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Abstract

Thyroid hormones are biologically active small molecules responsible for growth and development regulation, basal metabolic rate, and lipid and carbohydrate metabolism. Liquid chromatography mass spectrometry (LC–MS) can be used to quantify thyroid hormones blood level with high speed and selectivity, aiming to improve the diagnosis and treatment of the severe pathological conditions in which they are implicated, i.e., hypo- and hyperthyroidism. In this work, the gas-phase behavior of the isomeric thyroid hormones triiodothyronine (T3) and reverse triiodothyronine (rT3) in their deprotonated form was studied at a molecular level using MS-based techniques. Previously reported collision-induced dissociation experiments yielded distinct spectra despite the high structural similarity of the two compounds, suggesting different charge sites to be responsible. Infrared multiple photon dissociation spectroscopy on [T3-H]− and [rT3-H]− was performed, and the results were interpreted using DFT and MP2 calculations, assessing the prevalence of T3 in the carboxylate form and rT3 as a phenolate isomer. The different deprotonation sites of the two isomers were also found to drive their ion-mobility behavior. In fact, [T3-H]− and [rT3-H]− were successfully separated. Drift times were correlated with collisional cross section values of 209 and 215 Å2 for [T3-H]− and [rT3-H]−, respectively. Calculations suggested the charge site to be the main parameter involved in the different mobilities of the two anions. Finally, bare [T3-H]− and [rT3-H]− were made to react with neutral acetylacetone and trifluoroacetic acid, confirming rT3 to be more acidic than T3 in agreement with the calculated gas-phase acidities of T3 and rT3 equal to 1345 and 1326 kJ mol–1, respectively.

Sample Preparation

Triiodothyronine (3,5,3′-triiodothyronine, T3) and reverse triiodothyronine (3,3′,5′-triiodothyronine, rT3) were bought from Sigma-Aldrich, Milan, IT and used without further purification. T3 was dissolved in methanol of MS-grade to a final concentration of 10–5 M with the addition of ammonia (0.1% v/v). rT3 is provided as a methanolic solution already basified with ammonia, which was diluted in methanol to reach the same concentration as T3, i.e., 10–5 M. Acetylacetone, trifluoroacetic acid (TFA), and diethylmethoxyborane (DEMB) were commercial products obtained from Sigma-Aldrich to be used as neutral reagents for ion–molecule reactions.

IRMPD Analysis

IRMPD spectra in the 800–1800 cm–1 frequency range were recorded at the free-electron laser (FEL) beamline of the Centre Laser Infrarouge d’Orsay (CLIO). The FEL beamline (operated at 42.3 MeV for the present experiments) is coupled with a hybrid FT-ICR tandem mass spectrometer (APEX-Qe Bruker Daltonics) equipped with a 7.0 T actively shielded magnet and a quadrupole–hexapole interface. The ions of interest were generated by direct injection of the T3 and rT3 solutions into the ESI source of the mass spectrometer, mass isolated in the quadrupole sector, and accumulated in the hexapole for 1 ms before being irradiated in the FT-ICR cell with the IR FEL light operating at a repetition rate of 25 Hz. Irradiation times were 220 and 300 ms for [T3-H]− and [rT3-H]−, respectively. The photofragmentation products were mass analyzed, and IRMPD spectra were obtained by plotting the photofragmentation yield R (corresponding to −log[Ip/(Ip + ΣIf)], where Ip and ΣIf are the integrated abundances of the precursor ion and the sum of the fragments, respectively) as a function of the photon energy.