Analysis of thyroid hormones in serum by liquid chromatography-tandem mass spectrometry

Abstract

Thyroid hormones are essential hormones for regulating growth and development in humans and wildlife. Methods to monitor precise and low levels of these hormones in serum and tissues are needed to assess overall health, whether from disease considerations or possibly from environmental contaminant exposures. Common and routine methods typically rely upon radio-immunoassays, which can be expensive, and typically only measure T4 and T3, which can be a limitation in fully evaluating impacts on thyroid regulation. In this study we developed an liquid chromatography-tandem mass spectrometry method for the simultaneous analysis of five thyroid hormones including thyroxine (T4), 3,3′,5-triidothyronine (T3), 3,3′,5′-triiodothyronine (rT3; reverse T3), 3,3′-diiodothyronine (3,3′-T2), and 3,5-diiodothyronine (3,5-T2) in serum samples. The LC/MS-MS parameters were optimized and calibrated over a wide concentration range (1.0 to 500 ng/mL) with on-column detection limits of 1.5-7.0 pg. Using spiked bovine serum samples, mean method recoveries were calculated to be 81.3-111.9 % with RSDs of 1.2-9.6 % at spiking levels ranging from 10 to 100 ng/mL. This method was compared with measurements made by standard RIAs and to measurements made in a serum Standard Reference Material (SRM 1951b). Development of this method expands the capacity to measure thyroid hormones by including a larger suite of thyroid hormones, and has promising applications for measuring catabolism of thyroid hormones in vitro.

Quantification of 11 thyroid hormones and associated metabolites in blood using isotope-dilution liquid chromatography tandem mass spectrometry

“…All analytes yielded two SRM-ion transitions; however, it was only possible to obtain a single high abundance ion-transition for T 0 and T 1 AM. Our MS/MSfragmentation is in agreement with other studies that also found similar ion fragments caused by loss of carboxylic acid for T 4 , T 3 , rT 3 [8,[21][22][23], 3,5-T 2 , and 3,3′-T 2 [16,24]. For T 1 AM, we observed a MS/MS-fragment at m/z 212, corresponding to loss of aromatic hydroxyl and iodine, which agrees with other findings [25,26].…”

“…Wang and Stapleton described the analysis of T 4 , T 3 , rT 3 , 3,3'-T 2 and 3,5-T 2 in serum [16], and later Kunisue et al included 3-T 1 [17]. Recently, Noyes et al described sample preparation optimizations for analyzing T 4 and T 3 in fish serum [8].…”

“…Detection limits for these methods, even though not explicitly stated, ranged from 0.25 to 1.4 ng mL -1 [8,16,17]. Inspired by current LC-MS/MS methodologies analyzing two or more thyroid hormones [8,16,17], we Fig. 1 Molecular structures of the investigated thyroid hormones and associated metabolites.…”

Translating Pharmacological Findings from Hypothyroid Rodents to Euthyroid Humans: Is There a Functional Role of Endogenous 3,5-T2?

“…For this purpose, we used a newly developed immunoassay (20), representing the only currently available method to determine serum 3,5-T2 concentrations in humans (26)(27)(28). Our data revealed significant bell-shaped associations of serum 3,5-T2 concentrations with serum glucose concentration and TSH concentrations in the fasting state, pointing toward a versatile role of circulating 3,5-T2 in human metabolism.…”

Quantitative analysis of thyroid hormone metabolites in cell culture samples using LC-MS/MS

“…Classic TH concentrations are typically measured using antibody-based immunoassays specific for an individual analyte, thus requiring significant sample volumes and multiple assays if the full spectrum of the TH and TAM family is of analytical interest. Technical advances in HPLC liquid chromatography and its subsequent combination with tandem mass spectrometry (LC-MS/MS) provided a highly sensitive method to measure part -most often the classic metabolites -of the TH spectrum in human serum or plasma and selected animal tissue specimens [4,[9][10][11][12][13][14][15] . This analytical technique identifies the molecule of interest in a complex sample by its retention time (RT) and the mass-to-charge ratios of parent and fragmentation ions.…”