STM Journals Peer-reviewed research from Consortium e-Learning Network

Research & Reviews: A Journal of Drug Formulation, Development and Production Original Research

Bioanalytical Method Development and Validation for the Estimation of Levothyroxine in Human K₃EDTA Plasma by Using UPLC-MS/MS

  1. Shahzeb Ahmad Department of Pharmacy, Millennium College of Pharmacy, Bhopal
  2. Ankita Shukla Department of Pharmacy, Millennium College of Pharmacy, Bhopal
  3. Bala Subramanyam Department of Pharmacy, Millennium College of Pharmacy, Bhopal

Abstract

A rapid, sensitive, and highly selective UPLC-MS/MS method was developed and validated for the quantitative estimation of Levothyroxine in human plasma using Levothyroxine-D₃ as the internal standard (IS). Chromatographic separation was achieved on a Gemini NX-C18 column (50 × 3.0 mm, 3 µm) with a mobile phase consisting of acetonitrile and water (70:30, v/v) containing 0.015% formic acid at a flow rate of 0.5 mL/min. The total run time was 0.9 minutes per sample. Detection was carried out in positive electrospray ionization mode using multiple reaction monitoring (MRM) with transitions m/z 777.60→731.65 for Levothyroxine and m/z 780.70→734.60 for the IS. The method was validated over a linear range of 0.5–2000 ng/mL, with correlation coefficients (r²) consistently greater than 0.998. Intra- and inter-day precision (%CV) and accuracy values were within the acceptable limits of ±15% as per USFDA and ICH M10 guidelines. High and consistent recoveries were obtained using solid-phase extraction (SPE), with negligible matrix effects across six plasma lots. The analyte exhibited acceptable stability under all tested conditions including freeze–thaw, bench-top, autosampler, and long-term storage. Dilution integrity and system suitability parameters confirmed the robustness and reliability of the method. The validated method is simple, reproducible, and suitable for high-throughput analysis of Levothyroxine in clinical and pharmacokinetic studies, making it a strong candidate for application in therapeutic drug monitoring and bioequivalence evaluations.

Keywords

References (20)

  1. Shah RP, Shah NA, Patel SA. A validated LC-MS/MS method for the quantification of levothyroxine in human plasma and its application to a bioequivalence study. Biomed Chromatogr. 2020;34(10):e4898. doi:10.1002/bmc.4898.
  2. Kumar S, Saha S, Singh K, Singh T, Mishra AK, Dubey BN, et al. Beneficial Effects of Spirulina on Brain Health: A Systematic Review. Current Functional Foods. 2025;3(1). doi:10.2174/0126668629269256231222092721
  3. Professor and Head, Dept. of Pharmacognosy, The Erode College of Pharmacy, Perundurai Main Road, Veppampalayam, Erode-638112, Tamilnadu, India, RaviKKumar VR, Rathi S, Professor & Principal, School of Pharmacy, Rai University, Ahmedabad, Gujarat, India, Singh S, Assistant Professor, School of Pharmacy, Rai University, Ahmedabad, Gujarat, India, et al. A Comprehensive Review on Ulcer and Their Treatment. Chinese Journal of Applied Physiology. 2023;39:e20230006. doi:10.62958/j.cjap.2023.006
  4. Singh V, Arora S, Akram W, Alam S, Kumari L, Kumar N, et al. Involvement of Molecular Mechanism and Biological Activities of Pemirolast: A Therapeutic Review. New Emirates Medical Journal. 2024;05. doi:10.2174/0102506882308410240607053814
  5. Professor, Patel College of Pharmacy, Madhyanchal Professional University, Ratibad, Bhopal, MP. India, Rajput DS, Gupta N, Professor and Dean, Patel College of Pharmacy, Madhyanchal Professional University, Ratibad, Bhopal, MP. India, Singh S, Ph.D. Research Scholar, Patel College of Pharmacy, Madhyanchal Professional University, Ratibad, Bhopal, MP. India, et al. A Comprehensive Review: Personalized Medicine for Rare Disease Cancer Treatment. Chinese Journal of Applied Physiology. 2023;39:e20230008. doi:10.62958/j.cjap.2023.008
  6. Patel College of Pharmacy, Madhyanchal Professional University, Bhopal, MP. India, Singh S, Chaurasia A, Patel College of Pharmacy, Madhyanchal Professional University, Bhopal, MP. India, Rajput DS, Patel College of Pharmacy, Madhyanchal Professional University, Bhopal, MP. India, et al. Mucoadhesive Drug Delivery System and There Future Prospective: Are a Promising Approach for Effective Treatment? Chinese Journal of Applied Physiology. 2023;39:e20230005. doi:10.62958/j.cjap.2023.005
  7. Kumar S, Saha S, Sharma B, Singh S, Shukla P, Mukherjee S, et al. The Role of Resveratrol in Alzheimer's Disease: A Comprehensive Review of Current Research. Current Functional Foods. 2024;2(2). doi:10.2174/0126668629269244231127071411
  8. Sigmapharm laboratories llc, Bensalem, Pennsylvania, USA, Patel S, Y I, Crescent School of Pharmacy, B.S. Abdur Rahman Crescent Institute of Science and Technology, Chennai-600048, Tamilnadu, India, Singh S, GSVM Medical College (MBBS 3rd year Student), Kanpur-208002, Uttar Pradesh, India, et al. Recent Innovations and Future Perspectives in Transferosomes for Transdermal Drug Delivery in Therapeutic and Pharmacological Applications. Chinese Journal of Applied Physiology. 2024:e20240031. doi:10.62958/j.cjap.2024.031
  9. Assistant Professor, Department of Pharmaceutical Sciences, Faculty of Health Sciences, Marwadi University, Rajkot, India, Vaghela MC, Rathi S, Professor and Principal, School of Pharmacy, Rai University, Ahmedabad, Gujarat, India, Shirole RL, Associate Professor, Department of Pharmacology, DCS’s A.R.A. College of Pharmacy, Nagaon, Dhule (MS) India, et al. Leveraging AI and Machine Learning in Six-Sigma Documentation for Pharmaceutical Quality Assurance. Chinese Journal of Applied Physiology. 2024;40:e20240005. doi:10.62958/j.cjap.2024.005
  10. Sigmapharm laboratories llc, Bensalem, Pennsylvania, USA, Patel S, Y I, Crescent School of Pharmacy, B.S. Abdur Rahman Crescent Institute of Science and Technology, Chennai-600048, Tamilnadu, India, Singh S, GSVM Medical College (MBBS 3rd year Student), Kanpur-208002, Uttar Pradesh, India, et al. Recent Innovations and Future Perspectives in Transferosomes for Transdermal Drug Delivery in Therapeutic and Pharmacological Applications. Chinese Journal of Applied Physiology. 2024:e20240031. doi:10.62958/j.cjap.2024.031
  11. Kumar S, Saha S, Pathak D, Singh T, Kumar A, Singh K, et al. Cholesterol Absorption Inhibition by Some Nutraceuticals. Recent Advances in Food, Nutrition & Agriculture. 2024;16(1):2-11. doi:10.2174/012772574x285280240220065812
  12. Singh, S., Chaurasia, A., Rajput, D. S., & Gupta, N. (2024). An overview on mucoadhesive buccal drug delivery systems & approaches: A comprehensive review. African Journal of Biological Sciences (South Africa), 6(5), 522–541, doi:10.33472/AFJBS.6.5.2024.522-541
  13. Kumar S, Singh S, Rajput D, Sharma B, Chaturvedi K, Singh N, et al. Pharmacological Approaches and Herbal Interventions for Alzheimer's Disease. The Natural Products Journal. 2024;14(8). doi:10.2174/0122103155275266231123090138
  14. Department of Pharmacognosy, The Erode College of Pharmacy,Erode, Tamilnadu, India-638112, Ravikkumar VR, Patel BD, Department of Pharmaceutical Chemistry & Quality Assurance, School of Pharmacy, Rai University, Saroda, Dholka Road, Ahmedabad, Gujarat, India-382260, Rathi S, Department of Pharmaceutics, School of Pharmacy, Rai University, Saroda, Dholka Road, Ahmedabad, Gujarat, India-382260, et al. Formulation and Evaluation of Drumstick Leaves Tablet as An Immunomodulator. Chinese Journal of Applied Physiology. 2024;40:e20240004. doi:10.62958/j.cjap.2024.004
  15. Sharma, A., Bara, G., Keshamma, E., Sharma, B., Singh, S., Singh, S. P., Parashar, T., Rathore, H. S., Sarma, S. K., & Rawat, S. (2023). Cancer biology and therapeutics: A contemporary review. Journal of Cardiovascular Disease , 14(10), 1229-1247.
  16. DEWANGAN HK, SINGH S, MISHRA R, DUBEY RK. A REVIEW ON APPLICATION OF NANOADJUVANT AS DELIVERY SYSTEM. International Journal of Applied Pharmaceutics. 2020:24-33. doi:10.22159/ijap.2020v12i4.36856
  17. Patel College of Pharmacy, Madhyanchal Professional University, Ratibad, Bhopal, MP. India, Singh S, Chaurasia A, Patel College of Pharmacy, Madhyanchal Professional University, Ratibad, Bhopal, MP. India, Gupta N, Patel College of Pharmacy, Madhyanchal Professional University, Ratibad, Bhopal, MP. India, et al. Effect of Formulation Parameters on Enalapril Maleate Mucoadhesive Buccal Tablet Using Quality by Design (QbD) Approach. Chinese Journal of Applied Physiology. 2024;40:e20240003. doi:10.62958/j.cjap.2024.003
  18. Patel SK, Prathyusha S, Kasturi M, Godse KC, Singh R, Rathi S, et al. Optimizing Irbesartan Fast Dissolving Tablets Using Natural Polysaccharides for Enhanced Drug Delivery and Patient Compliance. International Research Journal of Multidisciplinary Scope. 2025;06(01):1181-1190. doi:10.47857/irjms.2025.v06i01.02542
  19. Formulation, optimization and evaluation of mucoadhesive buccal tablets of ondansetron for enhanced bioavailability and sustained drug release. Biochemical and Cellular Archives. 2025;25(1). doi:10.51470/bca.2025.25.1.1063
  20. Tagawa N, Oashi K, Okuno A, Kubota A, Shirokura Y. Simultaneous determination of levothyroxine and liothyronine in human serum by liquid chromatography–tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 2011;879(21):2069–74. doi:10.1016/j.jchromb.2011.05.022
Support