Endocrine Abstracts

JOINT522

Introduction: Thyroid hormones (TH) are critical for numerous biological processes including development, growth, and metabolism. The thyroid gland primarily secretes thyroxine (T4) and a small amount of triiodothyronine (T3), with T3 being predominantly produced through the deiodination of T4 in peripheral tissues. Deiodinase enzymes both activate T4 to T3 and facilitate the deactivation/clearance of T3 into various metabolites, including rT3, 3, 5-T2, 3, 3’-T2, 3’5’-T2, 3-T1, 3’-T1, and T0.

Methods and Results: We developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) method capable of analysing nine thyroid hormone metabolites (THM). Quantitation was achieved by comparison to reference standards following addition of internal standards (T3, rT3, 3-T1, and T4, all labelled with ¹³C6). Serum, calibrator, or quality control (QC) samples were treated with 1.25% NH4OH/acetonitrile (1:1) and centrifuged. The THM were then extracted using an Evolute Express AX 30mg solid phase extraction (SPE) plate, with the final elution using formic acid in methanol. After evaporation, samples were reconstituted in a methanol-water mixture before LC-MS/MS analysis using a Waters Acquity system with a Xevo-MS detector and a Luna Omega 1.6μm Polar C₁₈ column. Full chromatographic separation of nine THM was achieved within a 7-minute run time. The method demonstrated recovery rates ranging from 96-107%. Application of the method to male serum enabled the quantification of all nine THM (T0, 3-T1, 3’-T1, 3, 5-T2, 3, 3’-T2, 3’, 5’-T2, T3, rT3, T4).

Conclusions: This high-throughput LC-MS/MS method, provides a comprehensive tool for thyroid hormone profiling that will significantly advance THM research. Method validation is ongoing and will facilitate THM profiling in healthy and disease populations. This method has the potential to enhance understanding of TH metabolism in multiple contexts, supporting clinical diagnosis and management.