Cover Page

Cite item


Thyroid hormones play an integral role in growth, homeostasis, and maintenance of physiological functions and are necessary for normal development. Therefore, an accurate assessment of thyroid function is important for both diagnosis and treatment of thyroid disorders. Thyroid function is assessed by measuring thyroid-stimulating hormone (TSH) and thyroid hormones: thyroxine (T4), triiodothyronine (T3), total and free fractions. The concentration of thyroid hormones in patients without pathology of the thyroid gland remains constant and correlates with the level of hormones in tissues and their biological effects. However, most circulating hormones are bound to plasma proteins and only a small part is in free form, the most commonly used methods of immunoassay in this connection have limitations. The method of high performance liquid chromatography − tandem mass spectrometry (HPLC-MS/MS) successfully copes with this problem, which allows improving the specificity and accuracy of measurement of thyroid hormones. High performance liquid chromatography − tandem mass spectrometry allows to achieve specificity, accuracy for reliable determination of the level of thyroid hormones, increasing diagnostic capabilities. This method is especially important in the presence of thyroid gland pathology and factors affecting the binding of hormones to the protein.

About the authors

Margarita S. Mikhina

Endocrinology Research Centre, Moscow

Author for correspondence.
ORCID iD: 0000-0002-4382-0514


11 Dm.Ulyanova street, 117036 Moscow.

SPIN-код: 3172-5538

Russian Federation

Vitaliy A. Ioutsi

Endocrinology Research Centre, Moscow

ORCID iD: 0000-0001-9002-1662


11 Dm.Ulyanova street, 117036 Moscow.

SPIN-код: 9734-0997

Russian Federation

Ekaterina A. Troshina

Endocrinology Research Centre, Moscow

ORCID iD: 0000-0002-8520-8702

MD, PhD, Professor.

11 Dm.Ulyanova street, 117036 Moscow.

SPIN-код: 8821-8990

Russian Federation


  1. Little AM. Local regulation of thyroid hormone signaling. Vitam Horm. 2018;106:1−17. doi: 10.1016/bs.vh.2017.06.004.
  2. Oetting A, Yen PM. New insights into thyroid hormone action. Best Pract Res Clin Endocrinol Metab. 2007;21(2):193−208. doi: 10.1016/j.beem.2007.04.004.
  3. Al-azzam SI, Alkhateeb AM, Al-Azzeh O, et al. The role of type II deiodinase polymorphisms in clinical management of hypothyroid patients treated with levothyroxine. Exp Clin Endocrinol Diabetes. 2013;121(05):300−305. doi: 10.1055/s-0032-1331695.
  4. Maia AL, Kim BW, Huang SA, et al. Type 2 iodothyronine deiodinase is the major source of plasma T3 in euthyroid humans. J Clin Invest. 2005;115(9):2524−2533. doi: 10.1172/jci25083.
  5. Schussler GC. The thyroxine-binding proteinss. Thyroid. 2000;10(2):141−149. doi: 10.1089/thy.2000.10.141.
  6. Mendel CM, Weisiger RA. Thyroxine uptake by perfused rat liver. No evidence for facilitation by five different thyroxine-binding proteins. J Clin Invest. 1990;86(6):1840−1847. doi: 10.1172/jci114914.
  7. Christofides ND, Midgley JE. Inaccuracies in free thyroid hormone measurement by ultrafiltration and tandem mass spectrometry. Clin Chem. 2009;55(12):2228−2229. doi: 10.1373/clinchem.2009.134593.
  8. Symons RG, Wellby ML. Assay of serum free T4, free T3 and free rT3 by equilibration dialysis and radioimmunoassay. Pathology. 1979;11(2):321−322. doi: 10.1016/s0031-3025(16)39965-2.
  9. Weeke J, Orskov H. Ultrasensitive radioimmunoassay for direct determination of free triiodothyronine concentration in serum. Scand J Clin Lab Invest. 1975;35(3):237−244. doi: 10.1080/00365517509095735.
  10. Stockigt JR. Free thyroid hormone measurement. A critical appraisal. Endocrinol Metab Clin North Am. 2001;30(2):265−289. doi: 10.1016/s0889-8529(05)70187-0.
  11. Baloch Z, Carayon P, Conte-Devolx B, et al. Laboratory medicine practice guidelines. Laboratory support for the diagnosis and monitoring of thyroid disease. Thyroid. 2003;13(1):3−126. doi: 10.1089/105072503321086962.
  12. Nelson JC, Wang R, Asher DT, Bruce Wilcox RB. The nature of analogue-based free thyroxine estimates. Thyroid. 2004;14(12):1030−1036. doi: 10.1089/thy.2004.14.1030.
  13. Wilcox RB, Nelson JC. Counterpoint: legitimate and illegitimate tests of free-analyte assay function: we need to identify the factors that influence free-analyte assay results. Clin Chem. 2009;55(3):442−444. doi: 10.1373/clinchem.2008.120154.
  14. Soldin OP, Soldin SJ. Thyroid hormone testing by tandem mass spectrometry. Clin Biochem. 2011;44(1):89−94. doi: 10.1016/j.clinbiochem.2010.07.020.
  15. Jonklaas J, Kahric-Janicic N, Soldin OP, Soldin SJ. Correlations of free thyroid hormones measured by tandem mass spectrometry and immunoassay with thyroid-stimulating hormone across 4 patient populations. Clin Chem. 2009;55(7):1380−1388. doi: 10.1373/clinchem.2008.118752.
  16. Hoermann R, Eckl W, Hoermann C, Larisch R. Complex relationship between free thyroxine and TSH in the regulation of thyroid function. Eur J Endocrinol. 2010;162(6):1123−1129. doi: 10.1530/eje-10-0106.
  17. Hansen PS, Brix TH, Sørensen TI, et al. Major genetic influence on the regulation of the pituitary-thyroid axis: a study of healthy Danish twins. J Clin Endocrinol Metab. 2004;89(3):1181−1187. doi: 10.1210/jc.2003-031641.
  18. van Deventer HE, Mendu DR, Remaley AT, Soldin SJ. Inverse log-linear relationship between thyroid-stimulating hormone and free thyroxine measured by direct analog immunoassay and tandem mass spectrometry. Clin Chem. 2010;57(1):122−127. doi: 10.1373/clinchem.2010.154088.
  19. Clark PM, Holder RL, Haque SM, et al. The relationship between serum TSH and free T4 in older people. J Clin Pathol. 2012;65(5):463−465. doi: 10.1136/jclinpath-2011-200433.
  20. Serdar MA, Ozgurtas T, Ispir E, et al. Comparison of relationships between FT4 and log TSH in Access DXI 800 Unicel, Modular E170 and ADVIA Centaur XP Analyzer. Clin Chem Lab Med. 2012;50(10):1849–1852. doi: 10.1515/cclm-2012-0193.
  21. Fritz KS, Wilcox RB, Nelson JC. A direct free thyroxine (T4) immunoassay with the characteristics of a total T4 immunoassay. Clin Chem. 2007;53(5):911−915. doi: 10.1373/clinchem.2006.083915.
  22. Loh TP, Kao SL, Halsall DJ, et al. Macro-thyrotropin: a case report and review of literature J Clin Endocrinol Metab. 2012;97(6):1823−1828. doi: 10.1210/jc.2011-3490.
  23. Soukhova N, Soldin OP, Soldin SJ. Isotope dilution tandem mass spectrometric method for T4/T3. Clin Chim Acta. 2004;343(1–2):185−190. doi: 10.1016/j.cccn.2004.01.007.
  24. Jonklaas J, Davidson B, Bhagat S, Soldin SJ. Triiodothyronine levels in athyreotic individuals during levothyroxine therapy. JAMA. 2008;299(7):769−777. doi: 10.1001/jama.299.7.769.
  25. Midgley JE. Spurious conclusions on analog free thyroxine assay performance. Clin Chem. 2007;53(9):1714−1714. doi: 10.1373/clinchem.2007.090878.
  26. Sapin R, d’Herbomez M. Free thyroxine measured by equilibrium dialysis and nine immunoassays in sera with various serum thyroxine-binding capacities. Clin Chem. 2003;49(9):1531−1535. doi: 10.1373/49.9.1531.
  27. Wang R, Nelson JC, Weiss RM, Wilcox RB. Accuracy of free thyroxine measurements across natural ranges of thyroxine binding to serum proteins. Thyroid. 2000;10(1):31−39. doi: 10.1089/thy.2000.10.31.
  28. Christofides ND, Wilkinson E, Stoddart M, et al. Serum thyroxine binding capacity-dependent bias in an automated free thyroxine assay. J Immunoassay. 1999;20(4):201−221. doi: 10.1080/01971529909349351.
  29. Vaidya B, Anthony S, Bilous M, et al. Detection of thyroid dysfunction in early pregnancy: universal screening or targeted high-risk case finding? J Clin Endocrinol Metab. 2007;92(1):203−207. doi: 10.1210/jc.2006-1748.
  30. Utiger R. Maternal hypothyroidism and fetal development. N Engl J Med. 1999;341(8):601−602. doi: 10.1056/nejm199908193410809.
  31. Escobar G, Obregón M, Rey F. Maternal thyroid hormones early in pregnancy and fetal brain development. Best Pract Res Clin Endocrinol Metab. 2004;18(2):225−248. doi: 10.1016/j.beem.2004.03.012.
  32. Lazarus JH, Premawardhana LD. Screening for thyroid disease in pregnancy. J Clin Pathol. 2005;58(5):449−452. doi: 10.1136/jcp.2004.021881.
  33. Pop VJ, Brouwers EP, Vader HL, et al. Maternal hypothyroxinaemia during early pregnancy and subsequent child development: a 3-year follow-up study. Clin Endocrinol (Oxf). 2003;59(3):282−288. doi: 10.1046/j.1365-2265.2003.01822.x.
  34. Li Y, Shan Z, Teng W, et al. Abnormalities of maternal thyroid function during pregnancy affect neuropsychological development of their children at 25−30 months. Clin Endocrinol (Oxf). 2009;72(6):825−829. doi: 10.1111/j.1365-2265.2009.03743.x.
  35. Casey B, Dashe J, Wells C, et al. Subclinical hypothyroidism and pregnancy outcomes. Obstet Gynecol. 2005;105(2):239−245. doi: 10.1097/01.aog.0000152345.99421.22.
  36. Leung AS, Millar LK, Koonings PP, et al. Perinatal outcome in hypothyroid pregnancies. Int J Gynaecol Obstet. 1993;43(2):230−230. doi: 10.1016/0020-7292(93)90343-u.
  37. Lee RH, Spencer CA, Mestman JH, et al. Free T4 immunoassays are flawed during pregnancy. Am J Obstet Gynecol. 2009;200(3):260.e1−260.e6. doi: 10.1016/j.ajog.2008.10.042.
  38. Huang SA, Dorfman DM, Genest DR, et al. Type 3 iodothyronine deiodinase is highly expressed in the human uteroplacental unit and in fetal epithelium. J Clin Endocrinol Metab. 2003;88(3):1384−1388. doi: 10.1210/jc.2002-021291.
  39. Soldin O, Hilakivi-Clarke L, Weiderpass E, Soldin S. Trimester-specific reference intervals for thyroxine and triiodothyronine in pregnancy in iodine-sufficient women using isotope dilution tandem mass spectrometry and immunoassays. Clin Chim Acta. 2004;349(1−2):181−189. doi: 10.1016/j.cccn.2004.06.021.
  40. Soldin O, Tractenberg R, Soldin S. Differences between measurements of T4 and T3 in pregnant and nonpregnant women using isotope dilution tandem mass spectrometry and immunoassays: are there clinical implications? Clin Chim Acta. 2004;347(1−2):61−69. doi: 10.1016/j.cccn.2004.03.033.
  41. Lim VS. Thyroid function in patients with chronic renal failure. Am J Kidney Dis. 2001;38(4 Suppl 1):S80−S84. doi: 10.1053/ajkd.2001.27410.
  42. Kaptein E, Macintyre S, Weiner J, et al. Free thyroxine estimates in nonthyroidal illness: comparison of eight methods. J Clin Endocrinol Metab. 1981;52(6):1073−1077. doi: 10.1210/jcem-52-6-1073.
  43. Ross HA, de Rijke YB, Sweep FC. Spuriously high free thyroxine values in familial dysalbuminemic hyperthyroxinemia. Clin Chem. 2010;57(3):524−525. doi: 10.1373/clinchem.2010.158170.
  44. Cartwright D, O’Shea P, Rajanayagam O, et al. Familial dysalbuminemic hyperthyroxinemia: a persistent diagnostic challenge. Clin Chem. 2009;55(5):1044−1046. doi: 10.1373/clinchem.2008.120303.
  45. Martel J, Després N, Ahnadi C, et al. Comparative multicentre study of a panel of thyroid tests using different automated immunoassay platforms and specimens at high risk of antibody interference. Clin Chem Lab Med. 2000;38(8):785−793. doi: 10.1515/cclm.2000.112.
  46. Sakata S. Autoantibodies against thyroid hormones or lodothyronine. Ann Intern Med. 1985;103(4):579−589. doi: 10.7326/0003-4819-103-4-579.
  47. Sapin R. Serum thyroxine binding capacity-dependent bias in five free thyroxine immunoassays: assessment with serum dilution experiments and impact on diagnostic performance. Clin Biochem. 2001;34(5):367−371. doi: 10.1016/s0009-9120(01)00241-7.
  48. Rotondi M, Leporati P, La Manna A, et al. Raised serum TSH levels in patients with morbid obesity: is it enough to diagnose subclinical hypothyroidism? Eur J Endocrinol. 2008;160(3):403−408. doi: 10.1530/eje-08-0734.
  49. Marzullo P, Minocci A, Tagliaferri MA, et al. Investigations of thyroid hormones and antibodies in obesity: leptin levels are associated with thyroid autoimmunity independent of bioanthropometric, hormonal, and weight-related determinants. J Clin Endocrinol Metab. 2010;95(8):3965−3972. doi: 10.1210/jc.2009-2798.
  50. Holm SS, Hansen SH, Faber J, Staun-Olsen P. Reference methods for the measurement of free thyroid hormones in blood. Clin Biochem. 2004;37(2):85−93. doi: 10.1016/j.clinbiochem.2003.09.009.
  51. Gu J, Soldin OP, Soldin SJ. Simultaneous quantification of free triiodothyronine and free thyroxine by isotope dilution tandem mass spectrometry. Clin Biochem. 2007;40(18):1386−1391. doi: 10.1016/j.clinbiochem.2007.08.007.
  52. Tikanoja S. Ultrafiltration devices tested for use in a free thyroxine assay validated by comparison with equilibrium dialysis. Scand J Clin Lab Invest. 1990;50(6):663−669. doi: 10.3109/00365519009089185.
  53. Langton JE, Brent GA. Nonthyroidal illness syndrome: evaluation of thyroid function in sick patients. Endocrinol Metab Clin North Am. 2002;31(1):159−172. doi: 10.1016/s0889-8529(01)00008-1.
  54. Gounden V, Jonklaas J, Soldin S. A pilot study: subclinical hypothyroidism and free thyroid hormone measurement by immunoassay and mass spectrometry. Clin Chim Acta. 2014;430:121−124. doi: 10.1016/j.cca.2013.12.034.

Copyright (c) 2018 "Paediatrician" Publishers LLC

This website uses cookies

You consent to our cookies if you continue to use our website.

About Cookies