title: Determination of thyroid hormones in mouse tissues by isotope-dilution microflow liquid chromatography-mass spectrometry method.
abstract: Thyroid hormones (THs) play a critical role in the regulation of many biological processes such as growth, metabolism and development both in humans and wildlife. In general, TH levels are measured by immunoassay (IA) methods but the specificity of the antibodies used in these assays limits selectivity. In the last decade, several analytical methods using liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectrometry (LC-MS/MS) have been developed to measure THs. These new techniques proved to be more accurate than the IA analysis and they were widely used for the determination of TH level in different human and animal tissues. A large part of LC-MS/MS methods described in literature employed between 200 and 500mg of sample, however this quantity can be considered too high especially when preclinical studies are conducted using mice as test subjects. Thus an analytical method that reduces the amount of tissue is essential. In this study, we developed a procedure for the analysis of six THs; L-thyroxine (T4), 3,3',5-triiodo-l-thyronine (T3), 3,3',5'-triiodo-l-thyronine (rT3), 3,5-diiodo-l-thyronine (rT2), 3,3'-diiodo-l-thyronine (T2), 3-iodo-l-thyronine (T1) using isotope ((13)C6-T4, (13)C6-T3, (13)C6-rT3, (13)C6-T2) dilution liquid chromatography-mass spectrometry. The major difference with previously described methods lies in the utilization of a nano-UPLC (Ultra Performance Liquid Chromatography) system in micro configuration. This approach leads to a reduction compared to the published methods, of column internal diameter, flow rate, and injected volume. The result of all these improvements is a decrease in the amount of sample necessary for the analysis. The method was tested on six different mouse tissues: liver, heart, kidney, muscle, lung and brown adipose tissue (BAT). The nano-UPLC system was interfaced with a quadrupole time-of-flight mass spectrometer (Q-TOF2-MS) using the positive ion mode electrospray ionization. In our analytical method the instrumental calibration curves were constructed from 0 to 100pgμL(-1) and all of them showed good linearity (r(2)>0.99). The limit of quantification was from 2.5 to 5pg injected into the column. The method recoveries calculated using spiked mouse liver and spiked mouse muscle were between 83% and 118% (except T1 and rT2 at high concentration) with a coefficient of variation (CV) of <10% for all derivatives. The new methodology allows us to measure T4 and T3 concentrations in a range from 21 to about 100mg and give a more extensive insight on thyroid hormone concentration in different mouse tissue.
authors.affiliation: Helmholtz Zentrum München - German Research Center for Environmental Health (GmbH), Molecular EXposomics, Ingolstädter Landstr. 1, 85764, Neuherberg, Germany. Electronic address: meri.deangelis@helmholtz-muenchen.de.
authors.forename: Meri
authors.initials: M
authors.lastname: De Angelis
authors.affiliation: Technische Universität München-Weihenstephan, Lehrstuhl für Entwicklungsgenetik, c/o Helmholtz Zentrum München, Ingolstädter Landstr. 1, 85764, Neuherberg, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Giesert
authors.affiliation: Institute for Diabetes and Obesity (IDO) and Helmholz Diabetes Center, Helmholtz Zentrum München-German Research Center for Environmental Health (GmbH), Ingolstädter Landstr. 1, 85764, Neuherberg, Germany; Division of Metabolic Diseases, Department of Medicine, Technische Universität München, 85748, Munich, Germany; German Center for Diabetes Research (DZD), Ingolstädter Landstr. 1, 85764, Neuherberg, Germany.
authors.forename: Brian
authors.initials: B
authors.lastname: Finan
authors.affiliation: Institute for Diabetes and Obesity (IDO) and Helmholz Diabetes Center, Helmholtz Zentrum München-German Research Center for Environmental Health (GmbH), Ingolstädter Landstr. 1, 85764, Neuherberg, Germany; Division of Metabolic Diseases, Department of Medicine, Technische Universität München, 85748, Munich, Germany; German Center for Diabetes Research (DZD), Ingolstädter Landstr. 1, 85764, Neuherberg, Germany.
authors.forename: Christoffer
authors.initials: C
authors.lastname: Clemmensen
authors.affiliation: Institute for Diabetes and Obesity (IDO) and Helmholz Diabetes Center, Helmholtz Zentrum München-German Research Center for Environmental Health (GmbH), Ingolstädter Landstr. 1, 85764, Neuherberg, Germany; Division of Metabolic Diseases, Department of Medicine, Technische Universität München, 85748, Munich, Germany; German Center for Diabetes Research (DZD), Ingolstädter Landstr. 1, 85764, Neuherberg, Germany.
authors.forename: Timo D
authors.initials: TD
authors.lastname: Müller
authors.affiliation: Technische Universität München-Weihenstephan, Lehrstuhl für Entwicklungsgenetik, c/o Helmholtz Zentrum München, Ingolstädter Landstr. 1, 85764, Neuherberg, Germany; Helmholtz Zentrum München - German Research Center for Environmental Health (GmbH), Institute of Developmental Genetics, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany.
authors.forename: Daniela
authors.initials: D
authors.lastname: Vogt-Weisenhorn
authors.affiliation: Institute for Diabetes and Obesity (IDO) and Helmholz Diabetes Center, Helmholtz Zentrum München-German Research Center for Environmental Health (GmbH), Ingolstädter Landstr. 1, 85764, Neuherberg, Germany; Division of Metabolic Diseases, Department of Medicine, Technische Universität München, 85748, Munich, Germany; German Center for Diabetes Research (DZD), Ingolstädter Landstr. 1, 85764, Neuherberg, Germany.
authors.forename: Matthias H
authors.initials: MH
authors.lastname: Tschöp
authors.affiliation: Helmholtz Zentrum München - German Research Center for Environmental Health (GmbH), Molecular EXposomics, Ingolstädter Landstr. 1, 85764, Neuherberg, Germany; Technische Universität München, Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt, Department für Biowissenschaften, Weihenstephaner Steig 23, 85350 Freising, Germany.
authors.forename: Karl-Werner
authors.initials: KW
authors.lastname: Schramm
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D007553:Isotope Labeling / Q000379:methods*; D057230:Limit of Detection; D008099:Liver / Q000378:metabolism; D008810:Mice, Inbred C57BL; D009132:Muscles / Q000378:metabolism; D012015:Reference Standards; D012996:Solutions; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013963:Thyroid Hormones / Q000032:analysis* / Q000737:chemistry
chemical_list: D012996:Solutions; D013963:Thyroid Hormones
keywords: Mass spectrometry; Microflow liquid chromatography; Mouse tissues; Thyroid hormones