Thienpont L M, De Brabandere V I, Stöckl D, De Leenheer A P
Laboratoria voor Medische Biochemie en Klinische Analyse, University of Ghent, Belgium.
Biol Mass Spectrom. 1994 Aug;23(8):475-82. doi: 10.1002/bms.1200230804.
A new gas chromatographic/mass spectrometric method in combination with isotope dilution for the determination of thyroxine in serum is described. Special attention was paid to the methylation step of thyroxine, which was investigated using methanolic HCl, dimethylformamide/dimethylacetal and diazomethane, the latter giving the best results in terms of reproducible isotope ratios. For internal standardization, (13C6)-thyroxine was dissolved in fraction V human albumin solution (70 g l-1). The internal standard-in-albumin solution was mixed with known amounts of thyroxine standard, dissolved in 0.05 M Na2HPO4 buffer at pH 11.6, to give isotope ratios of 0.75, 1.00 and 1.25. The same internal standard solution was also used for isotope dilution of the unknown serum samples. The volume of serum was adapted to give a 1:1 isotope ratio. Sample pretreatment consisted of protein precipitation and a two-step liquid/liquid extraction procedure. After methylation of unlabelled and labelled thyroxine with diazomethane and perfluoroacylation with pentafluoropropionic anhydride and heptafluorobutyric anhydride, respectively, mass spectrometric monitoring was done at m/z 951/957 and 1001/1007. Quantitative determination of thyroxine in five serum samples in duplicate, during three consecutive days, showed a mean overall imprecision of 1.0% and a deviation of +0.4% from the target value as determined by a definitive method.
本文描述了一种结合同位素稀释的气相色谱/质谱新方法，用于测定血清中的甲状腺素。特别关注了甲状腺素的甲基化步骤，分别使用甲醇盐酸、二甲基甲酰胺/二甲基乙缩醛和重氮甲烷对其进行研究，就可重现的同位素比率而言，重氮甲烷给出了最佳结果。为进行内标，将(13C6)-甲状腺素溶解于V组分人白蛋白溶液(70 g l-1)中。将内标白蛋白溶液与已知量的甲状腺素标准品混合，后者溶解于pH 11.6的0.05 M Na2HPO4缓冲液中，以得到0.75、1.00和1.25的同位素比率。同样的内标溶液也用于未知血清样品的同位素稀释。调整血清体积以得到1:1的同位素比率。样品预处理包括蛋白质沉淀和两步液/液萃取程序。分别用重氮甲烷对未标记和标记的甲状腺素进行甲基化，并用五氟丙酸酐和七氟丁酸酐进行全氟酰化后，在m/z 951/957和1001/1007处进行质谱监测。连续三天对五个血清样品中的甲状腺素进行重复定量测定，结果显示总体平均不精密度为1.0%，与用权威方法测定的目标值偏差为+0.4%。
