (LC)/tandem mass spectrometry (MS/MS) method for FT4 and FT3.

Methods: ED was selected as the separation step. Serum samples were dialyzed 1:1 against a simple protein-free buffer for 20 h at 37 °C. Thyroid hormones in dialysates were purified by online solid-phase extraction (SPE), then chromatographically separated and quantified in positive ion and multiple reaction monitoring modes.

Results: For FT4 and FT3, the lower and upper limits of quantification were 1 ng/L (pg/mL) and 400 ng/L with total imprecision <10%. The method correlated well with an ED-RIA, 2 direct immunoassay methods for FT4, and 1 direct immunoassay and 1 tracer dialysis method for FT3. The adult reference intervals were 12.8–22.2 ng/L for FT4 and 3.62–6.75 ng/L for FT3. Reference intervals for the second trimester of pregnancy (14–20 weeks of gestation) were also established.

Conclusions: We developed a simple protein-free buffer and ED procedure. The performance characteristics and high throughput of the LC-MS/MS method with online SPE for FT4 and FT3 (also reverse T3) are sufficient for the intended clinical use.




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