Development and Evaluation of a Reference Measurement Procedure for the Determination of Total 3,3‘,5-Triiodothyronine in Human Serum Using Isotope-Dilution Liquid Chromatography−Tandem Mass Spectrometry

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Abstract

3,3',5-Triiodothyronine (T3) is an important diagnostic marker for thyroid function. A reference measurement procedure (RMP) for total T3 in serum involving isotope dilution coupled with liquid chromatography-tandem mass spectrometry (LC/MS/MS) has been developed and critically evaluated. The method uses solid-phase extraction with mixed-mode retention mechanisms of reversed phase and ion exchange prior to reversed-phase LC/MS/MS. In addition to a labeled T3 internal standard (T3-13C9), labeled thyroxine (T4-d5) is also added to serum samples in order to monitor the degradation of T4 to T3. The accuracy of the measurement was evaluated by a recovery study for added T3 and was supported by a comparison study with the other RMP. The recovery of the added T3 ranged from 98.9% to 99.4%. The results of this method and the other RMP agreed to within 1%. Samples of frozen serum pools were prepared and measured in three separate sets. Excellent reproducibility was obtained with within-set coefficients of variation (CVs) ranging from 0.8% to 1.6% and between-set CVs ranging from 1.9% to 2.6%. Excellent linearity was also obtained with correlation coefficients of all linear regression lines (measured intensity ratios vs mass ratios) ranging from 0.9995 to 0.9996. The detection limit at a signal-to-noise ratio of approximately 3 was 1 pg of T3. The T4 degradation during sample preparation was minimized to a small percentage (no more than 3% of the T3 values) by use of antioxidants (ascorbic acid, dithiothreitol, citric acid) and can be accounted for in the T3 measurement process. This well-characterized LC/MS/MS method for total serum T3, which demonstrates good accuracy and precision, low susceptibility to interferences, accountability of the conversion of T4 to T3, and comparability with the other RMP, qualifies as a reference measurement procedure and can be used to provide an accuracy base to which routine methods for T3 can be compared.

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