Quantification of 11 thyroid hormones and associated metabolites in blood using isotope-dilution liquid chromatography tandem mass spectrometry

This paper describes a novel analytical methodology for the simultaneous determination of absolute and total concentrations of 11 native thyroid hormones and associated metabolites, viz. thyroxine (T-4), 3,3', 5-triiodothyronine (T-3), 3,3', 5'-triiodothyronine (rT(3)), 3,5-diiodothyronine (3,5-T-2), 3,3'- diiodothyronine (3,3'-T-2), 3-iodothyronine (T-1), thyronine (T-0), 3-iodothyronamine (T(1)AM), tetraiodothyroacetic acid (Tetrac), triiodothyroacetic acid (Triac), and diiodothyroacetic acid (Diac), in 50-mu L of plasma or serum. The method was optimized using four isotopic labeled surrogate and internal standards in combination with solid-phase extraction and LC-MS/MS. The methodology was further evaluated using amphibian plasma and serum with matrix-matched calibration applied for quantification. Method detection limits are 3.5 pg T-4, 1.5 pg T-3, 2.9 pg rT(3), 1.7 pg 3,3'-T-2, 2.3 pg 3,5-T-2, and between 0.3 and 7.5 pg for the remaining six metabolites in 50 mu L aliquots of blood sera or plasma. Accuracies and repeatabilities for all analytes were between 88 and 103 % and 1.31 and 17.2 %, respectively. Finally, we applied the method on adult frog (Xenopus laevis) plasma and tadpole (Rana (Lithobates) catesbeiana) serum. We observed up to seven different thyroid hormones and associated metabolites in tadpole serum. This method will enable researchers to improve the assessment of thyroid homeostasis and endocrine disruption in animals and humans.