Rapid method for the measurement of circulating thyroid hormones in low volumes of teleost fish plasma by LC-ESI/MS/MS

Abstract

Thyroid hormones are critical regulators of normal development and physiological functioning in all vertebrates. Radioimmunoassay (RIA) approaches have been the method of choice for measuring circulating levels of thyroid hormones in vertebrates. While sensitive, RIA-based approaches only allow for a single analyte measurement per assay, can lack concordance across platforms and laboratories, and can be prone to analytical interferences especially when used with fish plasma. Ongoing advances in liquid chromatography tandem mass spectrometry (LC/MS/MS) have led to substantial decreases in detection limits for thyroid hormones and other biomolecules in complex matrices, including human plasma. Despite these advances, current analytical approaches do not allow for the measurement of native thyroid hormone in teleost fish plasma by mass spectrometry and continue to rely on immunoassay. In this study, we developed a new method that allows for the rapid extraction and simultaneous measurement of total T4 (TT4) and total T3 (TT3) in low volumes (50 μL) of fish plasma by LC/MS/MS. Methods were optimized initially in plasma from rainbow trout (Oncorhynchus mykiss) and applied to plasma from other teleost fishes, including fathead minnows (Pimephales promelas), mummichogs (Fundulus heteroclitus), sockeye salmon (Oncorhynchus nerka), and coho salmon (Oncorhynchus kisutch). Validation of method performance with T4- and T3-spiked rainbow trout plasma at 2 and 4 ng/mL produced mean recoveries ranging from 82 to 95 % and 97 to 105 %, respectively. Recovery of 13C12-T4 internal standard in plasma extractions was: 99±1.8 % in rainbow trout, 85±11 % in fathead minnow, 73±5.0 % in mummichog, 73±1.7 % in sockeye salmon, and 80±8.4 % in coho salmon. While absolute levels of thyroid hormones measured in identical plasma samples by LC/MS/MS and RIA varied depending on the assay used, T4/T3 ratios were generally consistent across both techniques. Less variability was measured among samples subjected to LC/MS/MS suggesting a more precise estimate of thyroid hormone homeostasis in the species targeted. Overall, a sensitive and reproducible method was established that takes advantage of LC/MS/MS techniques to rapidly measure TT4 and TT3 with negligible interferences in low volumes of plasma across a variety of teleost fishes.

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].…”

“…Washing the inside of polypropylene test tubes with methanol did not reveal any adsorbed residues. A number of protocols for extracting thyroid hormones from plasma use acids in combination with heat [8]. This prompted a more meticulous stability study to provide essential knowledge on these substances behavior ( Supplementary Fig.…”

“…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].…”

“…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.…”

“…Currently it is very difficult to capture the levels of all of these thyroid hormones. Enabling the scientific community to describe the thyroid hormone system state at an improved level would allow researchers to measure as many thyroid hormones and metabolites as possible, as metabolites are also highly bioactive [7][8][9][10]. Due to the characteristic lipophilic nature of thyroid hormones (e.g.…”