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

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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.

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.

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Acknowledgments

This work was financially supported by the European Commission Seventh Framework Program, grant no. PIOF-GA-2012-329996, and in part by the Engineering Research Center for Reinventing the Nation’s Water Infrastructure (ReNUWIt) EEC-1028968 at the University of California, Berkeley. T.H. and X.L. are supported by the Kapor Foundation, the Ceres Foundation, and Beyond Pesticides. C.C.H. is supported by the Natural Sciences and Engineering Research Council of Canada (NSERC). Dr. Anthony Iavarone at QB3 Chemistry Mass Spectrometry Facility, UC Berkeley and Professor Erland Björklund at Kristianstad University are gratefully acknowledged for fruitful discussions and contributions.

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Authors and Affiliations

  1. Molecular Toxicology, Energy and Resources Group, Endocrinology, Museum of Vertebrate Zoology, and Department of Integrative Biology, University of California, Berkeley, CA, 94720, USA

    Martin Hansen, Xuan Luong & Tyrone Hayes

  2. Department of Civil and Environmental Engineering, University of California, Berkeley, CA, 94720, USA

    Martin Hansen & David L. Sedlak

  3. Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, V8P 5C2, Canada

    Caren C. Helbing

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  1. Martin Hansen
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  2. Xuan Luong
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Correspondence to Martin Hansen.

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University of California and University of Victoria animal care and use programs, protocols, and procedures were followed and appropriate permits obtained.

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Hansen, M., Luong, X., Sedlak, D.L. et al. Quantification of 11 thyroid hormones and associated metabolites in blood using isotope-dilution liquid chromatography tandem mass spectrometry. Anal Bioanal Chem 408, 5429–5442 (2016). https://doi.org/10.1007/s00216-016-9614-9

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  • DOI: https://doi.org/10.1007/s00216-016-9614-9

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