You are here:
HPLC-ICP/MS Analysis of Thyroid Hormone and Related Iodinated Compounds in Tissues and Media
Citation:
BUTTERWORTH, B. C., D. E. HAMMERMEISTER, P. A. KOSIAN, J. HASELMAN, S. J. DEGITZ, AND J. E. TIETGE. HPLC-ICP/MS Analysis of Thyroid Hormone and Related Iodinated Compounds in Tissues and Media. Presented at SETAC Annual Meeting, Milwaukee, WI, November 11 - 15, 2007.
Impact/Purpose:
This abstract describes the development and application of a relatively novel analytical method for thyroid hormone and its synthetic precursors and metobolites.
Description:
Quantifying thyroid hormone (TH) and the synthetic precursors and metabolic products of TH is important for developing models of the hypothalamic-pituitary-thyroid (HPT) axis as well as for understanding the effects of xenobiotics on HPT axis function. In this study, the development of a high performance liquid chromatography-inductively coupled plasma/mass spectrometry (HPLC-ICP/MS) method is described that allows for the detection of 3-monoiodotyrosine (MIT), 3,5-diiodotyrosine (DIT), 3,5-diiodothyronine (T2), 3,5,3’-triiodothyronine (T3), 3,3’,5’-triiodothyronine (reverse T3), 3,3’,5,5’-tetratiodothyronine (T4 or TH), and the glucuronide conjugate of T4 (T4-G) in a single sample. This method has three main steps: sample preparation, chromatographic separation of analytes by HPLC, and detection of iodinated compounds by ICP/MS. Sample preparation varies according to the sample type. For serum and media samples, direct injection into the HPLC is generally used, while thyroid gland tissues require proteolytic treatment to liberate compounds covalently bound to thyroglobulin, such as MIT and DIT. The eluent from the HPLC is introduced directly to an ICP/MS operating in a time-resolve mode monitoring mass 127. The selectivity of the ICP/MS detector overlooks non-iodinated chemicals in an otherwise complex sample, allowing for quantification of the iodinated species only. This method was validated using analytical standards and detection limits are typically in the low ng/mL range. This method is quantitatively comparable to conventional radioimmunoassay (RIA) methods when analyzing total T4. The method has been used successfully to assess various samples from Xenopus laevis, including: serum, thyroid gland tissue, thyroid gland culture media, and media from hepatic microsomal assays for UDPGT activity.