Determination of Six Thyroid Hormones in the Brain and Thyroid Gland Using Isotope-Dilution Liquid Chromatography/Tandem Mass Spectrometry

摘要

Thyroid hormones (THs) play critical roles in the regula-ntion of growth and development, including brain develop-nment, in both humans and animals. Until recently, THnlevels were assayed with measurements in serum, usingnimmunoassay (IA)-based methods. IA methods are sensi-ntive but are compromised by the lack of adequate specific-nity. Furthermore, measurements of TH levels in blood donnot necessarily reflect the levels and profiles found inncritical organs such as the brain. Measurement of THnlevels in the brain is critical for studies that assess theneffects of environmental contaminants on TH homeostasis.nIn this study, we developed a selective and sensitivenmethod for the analysis of six THs, L-thyroxine (T4),n3,3′,5-triiodo-L-thyronine (T3), 3,3′,5′-triiodo-L-thyro-nnine (rT3), 3,5-diiodo-L-thyronine (3,5-T2), 3,3′-diiodo-nL-thyronine (3,3′-T2), and 3-iodo-L-thyronine (3-T1), innthe brain and thyroid gland (TG) using isotope ([13C]T4)-ndilution liquid chromatography (LC)/tandem massnspectrometry (MS/MS). Proteins in the (rat) brain andnTG were digested by pronase, and THs were extractednwith a solid-phase extraction method and analyzed bynLC/MS/MS. The instrumental calibration range forneach TH ranged from 0.5 to 200 ng/mL and showednexcellent linearity (r 0.9995). The instrumentalndetection limits for THs were in the range of 7.5 13.5npg, in positive ion mode, and 13.5 16.5 pg, in negativenion mode. The optimized procedural recoveries for THsn(except for 3-T1), spiked into a pig-brain matrix, werenbetween 97.6% and 109%, with a coefficient of variationn(CV) of 1.2 8.2%, for the brain, and between 96.4% andn101%, with a CV of 1.8 8.6%, for the TG. Concentrationsnof THs in the brain and TG of the five rats were 2.20 3.65nng/g T4, 1.56 2.20 ng/g T3, and below the limit ofndetection ( LOD) for the other THs, in the brain, and