Vike-Jonas Kristine, Gonzalez Susana Villa, Mortensen Åse-Karen, Ciesielski Tomasz Maciej, Farkas Julia, Venkatraman Vishwesh, Pastukhov Mikhail V, Jenssen Bjørn Munro, Asimakopoulos Alexandros G
Department of Chemistry, Norwegian University of Science and Technology (NTNU), 7491 Trondheim, Norway.
Department of Biology, Norwegian University of Science and Technology (NTNU), 7491 Trondheim, Norway.
J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Jan 1;1162:122447. doi: 10.1016/j.jchromb.2020.122447. Epub 2020 Nov 17.
A rapid hybrid solid phase extraction (HybridSPE®) protocol tailored to liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) analysis, was developed for the determination of four thyroid hormones, L-Thyroxine (T), 3,3',5-triiodo-L-thyronine (T), 3,3',5'-triiodo-L-thyronine (rT) and 3,3'-diiodo-L-thyronine (T) in blood plasma from Glaucous gulls (Larus hyperboreus) and Baikal seals (Phoca sibirica). The use of target analyte specific C internal standards allowed quantification to be performed through the standard solvent calibration curves and alleviated the need to perform quantification with matrix match curves. The relative recoveries were 100.0-110.1 % for T, 99.1-102.2 % for T, 100.5-108.0 % for rT, and 100.5-104.6 % for T. The matrix effects ranged from -1.52 to -6.10 %, demonstrating minor signal suppression during analysis. The method intra-day precision (method repeatability, RSD %, N = 5, k = 1 day) and inter-day precision (method reproducibility, RSD %, N = 10, k = 2 days) at the 1 ng/mL concentration of fortification were 8.54-15.4 % and 15.4-24.8 %, respectively, indicating acceptable chromatographic peak stabilities for all target THs even at trace level concentrations. The method limit of detection (LOD) for T, T, rT and T was 0.17, 0.16, 0.30 and 0.17 ng/mL, respectively. The HybridSPE® protocol was simple and rapid (<1 min) upon application, while the HybridSPE® cartridge did not require (as in classical SPE cartridges) any additional equilibration nor conditioning step prior sample loading. A total of 46 blood plasma samples, 30 samples collected from Glaucous gulls and 16 samples collected from Baikal seals, were analyzed for thyroid hormones to demonstrate the applicability of the developed method in these wildlife species. The concentrations of T and T in blood plasma from the Glaucous gulls were 5.95-44.2 and 0.37-5.61 ng/mL, respectively, whereas those from Baikal seals were 3.57-46.5 and 0.45-2.07 ng/mL, respectively. In both species, rT demonstrated low detection rate, while T was not detected. Furthermore, cross-array comparison between the HybridSPE®-LC-MS/MS protocol and an established routine radioimmunoassay (RIA) kit-based method was performed for T and T concentrations from selected Baikal seal plasma samples.
针对液相色谱 - 电喷雾电离串联质谱（LC - ESI - MS/MS）分析，开发了一种快速混合固相萃取（HybridSPE®）方案，用于测定来自白翅鸥（Larus hyperboreus）和贝加尔湖海豹（Phoca sibirica）血浆中的四种甲状腺激素，即L - 甲状腺素（T4）、3,3',5 - 三碘 - L - 甲状腺原氨酸（T3）、3,3',5'-三碘 - L - 甲状腺原氨酸（反T3）和3,3'-二碘 - L - 甲状腺原氨酸（T2）。使用目标分析物特异性碳内标物可通过标准溶剂校准曲线进行定量，并无需使用基质匹配曲线进行定量。T4的相对回收率为100.0 - 110.1%，T3为99.1 - 102.2%，反T3为100.5 - 108.0%，T2为100.5 - 104.6%。基质效应范围为 - 1.52%至 - 6.10%，表明分析过程中信号抑制较小。在1 ng/mL加标浓度下，该方法的日内精密度（方法重复性，RSD%，N = 5，k = 1天）和日间精密度（方法再现性，RSD%，N = 10，k = 2天）分别为8.54 - 15.4%和15.4 - 24.8%，这表明即使在痕量水平浓度下，所有目标甲状腺激素的色谱峰稳定性均可接受。T4、T3、反T3和T2的方法检测限（LOD）分别为0.17、0.16、0.30和0.17 ng/mL。HybridSPE®方案应用时简单快速（<1分钟），而HybridSPE®柱在进样前不需要（与传统固相萃取柱不同）任何额外的平衡或预处理步骤。总共分析了46份血浆样本，其中30份来自白翅鸥，16份来自贝加尔湖海豹，以证明所开发方法在这些野生动物物种中的适用性。白翅鸥血浆中T4和T3的浓度分别为5.95 - 44.2和0.37 - 5.61 ng/mL，而贝加尔湖海豹血浆中T4和T3的浓度分别为3.57 - 46.5和0.45 - 2.07 ng/mL。在这两个物种中，反T3的检出率较低，而未检测到T2。此外，针对选定的贝加尔湖海豹血浆样本中的T4和T3浓度，对HybridSPE® - LC - MS/MS方案和既定的基于常规放射免疫分析（RIA）试剂盒的方法进行了交叉阵列比较。
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