Gu Jianghong, Soldin Offie P, Soldin Steven J
Department of Laboratory Medicine, Children's National Medical Center, 111 Michigan Avenue, NW, Washington, DC, USA.
Clin Biochem. 2007 Dec;40(18):1386-91. doi: 10.1016/j.clinbiochem.2007.08.007. Epub 2007 Aug 24.
This study was designed to improve our previously developed tandem mass spectrometry (MS/MS) method for free thyroxine (FT4) by enhancing sensitivity and permitting simultaneous measurements of both free triidothyronine (FT3) and FT4 using a smaller plasma/serum sample.
An API-5,000 tandem mass spectrometer equipped with TurboIonSpray source and Shimadzu HPLC system was employed to perform the analysis using isotope dilution with deuterium labeled internal standard, T4-d(5). Four hundred microliters of human plasma/serum was filtered through a Centrifree YM-30 ultrafiltration device by centrifugation, and 450 microL of internal standard in methanol was then added to 150 microL of ultrafiltrate for deproteinization. After centrifugation, 500 microL of supernatant was diluted with 400 microL of distilled de-ionized water and a 650 microL aliquot was injected onto a C-18 column. After washing, the switching valve was activated and the analytes were eluted from the column with a water/methanol gradient into the MS/MS system. Quantification by multiple reaction-monitoring (MRM) analysis was performed in the negative mode.
The within-day and between-day coefficients of variation (CVs) were <or=9% for FT3 and <or=7% for FT4 at all concentrations tested. Accuracy ranged between 95% and 105%. The 2.5th-97.5th percentile for FT3 and FT4 was 0.09-0.4 ng/dL (1.4-6.2 pmol/L) and 0.8-2.1 ng/dL (10-26 pmol/L), respectively. The results correlated only moderately well with the immunoassays.
We describe an improved simple, accurate and fast isotope dilution tandem mass spectrometry method for the simultaneous determination of FT3 and FT4 in human serum/plasma samples.
本研究旨在改进我们先前开发的游离甲状腺素（FT4）串联质谱（MS/MS）方法，通过提高灵敏度并允许使用较小体积的血浆/血清样本同时测量游离三碘甲状腺原氨酸（FT3）和FT4。
采用配备TurboIonSpray源的API-5,000串联质谱仪和岛津HPLC系统，使用氘标记内标T4-d(5)进行同位素稀释分析。400微升人血浆/血清通过Centrifree YM-30超滤装置离心过滤，然后将450微升甲醇中的内标加入150微升超滤液中进行脱蛋白处理。离心后，500微升上清液用400微升蒸馏去离子水稀释，取650微升等分试样注入C-18柱。洗涤后，启动切换阀，分析物用水/甲醇梯度从柱上洗脱进入MS/MS系统。通过多反应监测（MRM）分析在负离子模式下进行定量。
在所有测试浓度下，FT3的日内和日间变异系数（CVs）≤9%，FT4的CVs≤7%。准确度在95%至105%之间。FT3和FT4的第2.5百分位数至第97.5百分位数分别为0.09 - 0.4 ng/dL（1.4 - 6.2 pmol/L）和0.8 - 2.1 ng/dL（10 - 26 pmol/L）。结果与免疫测定法的相关性仅为中等。
我们描述了一种改进的简单、准确且快速的同位素稀释串联质谱方法，用于同时测定人血清/血浆样本中的FT3和FT4。
