Objectives: Since T3 is mostly produced in peripheral tissues by T4 deio- dination, assaying tissue T3 and T4 would provide valuable information. We report a novel technique based on HPLC coupled to tandem mass spectrom- etry (MS-MS), which has been applied in rat liver and heart samples as well as in human myocardium. Methods: Samples were homogenized in phosphate buffer (pH 7.4). After centrifugation, supernatants were spiked with stable-isotope-labelled inter- nal standards (IS: 13C-T3 and 13C-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). Results: The yield of the esterification reaction was 80–100%, calibra- tion curves were linear in the range of 0.2 to 20 ng/ml (r > 0.99), and results were linear vs tissue mass in the range of 50–250 mg. Sample derivatization increased the sensitivity and accuracy of HPLC-MS-MS detection, and the minimum amount of tissue needed was close to 50 mg. In control rat myocar- dium, 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 mcg/Kg/day) or high-dose T3 (45 mcg/ Kg/day), cardiac 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 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 explanted hearts T3 and T4 averaged 1.51 ± 0.16 and 5.94 ± 0.63 pmol/g. Conclusions: An HPLC-MS-MS method based on derivatization with buthanol enables T3 and T4 assay in ≥50 mg samples. Tissue T3 and T4 assay may be critical to understand the role of thyroid hormones in physiological and pathophysiological conditions.
A NOVEL HPLC-MS-MS METHOD TO ASSAY TISSUE THYROID HORMONES
SABA, ALESSANDRO;CHIELLINI, GRAZIA;NANNIPIERI, MONICA;ZUCCHI, RICCARDO
2013-01-01
Abstract
Objectives: Since T3 is mostly produced in peripheral tissues by T4 deio- dination, assaying tissue T3 and T4 would provide valuable information. We report a novel technique based on HPLC coupled to tandem mass spectrom- etry (MS-MS), which has been applied in rat liver and heart samples as well as in human myocardium. Methods: Samples were homogenized in phosphate buffer (pH 7.4). After centrifugation, supernatants were spiked with stable-isotope-labelled inter- nal standards (IS: 13C-T3 and 13C-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). Results: The yield of the esterification reaction was 80–100%, calibra- tion curves were linear in the range of 0.2 to 20 ng/ml (r > 0.99), and results were linear vs tissue mass in the range of 50–250 mg. Sample derivatization increased the sensitivity and accuracy of HPLC-MS-MS detection, and the minimum amount of tissue needed was close to 50 mg. In control rat myocar- dium, 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 mcg/Kg/day) or high-dose T3 (45 mcg/ Kg/day), cardiac 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 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 explanted hearts T3 and T4 averaged 1.51 ± 0.16 and 5.94 ± 0.63 pmol/g. Conclusions: An HPLC-MS-MS method based on derivatization with buthanol enables T3 and T4 assay in ≥50 mg samples. Tissue T3 and T4 assay may be critical to understand the role of thyroid hormones in physiological and pathophysiological conditions.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
