采用同位素稀释串联质谱法测定游离甲状腺素。
The measurement of free thyroxine by isotope dilution tandem mass spectrometry.
作者信息
Soldin Steven J, Soukhova Nadia, Janicic Natasa, Jonklaas Jacqueline, Soldin Offie P
机构信息
Bioanalytical Core Laboratory, Georgetown Clinical Research Center, Georgetown University, Washington, DC 20007-2197, USA.
出版信息
Clin Chim Acta. 2005 Aug;358(1-2):113-8. doi: 10.1016/j.cccn.2005.02.010. Epub 2005 Apr 12.
BACKGROUND
Most clinical chemistry laboratories measure free thyroxine (FT4) by an analogue (direct) method. Nevertheless, the validity of analogue FT4 immunoassays has been questioned and patient's results using this approach frequently do not fit in with the clinical presentation. Because of these problems we routinely send out all direct free T4's that are < 2.5th percentile and many that are > 97.5th percentile for measurement by equilibrium dialysis, the gold standard method. In approximately half of these cases, the FT4 by equilibrium dialysis was normal. We developed a rapid, reliable free T4 method employing isotope dilution tandem mass spectrometry and compared our results with both the analogue (direct) free T4 and equilibrium dialysis procedures.
METHODS
An API-4000 tandem mass spectrometer (Sciex, Toronto, Canada) equipped with TurboIonSpray and Agilent HPLC system was used employing isotope dilution with deuterium labeled internal standard (IS=l-thyroxine-d2). Serum was filtered through the Centrifree YM-30 ultrafiltration device by centrifugation, IS added to the ultrafiltrate, and 400 microL injected onto a C-18 column. After washing, the switch valve is activated and a methanol gradient allows for elution of both T4 and the IS into the LC/MS/MS system. Quantitation was by MRM analysis in the negative mode. Equilibrium dialysis was performed by the Nichols method and analogue free T4 results were obtained on the Dade Dimension RxL.
RESULTS
The within-day and between-day CV's were < 7.1% at all concentrations tested. The results correlated well with equilibrium dialysis (Eq Dial=0.971 MS+0.041, n=68, Syx=1.381, r=0.954). A poor correlation was found with the analogue (direct) free T4 method (IA=0.326 MS+6.27, n=154, Syx=1.96, r=0.459).
CONCLUSIONS
Samples can be processed in batches of 30. The free T4 tandem MS method has a rapid turn-around-time vs the equilibrium dialysis procedure, with a chromatographic run time of 8 min per sample.
背景
大多数临床化学实验室通过类似物（直接）法测定游离甲状腺素（FT4）。然而，类似物FT4免疫测定的有效性受到质疑，使用这种方法得出的患者结果常常与临床表现不符。由于这些问题，我们常规将所有低于第2.5百分位数以及许多高于第97.5百分位数的直接游离T4送去采用平衡透析法（金标准方法）进行检测。在大约一半的此类病例中，通过平衡透析得出的FT4结果是正常的。我们开发了一种采用同位素稀释串联质谱法的快速、可靠的游离T4检测方法，并将我们的结果与类似物（直接）游离T4法以及平衡透析法的结果进行了比较。
方法
使用配备TurboIonSpray的API - 4000串联质谱仪（Sciex，加拿大多伦多）和安捷伦HPLC系统，采用氘标记内标（IS = 1 - 甲状腺素 - d2）进行同位素稀释。血清通过离心经Centrifree YM - 30超滤装置过滤，向超滤液中加入内标，然后将400微升注入C - 18柱。洗涤后，启动切换阀，甲醇梯度洗脱使T4和内标均进入LC/MS/MS系统。通过负模式下的MRM分析进行定量。采用Nichols法进行平衡透析，在Dade Dimension RxL上获得类似物游离T4结果。
结果
在所有测试浓度下，日内和日间CV均<7.1%。结果与平衡透析法相关性良好（平衡透析 = 0.971×质谱法 + 0.041，n = 68，Syx = 1.381，r = 0.954）。与类似物（直接）游离T4法相关性较差（免疫分析法 = 0.326×质谱法 + 6.27，n = 154，Syx = 1.96，r = 0.459）。
结论
样本可按30个一批进行处理。游离T4串联质谱法与平衡透析法相比周转时间更快，每个样本的色谱运行时间为8分钟。
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