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Endocrine Abstracts (2013) 32 P991 | DOI: 10.1530/endoabs.32.P991

1University of Pisa, Pisa, Italy; 2CNR Institute of Clinical Physiology, Pisa, Italy; 3University of Sydney, Sydney, New South Wales, Australia; 4VU University Medical Center, Amsterdam, The Netherlands.


Thyroid hormone (T3) is mostly produced in peripheral tissues by thyroxine (T4) deiodination, and its functional effects are directly related to tissue concentration, which is quite difficult to assay by conventional immunological methods. We report a novel technique, based on tandem mass spectrometry (MS/MS) coupled to HPLC, which was used in left ventricular myocardium obtained from rat and human hearts.

Samples were homogenized in phosphate buffer (pH 7.4). After centrifugation, supernatants were spiked with stable-isotope-labelled internal standards (IS: 13C6-T3 and 13C6-T4) and extracted by SPE. Dried residues were reconstituted and incubated with 3.0 N HCl in n-butanol, obtaining the butyl esters of T3, T4, and ISs. After removing excess reagents, residues were reconstituted with methanol/HCl 0.1 M (50:50 v:v) and injected in the HPLC-MS/MS system (AB-Sciex API 4000). Calibration curves were built with standard solutions containing both analytes at concentrations ranging from 1 to 50 ng/ml and the same amount of IS as put in the samples.

Sample derivatization increased method sensitivity and accuracy, and the minimum amount of tissue needed was ~100–150 mg. In control rat myocardium, tissue T3 and T4 averaged 1.59±0.11 and 2.24±0.22 pmol/g respectively (corresponding plasma free T3 and T4 were 3.69±0.39 and 17.09±1.56 pM). In animals treated with low-dose (6 μg/kg per day) or high-dose (45 μg/kg per day) T3, tissue T3 increased to 3.12±0.29 and 6.76±1.37 pmol/g (plasma free T3 was 8.06±0.83 and 19.59±3.80 pM), while tissue T4 decreased to 0.79±0.06 and 0.77±0.02 pmol/g (plasma free T4 was 6.34±1.41 and 3.49±0.43 pM). In human samples obtained from transplanted hearts T3 and T4 averaged 1.51±0.16 and 5.94±0.63 pmol/g.

In conclusion an HPLC-MS/MS method based on derivatisation with buthanol enables T3 and T4 assay in ≥150 mg myocardial samples. Tissue T3 and T4 assay may be critical to understand the role of thyroid hormones in physiological and pathophysiological conditions.

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