Exp Clin Endocrinol Diabetes 2007; 115 - P01_022
DOI: 10.1055/s-2007-972278

An LC-MS/MS based method for the simultaneous detection of thyroid hormones and their metabolites: thyronines and thyronamines

S Piehl 1, G Balizs 2, T Heberer 2, J Köhrle 1
  • 1Institut für Experimentelle Endokrinologie, Charité – Universitätsmedizin, Berlin, Germany
  • 2Bundesinstitut für Risikobewertung, Fachgruppe 55, Berlin, Germany

Novel metabolites of classical thyroid hormones have recently been shown to occur endogenously and to display unique effects. One example is 3-iodothyronamine (3-T1AM) which reduces body temperature in mice and activates the plasma membrane bound G-Protein coupled receptor TAAR1 (Scanlan et al., 2004). As thyroid hormone metabolites differ only slightly in structure, their distinction by immunological methods is difficult. Therefore, a sensitive and robust selected reaction monitoring (SRM) based liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed capable of unambiguously distinguishing between 13 analytes: thyronine (T0), 3-iodothyronine (3-T1), 3'-T1, 3,5-diiodothyronine (3,5-T2), 3,3'-T2, T3, rT3, T4, thyronamine (T0AM), 3-iodothyronamine (3-T1AM), 3,5-diiodothyronamine (3,5-T2AM), 3,5,3'-triiodothyronamine (3,5,3'-T3AM) and 3,5,3',5'-tetraiodothyronamine (T4AM).

Analyses were performed using an API 365 triple quadrupole tandem mass spectrometer (Applied Biosystems), equipped with TurboIonSpray, Agilent 1100 HPLC system and Bio Analyst Version 1.3.1 software.

All analytes showed the loss of ammonia in their product ion scans. Thus, each analyte was detected by the transition (M+H)+ →(M+H-NH3)+. Optimal HPLC based separation and symmetrical peak shapes were achieved employing a Synergi Polar-RP column (Phenomenex) and a multi-step 29min gradient elution program. Assay stability was verified by determining intra- and inter-assay variation of retention times (1.6s + 0.004s and 1.2s + 0.28s respectively) and area under the peak (4.9% + 3.3% and 8.0% + 2.7% respectively). R2 values >0.97 demonstrated linearity over a wide range of analyte amount (25 pg/ injection –50 ng/ injection). The limit of detection was 25 pg/ injection.

This method will be a useful tool to determine the complex thyroid hormone metabolite content in tissues and experimental set-ups.

Supported by Graduate College 1208, DFG