Nicholas School of the Environment, Duke University, Box 90328, Durham, NC, 2770, USA.
Anal Bioanal Chem. 2014 Jan;406(3):715-26. doi: 10.1007/s00216-013-7528-3. Epub 2013 Dec 18.
Thyroid hormones are critical regulators of normal development and physiological functioning in all vertebrates. Radioimmunoassay (RIA) approaches have been the method of choice for measuring circulating levels of thyroid hormones in vertebrates. While sensitive, RIA-based approaches only allow for a single analyte measurement per assay, can lack concordance across platforms and laboratories, and can be prone to analytical interferences especially when used with fish plasma. Ongoing advances in liquid chromatography tandem mass spectrometry (LC/MS/MS) have led to substantial decreases in detection limits for thyroid hormones and other biomolecules in complex matrices, including human plasma. Despite these advances, current analytical approaches do not allow for the measurement of native thyroid hormone in teleost fish plasma by mass spectrometry and continue to rely on immunoassay. In this study, we developed a new method that allows for the rapid extraction and simultaneous measurement of total T4 (TT4) and total T3 (TT3) in low volumes (50 μL) of fish plasma by LC/MS/MS. Methods were optimized initially in plasma from rainbow trout (Oncorhynchus mykiss) and applied to plasma from other teleost fishes, including fathead minnows (Pimephales promelas), mummichogs (Fundulus heteroclitus), sockeye salmon (Oncorhynchus nerka), and coho salmon (Oncorhynchus kisutch). Validation of method performance with T4- and T3-spiked rainbow trout plasma at 2 and 4 ng/mL produced mean recoveries ranging from 82 to 95 % and 97 to 105 %, respectively. Recovery of (13)C12-T4 internal standard in plasma extractions was: 99 ± 1.8 % in rainbow trout, 85 ± 11 % in fathead minnow, 73 ± 5.0 % in mummichog, 73 ± 1.7 % in sockeye salmon, and 80 ± 8.4 % in coho salmon. While absolute levels of thyroid hormones measured in identical plasma samples by LC/MS/MS and RIA varied depending on the assay used, T4/T3 ratios were generally consistent across both techniques. Less variability was measured among samples subjected to LC/MS/MS suggesting a more precise estimate of thyroid hormone homeostasis in the species targeted. Overall, a sensitive and reproducible method was established that takes advantage of LC/MS/MS techniques to rapidly measure TT4 and TT3 with negligible interferences in low volumes of plasma across a variety of teleost fishes.
甲状腺激素是所有脊椎动物正常发育和生理功能的关键调节剂。放射免疫分析（RIA）方法一直是测量脊椎动物循环中甲状腺激素水平的首选方法。虽然具有敏感性，但基于 RIA 的方法每次测定只能测量一种分析物，在不同平台和实验室之间可能缺乏一致性，并且在使用鱼类血浆时特别容易受到分析干扰。液相色谱串联质谱（LC/MS/MS）的不断进步，大大降低了复杂基质中甲状腺激素和其他生物分子的检测限，包括人血浆。尽管取得了这些进展，但目前的分析方法仍然无法通过质谱法测量硬骨鱼血浆中的天然甲状腺激素，并且仍然依赖于免疫测定。在这项研究中，我们开发了一种新方法，该方法允许通过 LC/MS/MS 从低体积（50 μL）的鱼血浆中快速提取和同时测量总 T4（TT4）和总 T3（TT3）。该方法最初在虹鳟（Oncorhynchus mykiss）的血浆中进行了优化，并应用于其他硬骨鱼的血浆中，包括黑头呆鱼（Pimephales promelas）、美洲蟾鱼（Fundulus heteroclitus）、红大麻哈鱼（Oncorhynchus nerka）和银大麻哈鱼（Oncorhynchus kisutch）。用 2 和 4 ng/mL 的 T4 和 T3 标记的虹鳟血浆对方法性能进行验证，得到的平均回收率分别为 82%至 95%和 97%至 105%。（13）C12-T4 内标在血浆提取中的回收率为：虹鳟为 99±1.8%，黑头呆鱼为 85±11%，美洲蟾鱼为 73±5.0%，红大麻哈鱼为 73±1.7%，银大麻哈鱼为 80±8.4%。尽管 LC/MS/MS 和 RIA 测量相同血浆样品中的甲状腺激素水平因所用的测定方法而异，但 T4/T3 比值在两种技术中通常一致。LC/MS/MS 测量的样品间变异性较小，表明目标物种的甲状腺激素稳态有更精确的估计。总的来说，建立了一种灵敏且可重复的方法，该方法利用 LC/MS/MS 技术，在低体积的血浆中快速测量 TT4 和 TT3，同时几乎没有干扰，可用于多种硬骨鱼。
