Received on ,
Accepted on ,

Endocrinology and Metabolism

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

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

^* To whom correspondence should be addressed. 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: We selected ED 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 on-line SPE for FT₄ and FT₃ (also reverse T₃) are sufficient for the intended clinical use.