Free thyroid hormones in serum by direct equilibrium dialysis and online solid-phase extraction--liquid chromatography/tandem mass spectrometry.
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 degrees 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.
Yue
Bingfang
B
ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT 84108-1221, USA. bingfang.yue@aruplab.com
Rockwood
Alan L
AL
Sandrock
Tanya
T
La'ulu
Sonia L
SL
Kushnir
Mark M
MM
Meikle
A Wayne
AW
D000328:Adult; D001798:Blood Proteins / Q000378:metabolism; D002247:Carbon Isotopes; D002853:Chromatography, Liquid; D003956:Dialysis; D005260:Female; D006801:Humans; D007118:Immunoassay; D007201:Indicator Dilution Techniques; D008297:Male; D011247:Pregnancy; D011262:Pregnancy Trimester, Second; D011485:Protein Binding; D012016:Reference Values; D044967:Serum; D052616:Solid Phase Extraction; D053719:Tandem Mass Spectrometry; D013974:Thyroxine / Q000097:blood*; D014284:Triiodothyronine / Q000097:blood*
D001798:Blood Proteins; D002247:Carbon Isotopes; D014284:Triiodothyronine; D013974:Thyroxine