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

Abstract

This paper describes a novel analytical methodology for the simultaneous determination of absolute and total concentrations of 11 native thyroid hormones and associated metabolites, viz. thyroxine (T4), 3,3', 5-triiodothyronine (T3), 3,3', 5'-triiodothyronine (rT3), 3,5-diiodothyronine (3,5-T2), 3,3'- diiodothyronine (3,3'-T2), 3-iodothyronine (T1), thyronine (T0), 3-iodothyronamine (T1AM), tetraiodothyroacetic acid (Tetrac), triiodothyroacetic acid (Triac), and diiodothyroacetic acid (Diac), in 50-μL of plasma or serum. The method was optimized using four isotopic labeled surrogate and internal standards in combination with solid-phase extraction and LC-MS/MS. The methodology was further evaluated using amphibian plasma and serum with matrix-matched calibration applied for quantification. Method detection limits are 3.5 pg T4, 1.5 pg T3, 2.9 pg rT3, 1.7 pg 3,3'-T2, 2.3 pg 3,5-T2, and between 0.3 and 7.5 pg for the remaining six metabolites in 50 μL aliquots of blood sera or plasma. Accuracies and repeatabilities for all analytes were between 88 and 103 % and 1.31 and 17.2 %, respectively. Finally, we applied the method on adult frog (Xenopus laevis) plasma and tadpole (Rana (Lithobates) catesbeiana) serum. We observed up to seven different thyroid hormones and associated metabolites in tadpole serum. This method will enable researchers to improve the assessment of thyroid homeostasis and endocrine disruption in animals and humans. Graphical Abstract Quantification of 11 thyroid hormones and metabolites from 50 μL plasma or serum using protein denaturation in combination with solid-phase extraction followed by LC-MS/MS.

Data collection in support of the Endocrine Disruption (ED) assessment for non‐target vertebrates

“…Luna et al (2013) used ultrahighperformance liquid chromatography and isotope dilution tandem mass spectrometry (UPLC-ID-MS-MS) to quantify total T4 in a small volume (10 μL) of plasma also from tadpoles and adult Xenopus laevis (reference 3463 in Annex B -). Hansen et al (2016) validated a LC-MS/MS method to quantify absolute and total concentrations of eleven thyroid hormones and associated metabolites in both 50 μL plasma of adult Xenopus or 50 μL serum of tadpoles (American bullfrog, Rana (Lithobates) catesbeiana) (reference 8142 in Annex B -). The LC-MS/MS methods described by Luna et al (2013) and Hansen et al (2016) do not only differ in their scope.…”

“…Hansen et al (2016) validated a LC-MS/MS method to quantify absolute and total concentrations of eleven thyroid hormones and associated metabolites in both 50 μL plasma of adult Xenopus or 50 μL serum of tadpoles (American bullfrog, Rana (Lithobates) catesbeiana) (reference 8142 in Annex B -). The LC-MS/MS methods described by Luna et al (2013) and Hansen et al (2016) do not only differ in their scope. The method described by Luna et al (2013) allowed quantification of T4 in smaller plasma sample volumes, that as a result did not need to be pooled even when handling tadpoles.…”

“…As detailed in Table 16: , while the method by Hansen et al (2016) requires higher sample volumes, overall it was shown to perform better in terms of its absolute recoveries, sensitivity and precision in The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following a tender procedure.…”

“…addition to allow the quantification of a wide range of relevant thyroid hormones and related compounds. Hansen et al (2016), and that their sample preparation method has also been applied in vitro in tadpole thyroid gland explant culture experiment as well as in vivo in tadpoles (Hornung et al 2015). A citation search (cited 20 times according to Web of Science), revealed that the method has been cited by authors developing their own methods in non-mammalian species (e.g.…”

Simultaneous quantification of T4, T3, rT3, 3,5‐T2 and 3,3′‐T2 in larval zebrafish (Danio rerio) as a model to study exposure to polychlorinated biphenyls

“…In the past decade, UPLC‐MS/MS has been successfully implemented to overcome some of the analytical difficulties recognized with immunoassay approaches (S. J. Soldin, Soukhova, Janicic, Jonklaas, & Soldin, ). Several methods have been validated to measure T3 and T4 in serum by LC‐MS (Hansen, Luong, Sedlak, Helbing, & Hayes, ; ; Tai, Bunk, White, & Welch, ; Tai, Sniegoski, & Welch, ; Wang & Stapleton, ) and GC‐MS (Hantson, De Meyer, & Guerit, ; Thienpont, Fierens, De Leenheer, & Przywara, ), and for measuring T2 and T3 levels in fish muscle (Little, Kunisue, Kannan, & Seebacher, ). There are no published methods assessing T4, T3, rT3, 3,5‐T2 and 3,3′‐T2 simultaneously in zebrafish larvae.…”