Development and validation of a candidate reference measurement procedure for free triiodothyronine and free thyroxine in human serum using equilibrium dialysis isotope-dilution liquid chromatography-tandem mass spectrometry
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Min Zhan
und
Xianzhang Huang
Abstract
Objectives
Free triiodothyronine (FT3) and free thyroxine (FT4) are important diagnostic markers for assessing thyroid function. However, their accurate quantification remains challenging due to low serum concentrations. Significant variability exists among current assay methods for measuring FT3 and FT4. This study aims to establish a candidate Reference Measurement Procedure (cRMP) for simultaneous quantification of serum FT3 and FT4 based on isotope-dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) technology.
Methods
A convenient and reusable equilibrium dialysis (ED) device was utilized to separate free thyroid hormones from their protein-bound counterparts in serum. Key dialysis parameters, including temperature, pH, membrane type, and duration, were optimized to ensure consistent and reliable performance. The dialysate containing FT3 and FT4 was directly quantified by ID-LC-MS/MS. The method underwent systematic validation, comparative analysis with existing assays, and a comprehensive uncertainty assessment.
Results
The developed cRMP demonstrated limits of quantification (LoQ) of 1.54 pmol/L for FT3 and 3.22 pmol/L for FT4, and with an imprecision of less than 3 %. No interference from endogenous analogs was observed, and the method showed good consistency in interlaboratory comparison. In contrast, chemiluminescent immunoassay results exhibited poor agreement with and the cRMP.
Conclusions
This study developed a highly precise, accurate, specific, and sensitive ID-LC-MS/MS-based cRMP for the simultaneous measurement of FT3 and FT4 in human serum. This method provides a reliable tool for standardizing routine thyroid function tests.
Funding source: The National Key Technologies R&D Program of China
Award Identifier / Grant number: 2022YFC3602302
Funding source: National High Level Hospital Clinical Research Funding
Award Identifier / Grant number: BJ-2025-136
Funding source: the Specific Research Fund for TCM Science and Technology of Guangdong Provincial Hospital of Chinese Medicine
Award Identifier / Grant number: YN2023QN07
Funding source: Guangdong Provincial Clinical Research Center for Laboratory Medicine
Award Identifier / Grant number: 2023B110008
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Research ethics: The collection of leftover serum samples was approved by the Ethics Committee of the Second Affiliated Hospital of Guangzhou University of Chinese Medicine (No. ZE2022-331-01).
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Informed consent: Because leftover samples involved in this study were anonymous, the ethics committee allowed the exemption of informed consent.
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Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
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Use of Large Language Models, AI and Machine Learning Tools: None declared.
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Conflict of interest: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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Research funding: This work was supported by the National Key Technologies R&D Program of China (2022YFC3602302); National High Level Hospital Clinical Research Funding (BJ-2025-136); the Specific Research Fund for TCM Science and Technology of Guangdong Provincial Hospital of Chinese Medicine (YN2023QN07); Guangdong Provincial Clinical Research Center for Laboratory Medicine (2023B110008).
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Data availability: The raw data can be obtained on request from the corresponding author.
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Supplementary Material
This article contains supplementary material (https://doi.org/10.1515/cclm-2025-0874).
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