Endocrine Abstracts

Thyroid hormones (TH) are vital for many biological processes, including development, growth, and metabolism. Despite the existence of numerous thyroid hormone metabolites (THM) with potential biological significance, research and clinical evaluation has been largely limited to the measurement of pro-hormone thyroxine (T4) and the active hormone triiodothyronine (T3) by immunoassays. These assays are not suitable for detecting multiple THM as their similarity leads to cross-reactivity, therefore more accurate quantification methodologies are required. We developed a novel liquid chromatography tandem-mass spectrometry (LC-MS/MS) method for an extensive panel of twelve THM, enabling investigation into the thyroid hormone metabolic pathway. We determined the quantification range and investigated pooled male serum (Merck) after the addition of a mixture of internal standards (T3, rT3, 3-T1, and T4, all 13C₆). Extraction was optimised using an Evolute Express AX 30 mg SPE plate, and samples were analysed on a Waters Acquity with Xevo-XS on a Luna Omega 1.6μm Polar C18 column. Complete chromatographic separation of all twelve THM was achieved within a 7-minute method (T0, 3-T1, 3’-T1, 3,5-T2, 3,3’-T2, 3’,5’-T2, T3, rT3, T4, TA2, TA3, TA4). Average recovery for nine THM was 101% (range 95.96-107.11%). Further optimisation is needed to improve recovery of thyroacetic acids (TA2, TA3, TA4). We were able to detect nine THM in pooled male serum. Our method provides a novel tool that will significantly advance THM research. Further optimisation and full method validation as well as determination of reference ranges in healthy populations is underway. This method will facilitate the simultaneous measurement of multiple THM to better understand TH metabolism in normal physiology and in thyroid disorders, leading to the potential development of THM signatures to support clinical diagnosis and management.