利用螢光高效能液相層析法分析白兔血清中四碘甲狀腺素及三碘甲狀腺素之含量

Determination of Thyroxine and Tri-iodothyronine by High-Performance Liquid Chromatography with Fluorescence Detection

黃昱綺(Yu-Chi Huang)

指導教授 ： 李仁愛

臺北醫學大學/藥學院/藥學研究所/碩士(2010年)

https://doi.org/10.6831/TMU.2010.00012

摘要

血中三碘甲狀腺素(T3)與四碘甲狀腺素(T4)的含量極微量，透過下視丘-腦垂體-甲狀腺的負回饋機制維持血中T3及T4的濃度於恆定狀態，血中濃度微量變化即能影響人體生長及新陳代謝等各種生理狀況，因此血中四碘甲狀腺素含量變化常被用來診斷甲狀腺機能狀況的一個參考指標。
為有效定量血中甲狀腺激素的濃度，本研究選用4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) 作為螢光衍生化試劑，以其對甲狀腺激素的反應作衍生化最佳化條件之分析。根據結果顯示，NBD-F衍生化試劑與T3、T4可在50℃、1分鐘內快速反應完成，以含13%異丙醇之氰甲烷溶液:水相(0.1%三氟乙酸) = 60:40為移動相時可有效分離T3及T4兩個成份。
血清檢品200 µL加入800 µL之氰甲烷去蛋白後，以每分鐘10,000 轉離心去除檢體中之蛋白質，取200 µL上清液與NBD-F反應後注入高效液相層析儀，以激發光波長460 nm及螢光波長530 nm定量T3及T4濃度，分析方法的準確度T3在100.49% ~ 112.14%間，T4在103.33% ~ 108.66%間，精密度之相對標準差T3在1.97% ~ 4.63%間，T4在3.51% ~ 7.13%。兔子血清T3及T4濃度分別為4.1 ~ 5.3 nM及6.7 ~ 11.4 nM。研究結果顯示此分析方法為一簡便有效地偵測血中T3及T4 含量之分析方法。

關鍵字

甲狀腺激素 ； 甲狀腺素 ； 三碘甲狀腺素 ； 螢光衍生化試劑 ； 白兔血清 ； 高效液相層析儀

並列摘要

The serum levels of Triiodothyronine (T3) and thyroxine (tetra-iodothyronine, T4) secreted by thyroid gland are extremely low, the serum levels of which are regulated by hypothalamic-pituitary-thyroid negative feedback control axis. Due to thyroid hormones influence body’s metabolism and growth, development, the levels of thyroid hormones in serum is often used as an indicator for evaluation of thyroid status.
To evaluate the concentration of thyroid hormones in rabbit serum effectively, a sensitive and precise method has been designed using 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). The parameters influencing the efficiency of derivatization of T3 and T4 with fluorescence detection (ex. = 460 nm; em. = 530 nm) were studied. The optimum conditions were as follows: 200 mM borate buffer (pH 8.0), 10 µM NBD-F, 50℃ of derivatization temperature, and 1 min of incubation time. A chromatographic method was used for separation and determination of T3 and T4 with a mixture of 13% isopropanol in acetonitrile and 0.1% aqueous solution of trifluoroacetic acid, 60:40(v/v) as mobile phase.
Eight hundred µL acetonitrile was added in 200 µL rabbit serum. After serum protein was precipitated by centrifugation at 10,000 rpm, the supernatant 200 µL was reacted with NBD-F. The validation of the method under optimal conditions showed a good linearity between the areas of the peaks and the concentrations. Intra-day relative standard deviations of the peak areas for T3 and T4 were 1.97% ~ 4.63% and 3.51% ~ 7.13%, respectively. A recovery of T3 and T4 was between 100.49% ~ 112.14% and 103.33% ~ 108.66%, respectively. Those results showed that the proposed method is a simple and effective method for determination and separation of T3 and T4 in rabbit serum.

並列關鍵字

T3 ； T4 ； NBD-F ； Rabbit serum ； Derivatization

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