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

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.