Abstract

Many environmental chemicals are known to disrupt thyroid function. Measurement of thyroid hormones in animal studies provides useful information to understand the effects of environmental chemicals on thyroid hormone metabolism. We report an efficient method, utilizing a protein precipitation followed by ultraperformance liquid chromatography-tandem mass spectrometry analysis, to quantitate total 3,3’,5-triiodo-L-thyronine (triiodothyronine, T3) and total 3,3’,5,5’-tetraiodo-L-thyronine (thyroxine, T4) in rodent serum. The use of synthetic serum for calibration standards eliminated the interferences from endogenous total T3 and T4 and allowed the experimental lower limits of quantitation (LOQ) to be set at the required concentration (T3, 20 ng/dL; T4, 0.5 µg/dL) to allow quantitation of endogenous concentrations. The method was linear (r > 0.99; range 20.0-600 ng/dL T3, 0.500-15 µg/dL T4) with good assay recoveries (90.4-107%) for both analytes. Intra- and inter-day accuracy, estimated as percent relative error, were ≤ ±7.6% and intra- and inter-day precision, estimated as the relative standard deviation, were ≤ 5.3% for both analytes. The method may easily be adapted to a well-plate format thereby further improving the efficiency. Total T3 and T4 concentrations were stable in male and female rat and mouse serum when stored in the freezer (∼−70 °C) for up to 62 d with determined values within 92.8-111% of day 0 for both analytes. The method can be extended to quantitate total T3 and T4 concentrations in humans or other species with minimal optimization.

 抽象的

已知许多环境化学物质会破坏甲状腺功能。动物研究中甲状腺激素的测量为了解环境化学物质对甲状腺激素代谢的影响提供了有用的信息。我们报告了一种有效的方法，利用蛋白质沉淀，然后进行超高效液相色谱-串联质谱分析，来定量总 3,3',5-三碘-L-甲状腺氨酸（三碘甲状腺原氨酸，T3）和总 3,3',5,啮齿动物血清中的 5'-四碘-L-甲状腺氨酸（甲状腺素，T4）。使用合成血清作为校准标准消除了内源性总 T3 和 T4 的干扰，并允许将实验定量下限 (LOQ) 设置为所需浓度（T3，20 ng/dL；T4，0.5 µg/dL）以允许内源浓度的定量。该方法呈线性（r > 0.99；范围 20.0-600 ng/dL T3、0.500-15 µg/dL T4），两种分析物的测定回收率良好 (90.4-107%)。两种分析物的日内和日间准确度（以百分比相对误差估计）≤ ±7.6%，日内和日间精密度（以相对标准偏差估计）≤ 5.3%。该方法可以很容易地适应孔板格式，从而进一步提高效率。当在冰箱（~−70°C）中保存长达 62 天时，雄性和雌性大鼠和小鼠血清中的总 T3 和 T4 浓度稳定，两种分析物的测定值在第 0 天的 92.8-111% 范围内。该方法可以扩展到对人类或其他物种的总 T3 和 T4 浓度进行最小优化的定量。