Perchlorate can competitively inhibit iodide uptake by the thyroid gland (TG) via the sodium/iodide symporter, consequently reducing the production of thyroid hormones (THs). Until recently, the effects of perchlorate on TH homeostasis are being examined through measurement of serum levels of TH, by immunoassay (IA)-based methods. IA methods are fast, but for TH analysis, they are compromised by the lack of adequate specificity. Therefore, selective and sensitive methods for the analysis of THs in TG are needed, for assessment of the effects of perchlorate on TH homeostasis. In this study, we developed a 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 TG, using liquid chromatography (LC)-tandem mass spectrometry (MS/MS). TGs used in this study were from rats that had been placed on either iodide-deficient diet or iodide-sufficient diet, and that had either been provided with perchlorate in drinking water (10 mg/kg/day) or control water. TGs were extracted by pronase digestion and then analyzed by LC-MS/MS. The instrumental calibration range for each TH ranged from 1 to 200 ng/ml and showed a high linearity (r > 0.99). The method quantification limits (LOQs) were determined to be 0.25 ng/mg TG for 3-T(1); 0.33 ng/mgTG for 3,3'-and 3,5-T(2); and 0.52 ng/mg TG for rT(3), T(3), and T(4). Rats were placed on an iodide-deficient or -sufficient diet for 2.5 months, and for the last 2 weeks of that period were provided either perchlorate (10 mg/kg/day) in drinking water or control water. Iodide deficiency and perchlorate administration both reduced TG stores of rT(3), T(3), and T(4). In iodide-deficient rats, perchlorate exacerbated the reduction in levels of THs in TG. With the advances in analytical methodology, the use of LC-MS/MS for measurement of hormone levels in TG will allow more comprehensive evaluations of the hypothalamic-pituitary-thyroid axis. (C) 2010 Elsevier B.V. All rights reserved.