Determination of six thyroid hormones in dog brain and liver using acidic extraction, mixed-mode cleanup, and liquid chromatography–tandem mass spectrometry

Abstract

Thyroid hormones (THs) play a critical role in the regulation of biological processes, such as growth, metabolism, and development, in various animal species. Prohormone L-thyroxine (T4) is secreted from the thyroid gland and carried to peripheral tissues. T4 is then biotransformed to several metabolites which play different roles, mainly by iodothyronine deiodinases. Determination of deiodinated TH metabolites in key organs such as liver and brain would help to understand tissue-specific TH metabolism and homeostasis. In this study, we thus developed a highly sensitive method for the determination of six THs [T4, 3,5,3′-triodo-L-thyronine (T3), 3,3′,5′-triiodo-L-thyronine (rT3), 3,5-diiodo-l-thyronine (3,5-T2), 3,3′-diiodo-l-thyronine (3,3′-T2), and 3-iodo-l-thyronine (3-T1)] in the brain and liver by using stored dog samples. The analytical method consisted of ultrasonic-assisted extraction in acetone acidified with formic acid, cleanup with a EVOLUTE® EXPRESS CX cartridge (reversed-phase combined with strong cation-exchange cartridge), and quantification with liquid chromatography-tandem mass spectrometry. Acceptable accuracy (internal standard-corrected recovery: 80%–120%) and intra- and inter-day precision (coefficient of variation: <6% and <15%, respectively) (n = 3/ batch, three days) were obtained for both brain and liver samples. In addition, low method detection limits were achieved for both brain (0.013–0.12 ng g–1) and liver (0.030–0.78 ng g–1), which resulted in the quantitation of not only T4, T3, and rT3, but also 3,3′-T2 in both dog brain and liver samples. The developed method was successfully applied to the analysis of THs in the brain and liver of dogs (Canis lupus familiaris) which were exposed to polychlorinated biphenyls (PCBs). As a result, concentration ratios of rT3/T4 and 3,3′-T2/T3 in the PCB-exposed dogs were significantly higher than those in the control groups, suggesting the enhanced inner (tyrosyl)-ring deiodination (5-deiodination) by PCB exposure. The analytical method developed in the present study enables comprehensive evaluation of alterations in peripheral TH metabolism which are caused by exposure to environmental pollutants.