Measurement of free thyroxine and triiodothyronine in human serum: Comparison between equilibrium dialysis, ultrafiltration and constant-volume diafiltration

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: MON 437-470-Non-neoplastic Thyroid Disorders
Monday, June 17, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board MON-462
Ashley Ribera*1, Julianne Cook Botelho2 and Hubert W Vesper3
1Centers for Disease Control and Prevention, 2Center for Disease Control and P, Atlanta, GA, 3Centers for Disease Control and, Atlanta, GA
The measurement of free triiodothyronine (FT3) and free thyroxine (FT4) levels in serum are an important part of the diagnosis and management of hypo- and hyperthyroidism. Existing clinical guidelines for the diagnosis and treatment of these thyroid disorders begin, in part, with the measurement of thyroid hormones not bound to thyroxine binding globulin, transthyretin and albumin: FT3 and FT4. Although FT3 and FT4 measurements are used extensively in research and clinical settings, available immunoassays are sensitive to alterations in binding proteins levels, which can occur in normal pregnancy and patients on estrogen treatment.  This can introduce bias in reported measurements.  The use of mass spectrometry, as the gold standard, to measure FT3 and FT4 require the isolation of the FT3 and FT4 while maintaining the free hormone equilibrium.  Equilibrium dialysis (ED) and ultrafiltration (UF) are two of the most common practices used for the isolation of FT3 and FT4 from serum. Diafiltration (DF) is a third method of separation that is designed to overcome long dialysis times and possible disruption of equilibrium and conversion of free to bound thyroid hormone during ultrafiltration. The proposed DF method maintains FT3 and FT4 equilibrium by allowing for constant sample volume while filtering FT3 and FT4 from the protein bound analytes.  After isolation by either ED, UF, or DF isotope-labeled internal standards (T3-13C6 and T4-13C12) are added to the serum material for quantification  and endogenous FT3 and FT4 is further isolated from the serum matrix using anion exchange solid phase extraction, followed by chromatographic separation from interfering compounds and quantitation by tandem mass spectrometry. A triple quadrupole mass spectrometer using electrospray ionization in the positive move coupled with HPLC is used for measurement. Chromatographic separation on a C18 column is performed using a gradient of water and acetonitrile with 0.1% formic acid. All analytes and internal standards are quantitated by selective reaction monitoring of a quantitation and confirmation ion peak. The performance of ED, UF, and DF methods has been assessed by comparing accuracy, recovery, repeatability and robustness of these methods.

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