背景：甲状腺激素是由下丘脑-垂体-甲状腺轴分泌，维持人体生长发育、调节代谢的重要激素，在血液中有结合态和游离态两种形式，游离甲状腺激素占据极少部分，主要包括游离甲状腺素（Free Thyroxine，FT4）和游离三碘甲状原氨酸（Free Triiodothyronine，FT3）。FT3、FT4 可以直接进入靶细胞发挥作用，因此可以更好反映甲状腺的功能状态，是临床甲状腺功能检测的首选指标。临床实验室常用免疫学方法检测 FT3、FT4，但2023年国家卫生健康委临床检验中心室间质量评价计划（External Quality Assessment，EQA）结果显示，免疫法在不同临床实验室间的检测结果一致性较差，变异系数（Coefficient Of Variation，CV）可达13.82%-21.42%，无法保证准确性。液相色谱串联质谱法（Liquid Chromatography-Tandem Mass Spectrometry，LC-MS/MS）特异、灵敏，已有部分实验室和厂家开发游离甲状腺激素的质谱法检测，国际检验医学溯源联合委员会列表中也收录了2种 FT4 的参考方法，均采用平衡透析结合质谱法检测，质谱法可能会在未来临床检测 FT3、FT4 中发挥优势并推进检测结果一致化。本研究旨在开发并验证基于平衡透析结合同位素稀释 LC-MS/MS 法的血清 FT4 准确定量参考测量程序，为实现 FT4 检测的一致化和标准化奠定基础。

方法：本研究使用目前可获得的最高等级的甲状腺素（Thyroxine，T4）标准物质IRMM-468作为标准品，保证方法的溯源性，配制标准溶液，探索并优化 T4 及其同位素内标色谱和质谱条件和参数。根据 CLSI 发布的 C62-A 等文件对 LC-MS/MS 法的检测限、定量限、基质效应、精密度和正确度等性能进行评价。

结果：本研究探索了最优的平衡透析条件，采用Scienova平衡透析装置，500 µL血清样本加到透析板样本侧，1400 µL平衡透析液加到透析板透析侧，37℃恒温箱环境下，透析板在摇床上以100 rpm/min速度平衡18 h。平衡完成后从透析侧取出含有游离激素的样本透析液，采用固相萃取法作为前处理方法来去除干扰达到目标物富集的目的。 LC-MS/MS 法中的液相色谱采用梯度洗脱，质谱采用电喷雾电离正离子模式和多反应监测模式对 T4 进行检测。本研究建立方法的检测限和定量限分别为0.71 pg/g和2.36 pg/g；内标可以有效校准基质效应，校准后的基质效应为1.65%；检测方法的批内、批间和总不精密度分别为2.34-2.78%，3.42-4.72%和4.14-5.51%；样本透析液的加标回收率为99.03%-104.26%；样本透析液检测的相对扩展不确定度为2.17%-3.95%。

结论：本研究探索了 FT4 的最优平衡条件，建立了基于平衡透析结合 LC-MS/MS 检测血清中 FT4 的候选参考方法，该方法具有很高的特异性、准确性、灵敏度和精密度，采用高等级的 T4 标准物质 IRMM-468 保证了方法的溯源性，能够为其他各实验室所建立的常规方法提供参考，有利于 FT4 检测结果的一致性和标准化。本研究建立的方法可以为 EQA 材料进行赋值，为 FT4 参考物质的研制奠定基础，是 FT4 检测标准化不可缺少的环节。

Background: Thyroid hormones are secreted by the hypothalamic-pituitary-thyroid axis and serve as crucial hormones for maintaining human growth, development, and metabolic regulation. In the bloodstream, they exist in two forms: bound and free, with free thyroid hormones constituting only a minimal fraction. These primarily include Free Thyroxine (FT4) and Free Triiodothyronine (FT3). FT3 and FT4 can directly enter target cells to exert their biological effects, making them superior indicators for assessing thyroid functional status and the preferred markers for clinical thyroid function testing. Clinical laboratories commonly employ immunoassays to measure FT3 and FT4. However, the 2023 External Quality Assessment (EQA) program conducted by the National Center for Clinical Laboratories revealed significant variability in immunoassay results across different clinical laboratories, with coefficients of variation (CV) ranging from 13.82% to 21.42%, indicating insufficient accuracy. In contrast, Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) offers high specificity and sensitivity. Several laboratories and manufacturers have developed mass spectrometry-based methods for free thyroid hormone measurement, and the Joint Committee for Traceability in Laboratory Medicine has listed two reference methods for FT4, both utilizing equilibrium dialysis combined with mass spectrometry. These advancements suggest that LC-MS/MS may emerge as a superior approach for future clinical FT3 and FT4 testing, promoting result harmonization. This study aims to develop and validate a reference measurement procedure for the accurate quantification of serum FT4 based on equilibrium dialysis coupled with isotope dilution LC-MS/MS, laying the foundation for achieving standardized and harmonized FT4 testing.

Methods: This study utilized the highest currently available grade thyroxine (T4) reference material, IRMM-468, as the calibrator to ensure method traceability. Standard solutions were prepared, followed by systematic optimization of chromatographic and mass spectrometric conditions and parameters for T4 and its isotopic internal standard. In accordance with documents such as CLSI C62-A, the performance characteristics of the LC-MS/MS method were rigorously evaluated, including limit of detection, limit of quantification, matrix effects, precision, and trueness.

Results: This study investigated the optimal equilibrium dialysis conditions using a Scienova dialysis device. For each dialysis unit, 500 μL of serum sample was loaded into the sample chamber and 1400 μL of dialysis buffer was added to the buffer chamber. The dialysis plate was then incubated at 37°C with constant shaking at 100 rpm for 18 hours. Following equilibrium, the dialysate containing free hormones was collected from the buffer chamber. Solid-phase extraction was employed as the pretreatment method to eliminate interferences and achieve target analyte enrichment. For LC-MS/MS analysis, gradient elution was applied in the liquid chromatography system, while electrospray ionization in positive ion mode with multiple reaction monitoring was used for T4 detection. The established method demonstrated a limit of detection of 0.71 pg/g and limit of quantification of 2.36 pg/g. The internal standard effectively corrected for matrix effects, yielding a normalized matrix effect of 1.65%. The method's precision was characterized by within-run, between-run, and total imprecision of 2.34-2.78%, 3.42-4.72%, and 4.14-5.51%, respectively. Spike recovery rates for dialysate samples ranged from 99.03% to 104.26%, with relative expanded uncertainty of measurement between 2.17% and 3.95% for dialysate analysis.

Conclusion: This study investigated the optimal equilibrium conditions for FT4 and established a candidate reference method for serum FT4 measurement based on equilibrium dialysis coupled with LC-MS/MS. The developed method demonstrates high specificity, accuracy, sensitivity, and precision. By employing the highest-grade T4 reference material IRMM-468, the method ensures metrological traceability and can serve as a reference for routine methods established in other laboratories, thereby facilitating the harmonization and standardization of FT4 measurements. The proposed method enables value assignment for EQA materials and lays the foundation for the development of FT4 reference materials. This represents an essential component in the standardization of FT4 testing, as it provides the necessary metrological basis for achieving comparable results across different measurement procedures.