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

Year : 2025 | Volume : 12 | 03 | Page :
    By

    Shahzeb Ahmad,

  • Ankita Shukla,

  • Bala Subramanyam,

  1. Research Scholar, Department of Pharmacy, Millennium College of Pharmacy, Bhopal, Madhya Pradesh, India
  2. Professor, Department of Pharmacy, Millennium College of Pharmacy, Bhopal, Madhya Pradesh, India
  3. Professor, Department of Pharmacy, Millennium College of Pharmacy, Bhopal, Madhya Pradesh, India

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: Levothyroxine, UPLC-MS/MS, Bioanalytical Method Validation, Human Plasma, ICH M10, USFDA, SPE Extraction, Pharmacokinetics

How to cite this article:
Shahzeb Ahmad, Ankita Shukla, Bala Subramanyam. Bioanalytical Method Development and Validation for the Estimation of Levothyroxine in Human K₃EDTA Plasma by Using UPLC-MS/MS. Research & Reviews: A Journal of Drug Formulation, Development and Production. 2025; 12(03):-.
How to cite this URL:
Shahzeb Ahmad, Ankita Shukla, Bala Subramanyam. Bioanalytical Method Development and Validation for the Estimation of Levothyroxine in Human K₃EDTA Plasma by Using UPLC-MS/MS. Research & Reviews: A Journal of Drug Formulation, Development and Production. 2025; 12(03):-. Available from: https://journals.stmjournals.com/rrjodfdp/article=2025/view=223210


References

  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, A. K., Dubey, B. N., & Singh, S. (2024). Beneficial effects of spirulina on brain health: A systematic review. Current Functional Foods, 3(1), Article e120124225622. https://doi.org/10.2174/0126668629269256231222092721
  3. RaviKKumar VR, Rathi S, Singh S, Patel B, Singh S, Chaturvedi K, Sharma B. A Comprehensive Review on Ulcer and Their Treatment. Zhongguo Ying Yong Sheng Li Xue Za Zhi. 2023 Dec 21;39:e20230006. doi: 10.62958/j.cjap.2023.006. PMID: 38755116.
  4. Singh, V., Arora, S., Akram, W., Alam, S., Kumari, L., Kumar, N., Kumar, B., Kumar, S., Agrawal, M., Singhal, M., Kumar, S., Singh, S., Singh, K., Saha, S., & Dwivedi, V. (2024). Involvement of molecular mechanism and biological activities of Pemirolast: A therapeutic review. New Emirates Medical Journal, 5, Article e02506882308410. https://doi.org/10.2174/0102506882308410240607053814
  5. Rajput DS, Gupta N, Singh S, Sharma B. A Comprehensive Review: Personalized Medicine for Rare Disease Cancer Treatment. Zhongguo Ying Yong Sheng Li Xue Za Zhi. 2023 Dec 23;39:e20230008. doi: 10.62958/j.cjap.2023.008. PMID: 38830754.
  6. Singh S, Chaurasia A, Rajput DS, Gupta N. Mucoadhesive Drug Delivery System and There Future Prospective: Are a Promising Approach for Effective Treatment? Zhongguo Ying Yong Sheng Li Xue Za Zhi. 2023 Dec 20;39:e20230005. doi: 10.62958/j.cjap.2023.005. PMID: 38751344.
  7. Kumar, S., Saha, S., Sharma, B., Singh, S., Shukla, P., Mukherjee, S., Agrawal, M., Singh, K., & Singh, T. (2023). The role of resveratrol in Alzheimer’s disease: A comprehensive review of current research. Current Functional Foods, 2(2), Article e121223224364, 13 pages. https://doi.org/10.2174/0126668629269244231127071411
  8. Patel S, Ismail Y, Singh S, Rathi S, Shakya S, Patil SS, Bumrela S, Jain PC, Goswami P, Singh S. Recent Innovations and Future Perspectives in Transferosomes for Transdermal Drug Delivery in Therapeutic and Pharmacological Applications. Zhongguo Ying Yong Sheng Li Xue Za Zhi. 2024 Oct 24;40:e20240031. doi: 10.62958/j.cjap.2024.031. PMID: 39442957.
  9. Vaghela MC, Rathi S, Shirole RL, Verma J, Shaheen, Panigrahi S, Singh S. Leveraging AI and Machine Learning in Six-Sigma Documentation for Pharmaceutical Quality Assurance. Zhongguo Ying Yong Sheng Li Xue Za Zhi. 2024 Jul 18;40:e20240005. doi: 10.62958/j.cjap.2024.005. PMID: 39019923.
  10. Patel S, Ismail Y, Singh S, Rathi S, Shakya S, Patil SS, Bumrela S, Jain PC, Goswami P, Singh S. Recent Innovations and Future Perspectives in Transferosomes for Transdermal Drug Delivery in Therapeutic and Pharmacological Applications. Zhongguo Ying Yong Sheng Li Xue Za Zhi. 2024 Oct 24;40:e20240031. doi: 10.62958/j.cjap.2024.031. PMID: 39442957.
  11. Kumar, S., Saha, S., Pathak, D., Singh, T., Kumar, A., Singh, K., Mishra, A. K., Singh, S., & Singh, S. (2024). Cholesterol absorption inhibition by some nutraceuticals. Recent Advances in Food, Nutrition & Agriculture, 16(1), 2–11. https://doi.org/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., Saha, S., Singh, K., & Mukherjee, S. (2024). Pharmacological approaches and herbal interventions for Alzheimer’s disease. The Natural Products Journal, 14(8), Article e220124225945. https://doi.org/10.2174/0122103155275266231123090138 
  14. Ravikkumar VR, Patel BD, Rathi S, Parthiban S, Upadhye MC, Shah AM, Rehan SSA, Samanta S, Singh S. Formulation and Evaluation of Drumstick Leaves Tablet as An Immunomodulator. Zhongguo Ying Yong Sheng Li Xue Za Zhi. 2024 Jun 21;40:e20240004. doi: 10.62958/j.cjap.2024.004. PMID: 38902996.
  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, H. K., Singh, S., Mishra, R., & Dubey, R. K. (2020). A review on application of nanoadjuvant as delivery system. International Journal of Applied Pharmaceutics, 12(4), 24–33. https://doi.org/10.22159/ijap.2020v12i4.36856 
  17. Singh S, Chaurasia A, Gupta N, Rajput DS. Effect of Formulation Parameters on Enalapril Maleate Mucoadhesive Buccal Tablet Using Quality by Design (QbD) Approach. Zhongguo Ying Yong Sheng Li Xue Za Zhi. 2024 Jun 27;40:e20240003. doi: 10.62958/j.cjap.2024.003. PMID: 38925868.
  18. Patel SK, Prathyusha S, Kasturi M, Godse KC, Singh R, Rathi S, Bumrela S, Singh S, Goswami P. Optimizing Irbesartan Fast Dissolving Tablets Using Natural Polysaccharides for Enhanced Drug Delivery and Patient Compliance. Int Res J Multidiscip Scope (IRJMS). 2025;6(1):1181-1190. https://doi.org/10.47857/irjms.2025.v06i01.02542  
  19. Prince Patel, Piyush Jain, Hetvarth Patel, Aman Tiwari, Sanjesh Rathi and Shubham Singh (2025) Formulation, optimization and evaluation of mucoadhesive buccal tablets of ondansetron for enhanced bioavailability and sustained drug release. Biochem. Cell. Arch. 25, 1063-1069. DOI: https://doi.org/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
Ahead of Print Subscription Original Research
Volume 12
03
Received 19/07/2025
Accepted 29/07/2025
Published 13/08/2025
Publication Time 25 Days
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