Determination of six thyroid hormones in the brain and thyroid gland using isotope-dilution liquid chromatography/tandem mass spectrometry.

Thyroid hormones (THs) play critical roles in the regulation of growth and development, including brain development, in both humans and animals. Until recently, TH levels were assayed with measurements in serum, using immunoassay (IA)-based methods. IA methods are sensitive but are compromised by the lack of adequate specificity. Furthermore, measurements of TH levels in blood do not necessarily reflect the levels and profiles found in critical organs such as the brain. Measurement of TH levels in the brain is critical for studies that assess the effects of environmental contaminants on TH homeostasis. In this study, we developed a selective and sensitive method for the analysis of six THs, l-thyroxine (T(4)), 3,3',5-triiodo-l-thyronine (T(3)), 3,3',5'-triiodo-l-thyronine (rT(3)), 3,5-diiodo-l-thyronine (3,5-T(2)), 3,3'-diiodo-l-thyronine (3,3'-T(2)), and 3-iodo-l-thyronine (3-T(1)), in the brain and thyroid gland (TG) using isotope ([(13)C]T(4))-dilution liquid chromatography (LC)/tandem mass spectrometry (MS/MS). Proteins in the (rat) brain and TG were digested by pronase, and THs were extracted with a solid-phase extraction method and analyzed by LC/MS/MS. The instrumental calibration range for each TH ranged from 0.5 to 200 ng/mL and showed excellent linearity (r > 0.9995). The instrumental detection limits for THs were in the range of 7.5-13.5 pg, in positive ion mode, and 13.5-16.5 pg, in negative ion mode. The optimized procedural recoveries for THs (except for 3-T(1)), spiked into a pig-brain matrix, were between 97.6% and 109%, with a coefficient of variation (CV) of 1.2-8.2%, for the brain, and between 96.4% and 101%, with a CV of 1.8-8.6%, for the TG. Concentrations of THs in the brain and TG of the five rats were 2.20-3.65 ng/g T(4), 1.56-2.20 ng/g T(3), and below the limit of detection (<LOD) for the other THs, in the brain, and 88.9-274 ng/mg T(4), 14.1-49.1 ng/mg T(3), 2.79-12.0 ng/mg rT(3), 0.176-0.535 ng/mg 3,5-T(2), 0.340-0.880 ng/mg 3,3'-T(2), and <LOD for 3-T(1), in the TG. This method can permit more comprehensive evaluation of TH homeostasis in the brain and other critical organs following exposure to environmental contaminants.