Analytical Methods for the Estimation of Levothyroxine and Levothyroxine Sodium in Pharmaceutical Formulations – A Review



  • Preethi G Department of Pharmaceutical Analysis,  Chalapathi Institute of Pharmaceutical Sciences,  Guntur
  • Siva Prasad M. Department of pharmaceutics,  Chalapathi Institute of Pharmaceutical Sciences
  • Prachet P. Department of Pharmaceutical Analysis,  Chalapathi Institute of Pharmaceutical Sciences,  Guntur
  • Rama Rao N. Department of pharmaceutics,  Chalapathi Institute of Pharmaceutical Sciences,Guntur


Levothyroxine (L-T4) is used in the treatment of hypothyroidism and is a synthetic levoisomer of the thyroid hormone thyroxine produced by the thyroid gland. Levothyroxine sodium (L-T4S) salt has a high clinical utility. There are numerous studies that have been published that describe analytical methods for the estimation of L-T4 and L-T4S in pharmaceutical formulations and also in various biological matrices like blood, plasma, serum, and urine. This review article focused on analytical methods for estimating L-T4 and L-T4S in pharmaceutical formulations such as Thin Layer Chromatography (TLC), UV-visible spectrophotometer, High-Performance Liquid Chromatography (HPLC), Capillary electrophoresis, Luminescence, Sequential Injection Analysis (SIA), Voltammetry, and Hyphenated techniques. All the analytical methods for the estimation of L-T4 and L-T4S are given in tabular form. Out of all the analytical methods reported, HPLC is the most commonly used method for the estimation of L-T4 and L-T4S with optimum separation and resolution.


Download data is not yet available.


Metrics Loading ...


L-T4, L-T4S, Hypothyroidism, Pharmaceutical, Analytical methods



How to Cite

G P, M. SP, P. P, N. RR. Analytical Methods for the Estimation of Levothyroxine and Levothyroxine Sodium in Pharmaceutical Formulations – A Review. Scopus Indexed [Internet]. 2023 Nov. 15 [cited 2024 Apr. 20];16(6):7104-13. Available from:


Layal Chakar.; Antanio C Bianco.; Jaqueline Jonklaas.; Robin P Peeters. Hypothyroidism. Lancet. 2017, 390(10101), 1550-1562.

Leslie Mendoza Temple.; Pooja Saigal. Chapter 34 – Hypothyroidism. Integrative Medicine (Fourth Edition). Elsevier. 2018, Pages 347-360.e3.

Funkhouser William K. “Pathology: The Clinical Description of Human Disease”. Essential Concepts in Molecular Pathology. 2020, 177–190.

Hypothyroidism research: A long term effort. MedlinePlus Magazine. National Library of Medicine. 2021.

Mincer DL,; Jialal I. Hashimoto Thyroiditis. [Updated 2022 Jun 21]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023 Jan

Chaker L.; Razvi S.; Bensenor I.M.; et al. Hypothyroidism. Nat Rev Dis Primers 8, 30, 2022

Mayo Clinic, Diseases and conditions, Symptoms and Causes, Hypothyroidism (underactive thyroid). 2022

Hypothyroidism (underactive thyroid). National Institute of Diabetes and Digestive and Kidney Diseases. 2021

Rashmi Mullur.; Yan-Yan Liu.; Gregory A. Brent. Thyroid Hormone Regulation of Metabolism. Physiol Rev. 2014, 92(2), 355-38.

A.N. Rajalakshmi.; Farghana A Begam. Thyroid Hormones in the Human Body: A review. JDDT. 2021, 11(5), 178-182.

Harington CR. Chemistry of Thyroxin: Isolation of Thyroxin from the Thyroid Gland. Biochem J. 1926, 20(2), 293-299.

Harington CR. Chemistry of Thyroxine: Constitution and Synthesis of Desiodo- Thyroxine. Biochem J. 1926, 20(2), 300-313.

World Health Organization model list of essential medicines: 22nd list 2021, pp44.

Meteo RCI.; Hennessey JV. Thyroxine and treatment of hypothyroidism: seven decades of experience. Endocrine. 2019, 66(1), 10-17.

Cooper DS.; Halpern R.; Wood LC.; Levin AA.; Ridgway EC. L-Thyroxine therapy in subclinical hypothyroidism. A double-blind, placebo-controlled trial. Ann Intern Med, 1984, 101(1), 18-24.

Haugen BR.; Alexander EK.; Bible KC.; Doherty GM.; Mandel SJ.; Nikiforov YE.; Pacini F.; Randolph GW.; Sawka AM.; Schlumberger M.; Schuff KG.; Sherman SI.; Sosa JA.; Steward DL.; Tuttle RM.; Wartofsky L. 2015 American Thyroid Association Management Guidelines for Adult Patients with Thyroid Nodules and Differentiated Thyroid Cancer: The American Thyroid Association Guidelines Task Force on Thyroid Nodules and Differentiated Thyroid Cancer. Thyroid. 2016, 26(1), 1-133.

Salim A.; Vassiliu P.; Velmahos GC.; Sava J.; Murray JA.; Belzberg H.; Asensio JA.; Demetriades D. The role of thyroid hormone administration in potential organ donors. Arch Surg. 2001, 136(12), 1377-80.

Calissendorff J.; Falhammar H. To Treat or Not to Treat Subclinical Hypothyroidism, What Is the Evidence? Medicina (Kaunas, Lithuania). 2020, 56(1), 40.

Government of India, Ministry of health and family welfare. Indian Pharmacopoeia. Assay of Thyroxine sodium. 2022, 9th edition, vol 3, pp 3787,3788.

Archana A. Bele.; Anubha Khale. An Overview on Thin Layer Chromatography, IJPSR, 2011, 2(2), 256-267.

Govinda Verma and Dr. Manish Mishra. Development and Optimization of UV-Vis Spectroscopy - A Review. WJPS. 2018, 7(11), 1170-1180.

Abdu Hussen Ali. High-Performance Liquid Chromatography (HPLC): A Review. Annals of Advances in Chemistry, 2022, 6(1), 10-20.

S. Gummadi and V. N. Kandula. A Review on Electrophoresis, Capillary Electrophoresis and Hyphenations. IJPSR. 2020, 11(12), 6038-6056.

Manisha Kumar Mishra.; P. M. Mishra.; Vikas Dubay.; Isharat Khan.; Taranjeet Sachdev. A Review for Luminescence Property of Materials, Its Detection and Probabilities for Embedding of Luminescence with MEMS Technology. Advanced Materials Letters. 2021, 12(5), 1-9.

Raluca-Ioana Stefan.; Jacobus F Staden.; Hassan Y About-Eneib. Simultaneous determination of l-thyroxine (l-T4), d-thyroxine (d-T4), d-triiodothyronine (l-T3) using a sensors/sequential injection analysis system. Talanta. 2004, 64(1), 151-155.

Shantonu Roy.; Soumya Pandit. Microbial Electrochemical Technology, chapter 1.2 - Microbial Electrochemical System: Principles and Application, Elsevier, 2019, 19-28.

Pallavi Phalke.; Shilpa Kavade. Review on Hyphenated Techniques. Int J Chem Stud. 2013, 1(3), 157-165.

Lipp HP.; Administration and Pharmacokinetics of Levothyroxine, In: Kahaly, G.J.(eds), 70 Years of levothyroxine, Springer, Cham, 2021

Engler D.; Merkelbach U.; Steiger G.; Burger AG. The monodeiodination of Triiodothyronine and reverse Triiodothyronine in man: a quantitative evaluation of the pathway by the use of turnover rate techniques. J Clin EndoceinolMetab. 1984, 58(1), 49-61.

Hassan Y Aboul-Enein.; Vince Serignese. Optical purity determination of thyroxine enantiomers in bulk materials by chiral thin layer chromatography. Biomedical Chromatography. 1994, 8(6), 317-318.

Gregorini A.; Ruiz M. E.; Volonte M G. A derivative UV spectrophotometric method for the determination of levothyroxine sodium in tablets. J. Anal. Chem. 2013, 68(6), 510-515.

Nabeel Othman.; Ali M Hussein. Determination of Thyroxine by Spectrophotometric and High Performance Liquid Chromatographic Methods. IJRR. 2018, 11(1), 30-38.

Smith DJ.; Biesemeyer M.; Yaciw C. The separation and determination of liothyronine and levothyroxine in tablets by reversed-phase high performance liquid chromatography. J Chromatogr Sci. 1981, 19(2), 72-8.

Rao S Rapaka.; Patrick W Knight.; Vadlamani K Prasad. Reverse-Phase High- Performance Liquid Chromatographic Analysis of Liothyronine Sodium and Levothyroxine Sodium in Tablets Formulation: Preliminary Studies on Dissolution and Content Uniformity. Journal of Pharmaceutical Sciences. 1981, 70(2), 131-134.

Richheimer SL.; Ameer TM. Stability-indicating assay, dissolution, and content uniformity of sodium levothyroxine in tablets. J Pharm Sci. 1983, 72(11), 1349-1351.

Brower JF.; Toler DY.; Reepmeyer JC. Determination of sodium levothyroxine in bulk, tablet, and injection formulations by high-performance liquid chromatography. J Pharm Sci. 1984, 73(9), 1315-1317.

Collier J.W.; Shah R.B.; Bryant A.R.; Habib J Muhammad.; Khan Mansoor.; Faustino J Patrick. Development and application of a validated HPLC method for the analysis of dissolution samples of levothyroxine sodium drug products. Journal of pharmaceutical and biomedical analysis. 2010, 54(3), 433-438.

Takahashi M.; Nagashima M.; Shigeoka S.; Kamimura H.; Kamata K. Determination of thyroid hormones in pharmaceutical preparations, after derivatization with 9-anthroylnitrile, by high-performance liquid chromatography with fluorescence detection. J Chromatogr A. 2002, 958(1-2), 299-303.

Shah RB.; Bryant A.; Collier J.; Habib MJ.; Khan MA. Stability indicating validated HPLC method for quantification of levothyroxine with eight degradations peaks in the presence of excipients. International Journal of Pharmaceutics. 2008, 360(1-2), 77-82.

Singars Pravin and Belamker Nikhil. Applying DMAIC principles for improving method performance of Quantitative determination of Levothyroxine sodium in tablet dosage form using high performance liquid chromatography technique. RJPBCS. 2016, 7(4), 2041-2052.

Steven L.; Richheimer.; Charlotte B Jensen.; Determination of liothyronine and levothyroxine in thyroid preparations by liquid chromatography. JPS. 1986, 75(2), 215-217.

R.L. Garnick.; G. F. Burt.; D. A. Long.; J. W. Bastian.; J. W. Bastian.; J. P. Aldred. High-performance liquid chromatographic assay for sodium levothyroxine in tablet formulations: Content uniformity applications. JPS. 1984, 73(1), 75-77.

Gondová Tat'ána.; Petrovaj Ján.; Suchá Mária.; Armstrong Daniel W. Stereoselective HPLC Determination of Thyroxine Enantiomers in Pharmaceuticals. Journal of Liquid Chromatography & Related Technologies. 2011, 34(19), 2304–2314.

So Hee Jeon.; Minkee Kim.; Hyo-Kyung Han.; Wonjae Lee. Direct enantiomer separation of thyroxine in pharmaceuticals using crown ether type chiral stationary phase. Arch Pharm Res. 2010, 33(9), 1419-1423.

C.N. Carducci.; S.E. Lucangioli.; V.G. RrodríG.C.; Farnández Otero. Application of extraction disks in dissolution test of clenbuterol and levothyroxine tablets by capillary electrophoresis. Journal of Chromatography. 1996, 730(1-2), 313-319.

Sarzamin Khan A.; Carneiro Leonardo S.A.; Romani Eric C.; DunieskysLarrudé G.; Ricardo Aucelio Q. Quantification of thyroxine by the selective photoluminescence quenching of L-cysteine-ZnS quantum dots in aqueous solutions containing hexadecyltrimethylammonium bromide. Journal of Luminescence. 2014, 156, 16-24.

Amir Waseem.; Mohammad Yaqoob,; Abdul Nabi.; Gillian M Greenway. Determination of Thyroxine Using Tris(2,2'-Bipyridyl)Ruthenium(III)-NADH Enhanced Electrochemiluminescence Detection. Analytical Letters. 2007, 40(6), 1071-1083.

Amir Waseem.; Mohammad Yaqoob.; Abdul Nabi. Determination of thyroxine in pharmaceuticals using flow injection with luminal chemiluminescence inhibition detection. Luminescence. 2006, 21(3), 174-178.

Amir Waseem.; Mohammad Yaqoob.; Abdul Nabi. Flow injection determination of thyroxine in pharmaceutical preparations using tris(2,2'-bipyridyl)ruthenium(III)-NADH chemiluminescence detection. Talanta. 2007, 71(1), 56-61.

Smajdor Joanna.; Piech Robert.; RuminMartyna.; Paczosa-Balor Beata.; Smajdor Zbigniew. High Sensitive Voltammetric Levothyroxine Sodium Determination on Renewable Mercury Film Silver Based Electrode. Journal of the Electrical Society. 2016, 163(7), H605-H609.

Sasi S Kannamkumarath.; Rodolfo G Wuilloud.; Apryll Stalcup.; Joseph A Caruso.; Himanshu Patel.; Adel Saka. Determination of levothyroxine and its degradation products in pharmaceutical tablets by HPLC-UV-ICP-MS. JAAS. 2004, 19(1), 107-113.

Dongri Jin.; Avvaru Praveen Kumar.; Goon-Cheol Song.; Yong-Ill Lee. Determination of thyroxine enantiomers in pharmaceutical formulation by high-performance liquid chromatography-mass spectrometer with precolumn derivatization. Microchemical Journal. 2008, 88(1), 62-66.

Jisun Lee.; Suraj Adhikari.; Wonjae Lee.; Hye-Ran Yoon. Devolepment of Ultra High-Performance Liquid Chromatography-Tandem Mass Spectrometry Method for Enantiomer Resolution of Thyroxine on a Chiral Crown Ether Derived Chiral Stationaru Phase. Chromatographia. 2022.

Gholamali Kazemifard.; Douglas Moore.; Ali A Habashi. Identification and quantification of sodium-thyroxine and its degradation products by LC using electrochemical and MS detection. JPBA. 2001, 25(5-6), 697-711.