Development of a new method for the determination of thyroxine in serum based on isotope dilution gas chromatography mass spectrometry

✍ Scribed by Linda M. Thienpont; Veronique I. De Brabandere; Dietmar Stöckl; André P. De Leenheer

• Publisher: John Wiley and Sons
• Year: 1994
• Tongue: English
• Weight: 668 KB
• Volume: 23
• Category: Article
• ISSN: 1076-5174
• DOI: 10.1002/bms.1200230804

✦ Synopsis

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 HCI, dimethylformamide/dimethylacetal and diazomethane, the latter giving the best results in terms of reproducible isotope ratios. For internal standardization, (13C,)-thyroxine was dissolved in fraction V human albumin solution (70 g I-'). The internal standard-in-albumin solution was mixed with known amounts of thyroxine standard, dissolved in 0.05 M Na,HPO, 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 liquidbiquid extraction procedure. After methylation of unlabelled and labelled thyroxine with diazomethane and perlluoroacylation 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.