Current assessment of patients with (a suspicion of) thyroid disease is predominantly based on the screening test, thyroid stimulating hormone (TSH). If TSH is out of range, patients are further characterized by measuring either free T4 (3,3’,5,5’-tetraiodothyronine (L-thyroxine, T4)), (free) 3,3’,5–triiodothyronine (T3) or reverse T3. Thyroid hormones are metabolized via several metabolic pathways which is predominantly by deiodination, but also by (oxidative) deamination, decarboxylation, glucuronidation and sulfation. Thyroid hormone metabolism results in the production of different thyroid hormone metabolites (THMs) belonging to the group of iodothyronines, iodothyroacetic acids, iodothyronamines, iodoglucuronides and iodosulfates (1). Potential roles for these THMs have been described, examples being the role of 3,5-diiodothyronine (3,5-T2) in energy metabolism and thyromimetic effects such as TSH inhibition of 3,3’,5-triiodothyroacetic acid (TA3) and 3,3’,5,5’-tetraiodothyroacetic acid (TA4) (2). Adequate analytical methods to measure THMs are lacking, limiting the interpretation of concentrations of THMs in thyroid hormone pathophysiology and metabolism. Until now, method development of liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for THMs has been limited to single-analyte assays or small multi-analyte assays of iodothyronines with similar chemical properties.

We developed a multi-analyte assay for seven iodothyronines (L-thyronine (T0), 3-monoiodothyronine (3-T1), 3,5-T2, 3,3’-diiodothyronine (3,3’-T2), T3, rT3 and T4) and two iodothyroacetic acids (TA3, TA4) in human serum (3). The sample preparation consists of a protein precipitation and solid phase extraction (SPE) before quantification of the THMs with LC-MS/MS.

LC-MS/MS analysis is selective and enables the determination of several compounds in a single analysis. These strengths of LC-MS/MS are valuable but the methods can still be subject to analytical interferences which include matrix, contamination from substances added during sample preparation and isobaric compounds. Optimization during method development, such as using a selective sample preparation, optimize chromatography and measurement of several fragments per component in the mass spectrometry, can minimize the effect of interference. In terms of method development, a different approach is needed for a multi-analyte assay compared to a single-analyte assay. For a multi-analyte assay, instead of choosing the optimal condition for just one compound, an optimal balance of all settings for the several compounds must be obtained. The nine THMs have diverse chemical properties as the logarithmic partition coefficients (Log P) varies from -0.7 for the most hydrophilic compound (T0) to 5.2 for the most hydrophobic compound (TA4). Finding the optimal balance is even more challenging for compounds with these diverse chemical properties.

Important considerations for the method development of our multi-analyte assay are stable internal standards and the reconstitution method after drying under nitrogen. For adequate quantification, proper correction for the losses during sample preparation and LC-MS/MS settings by stable internal standards is key. The reconstitution method is important as very hydrophilic compounds can only be retained on the SPE-cartridge and the column if the samples are reconstituted in a low percentage of organic solvent. A low percentage of organic solvent is however suboptimal for the reconstitution of hydrophobic compounds. Consecutively adding the organic solvent, mixing the sample and adding the inorganic solvent can optimize the reconstitution method.

After the method development and analytical validation, we determined reference intervals in 253 healthy individuals. We observed a broad dynamic range among the THMs T0, 3-T1, 3,3’-T2, T3, rT3, T4 and TA4 (range 0.06 ng/dL to 10.0 µg/dL), 3,5-T2 was below 0.13 ng/dL and TA3 was not detected in healthy individuals. Our mass spectrometry-based panel of nine thyroid hormone metabolites in human serum may help to better understand thyroid hormone metabolism in health, disease and comorbidity. At present, we are focused on the clinical validation of the LC-MS/MS method.


  1. Peeters RP, Visser TJ. Metabolism of Thyroid Hormone. 2000.
  2. Zucchi R, Rutigliano G, Saponaro F. Novel thyroid hormones. Endocrine. 2019;66(1):95-104.
  3. Jongejan RMS, Klein T, Meima ME, Visser WE, van Heerebeek REA, Luider TM, et al. A Mass Spectrometry-Based Panel of Nine Thyroid Hormone Metabolites in Human Serum. Clin Chem. 2020, in press.