Isotope dilution-liquid chromatography/electrospray ionization-tandem mass spectrometry for the determination of serum thyroxine as a potential reference method

Abstract

A new candidate reference method is presented for the determination of thyroxine in serum. The method is based on isotope dilution-liquid chromatography/tandem mass spectrometry using electrospray for ionization. The internal standard used was 13C6-thyroxine, sample pretreatment consisted of protein precipitation and a two-step liquid/liquid extraction procedure, HPLC was performed on a C-18 column with an eluent containing methanol/water/formic acid (60:40:0.1, by volume), and finally thyroxine and its isotopically labeled analogue were measured in the selected reaction monitoring mode for the transitions m/z 777.7--> 731.7 and m/z 783.7--> 737.7, respectively. The detection limit for thyroxine was 6 pg, the within-run coefficient of variation was 1.1%. The samples were measured in six-fold: in duplicate on three independent days. The mean overall coefficient of variation of the method was 1.6%. The new method was evaluated by measuring nine control sera previously determined by an existing ID-GC/MS method. The differences between the results of the two methods ranged from-1.6% to +3.3%, with a mean of +0.2%.

Isotope dilution tandem mass spectrometric method for T4/T3

“…For example, Table 1 Currently serum total T4 (TT4) and total serum T3 (TT3) concentrations are most commonly measured by immunoassay methods. Recently some reports of quantitative measurement of T4 and T3 by HPLC, gas chromatography mass spectrometry (GC-MS), liquid chromatography mass spectrometry (LC-MS) or tandem mass spectrometry (LC-MS/ MS) were published [15][16][17][18][19]. All those methods required extraction, derivatization and even prior chromatographic separation that are very time-consuming [20,21].…”

Quantitative analysis of thyroid hormone metabolites in cell culture samples using LC-MS/MS

“…The use of MS/MS and stable isotope IS permits the development of highly sensitive and specific assays [22][23][24] . Here, we report the validation of the first high-performance stable isotope dilution LC-MS/MS method to measure 9 TH and 6 TAM from a single cell culture media sample using a simple and fast liquid-liquid extraction.…”

Metrologic Traceability of Total Thyroxine Measurements in Human Serum: Efforts to Establish a Network of Reference Measurement Laboratories

“…Apart from the instruction to calibrate only with the T 4 candidate primary calibrator, no further constraints were applied with respect to the design of the cRMPs. The laboratories at Ghent University and NIST used their previously developed and recently modified procedures based on isotope dilution-liquid chromatography-tandem MS with electrospray ionization (21)(22)(23). The laboratory at the University of Bonn recently had transferred its isotope dilution-gas-liquid chromatography-MS cRMP (24 ) into an isotope dilution-liquid chromatography-electrospray ionization, single-stage MS variant (see the Data Supplement that accompanies the online version of this article at http://www.clinchem.…”