Simultaneous quantification of free triiodothyronine and free thyroxine by isotope dilution tandem mass spectrometry

Objectives: 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. Design and methods: An API-5000 tandem mass spectrometer equipped with TurbolonSpray 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 Centriftee YM-30 ultrafiltration device by centrifugation, and 450 mu L of internal standard in methanol was then added to 150 mu L of ultrafiltrate for deproteinization. After centrifugation, 500 mu L of supernatant was diluted with 400 mu L of distilled deionized water and a 650 mu L 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. Results: The within-day and between-day coefficients of variation (CVs) were <= 9% for FT3 and <= 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. Conclusions: 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. (c) 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.