Determination of Thyroid Hormones and 11 Metabolites in the Human Serum Using a Simple Derivatization Strategy and Analysis by Isotope-Dilution Liquid Chromatography Tandem Mass Spectrometry

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Abstract
Many analytical methods for thyroid hormone (TH) determination lack sensitivity and/or specificity. The thyroid hormone metabolites (THMs) are usually not measured at all. This study describes the development of sensitive high-throughput analytical methods for determining the total concentration and free fraction of TH and THM in the human serum. For the analysis of the TOTAL fraction, we employed protein precipitation and anionic exchanger solid-phase extraction. For the FREE fraction, ultrafiltration and salt-out liquid partitioning were used. Derivatization using dansyl chloride was employed to enhance the sensitivity of HPLC-ESI-MS/MS analysis. Both protocols were validated according to the European Analytical Guidelines (2002/657/EC). We obtained very good recoveries (73-115%) and precision. Interday coefficients of variation (CVs) for most of the analytes ranged from 1.2 to 16.4%. The sensitivity was excellent with detection limits in the sub ppt range for the majority of TH and THM. A significant enhancement in sensitivity (>10 fold) was achieved through derivatization. The applicability was proved on a set of samples from pregnant women enrolled in the CELSPAC cohort (n = 120). Our TH reference ranges are in good agreement with those reported in the literature. The methods also allowed us to quantify the levels of 11 THM, including some previously undetected THM in total and free fractions, and proved to be suitable for high-throughput routine TH and THM analyses. Our approach offers an important advancement in thyroid hormone analysis. To the best of our knowledge, it is for the first time that data for T1A and T2A as well as for free THM levels in the human serum are published in the literature. Moreover, our study also brings the first information about the levels of most of the THM in pregnant women.
Funding Information
  • Ministerstvo ?kolstv?, Ml?de?e a Telov?chovy (CZ.02.2.69/0.0/0.0/16_027/0008360, LM2023033, LM2023069)
  • Horizon 2020 Framework Programme (825753, 857560)
  • University Hospital Brno (65269705)

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