Thyroid hormones (THs), which mainly consist of 3, 3′, 5-triiodo-l-thyronine (T3) and L-thyroxine (T4), play a critical role in regulating biological processes such as growth and metabolism in various animal species. Thus, accurate measurement of T3 and T4, especially physiologically active free (protein-unbound) forms, in serum/plasma is needed for the evaluation of TH homeostasis. However, such high-precision determination of free THs is lacking for non-human species. The present study aimed to develop a highly sensitive and reliable liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for the determination of six free THs in serum/plasma, which is applicable to not only humans but also non-human species. Two different physical separation steps, ultrafiltration (UF) and equilibrium dialysis (ED), were examined to obtain the free TH fraction. Several experimental conditions were carefully optimized and validated for UF or ED using the commercially available bovine serum. As a result, UF at 1100 × g and 37 °C for 30 min with a 30 kDa ultrafiltration device (Centrifree YM-30, Millipore) yielded excellent precision (CV: <10%). The optimized ED step also yielded high precision (CV: <10%) and the measurement values were approximately equal to those of UF, but at least 16 h were required to reach equilibrium. Thus, UF combined with LC–MS/MS was finally chosen, in terms of the time needed for the measurement. Acceptable accuracy (recovery: 70%–110%) and intra- and inter-day precision (CV: <10% and <12%, respectively) were obtained, when triplicate analyses in three different days were conducted using the bovine serum. The developed analytical method was successfully applied to the determination of free THs in serum/plasma samples of humans, cats, and dogs. Furthermore, comparison with free T4 concentrations measured by a common immunoassay method evidently indicated that the ultrafiltration-LC–MS/MS method developed in this study can increase the specificity and accuracy of TH measurement.

甲状腺激素（THS），其主要由3,3'，5-三碘升-甲状腺素（T3）和L-甲状腺素（T4）在调节生物过程（例如各种动物的生长和代谢）中起关键作用。因此，需要准确测量血清/血浆中T3和T4的含量，尤其是生理活性的游离（未结合蛋白质的）形式，以评估TH稳态。然而，对于非人类物种而言，缺乏对游离TH的高精度测定。本研究旨在开发一种高度灵敏且可靠的液相色谱-串联质谱（LC-MS / MS）方法，用于测定血清/血浆中的六种游离TH，不仅适用于人类，而且适用于非人类物种。检查了两个不同的物理分离步骤，超滤（UF）和平衡透析（ED），以获得游离的TH馏分。仔细优化了几个实验条件，并使用市售牛血清对UF或ED进行了验证。结果，使用30 kDa超滤装置（Centrifree YM-30，Millipore）在1100×g和37°C下处理30分钟的超滤膜具有极好的精度（CV：<10％）。优化的ED步骤还产生了高精度（CV：<10％），并且测量值大约与UF相等，但是至少需要16小时才能达到平衡。因此，根据测量所需的时间，最终选择了超滤结合LC-MS / MS。当使用牛血清在三天内进行三次重复分析时，可获得可接受的准确性（回收率：70％–110％）和日内和日间精度（CV：分别为<10％和<12％）。所开发的分析方法已成功地用于测定人，猫和狗的血清/血浆样品中的游离TH。此外，与通过常规免疫测定法测量的游离T4浓度进行比较显然表明，本研究中开发的超滤LC-MS / MS方法可以提高TH测量的特异性和准确性。