A NOVEL REVERSE-PHASE ULTRA-PERFORMANCE LIQUID CHROMATOGRAPHIC METHOD WITH GRADIENT ELUTION FOR SIMULTANEOUS DETERMINATION OF DOLUTEGRAVIR, LAMIVUDINE AND TENOFOVIR DISOPROXIL FUMARATE IN FIXED-DOSE COMBINATION

GAMPA NAGAMALLESWARI; UMA SHANKAR MS

doi:10.22159/ajpcr.2025v18i5.54280

Authors

GAMPA NAGAMALLESWARI Department of Pharmaceutical Analysis, SRM College of Pharmacy, SRMIST, Kattankulathur, Chengalpattu, Tamil Nadu, India. https://orcid.org/0009-0003-2238-5597
UMA SHANKAR MS Department of Pharmaceutics, SRM College of Pharmacy, SRMIST, Kattankulathur, Chengalpattu, Tamil Nadu, India. https://orcid.org/0000-0002-2762-6148

DOI:

https://doi.org/10.22159/ajpcr.2025v18i5.54280

Keywords:

Dolutegravir, Lamivudine, Tenofovir disoproxil fumarate, Reverse phase ultra-performance liquid chromatographic, Gradient elution, Stability-indicating method, Method validation

Abstract

ABSTRACT

Objective: To develop a sensitive and rapid reverse phase ultra-performance liquid chromatographic method for the simultaneous determination of dolutegravir (DTG), lamivudine (LAM), and tenofovir disoproxil fumarate (TDF) with improved separation and stability-indicating features.

Methods: Chromatographic separation was performed on an acquity bridged ethyl siloxane/silica hybrid C18 (50 × 2.1 mm, 1.7 μm) column at 40°C using 0.05% trifluoroacetic acid in water (pH 3.0) and methanol as mobile phase, with a flow rate of 0.4 mL/min and ultraviolet detection at 265 nm. The method validation was performed per ICH guidelines and included stress degradation studies.

Results: Effective separation was achieved; retention times were 1.25 min for LAM, 2.87 min for TDF, and 3.65 min for DTG. Supporting validation, the limits of linearity were set at 15–90 μg/mL for both LAM and tenofovir, and 2.5–15 μg/mL for DTG, with R₂values exceeding 0.999.

Conclusion: The proposed technique provides high accuracy, precision, sensitivity, and specificity in detecting multiple antiretroviral drugs in fixed-dose drug combinations. Compared to previously published methods, the gradient elution profile offers better peak height, greater peak splitting, as well as decreased total time required for analysis. The method’s stability-indicating features, confirmed by forced degradation studies, demonstrate the method’s value for pharmaceutical analysis quality control and stability testing.

Downloads

Download data is not yet available.

References

World Health Organization. Updated Recommendations on First- Line and Second-Line Antiretroviral Regimens and Post-Exposure Prophylaxis and Recommendations on Early Infant Diagnosis of HIV. Geneva: World Health Organization; 2018.

Cihlar T, Ray AS. Nucleoside and nucleotide HIV reverse transcriptase inhibitors: 25 years after zidovudine. Antiviral Res. 2010 Jan;85(1):39-58. doi: 10.1016/j.antiviral.2009.09.014, PMID: 19887088

Wang X, Song M, Wang Z, Liu S, Wang Z, Dong Y, et al. Metabolism of dolutegravir, a novel HIV-1 integrase inhibitor, by UGT1A1: Influence of genetic polymorphisms on pharmacokinetic variability. Drug Metab Dispos. 2018 Oct;46(10):1445-56.

Kandagal PB, Manjunatha DH, Seetharamappa J, Kalanur SS. RP-HPLC method for the determination of tenofovir in pharmaceutical formulations and spiked human plasma. Anal Lett. 2008;41(4):561-70. doi: 10.1080/00032710801910742

Kaul N, Agrawal H, Paradkar AR, Mahadik KR. HPTLC method for determination of lamivudine in tablet dosage form. Talanta. 2004 Apr;62(4):843-52.

Bhavsar DS, Patel BN, Patel CN. RP-HPLC method for simultaneous estimation of tenofovir disoproxil fumarate, lamivudine, and efavirenz in combined tablet dosage form. Pharm Methods. 2012 Jul;3(2):73-8. doi: 10.4103/2229-4708.103876, PMID: 23781482

Mallikarjuna Rao N, Gowri Sankar D. Development and validation of stability-indicating HPLC method for simeltaneous determination of lamivudine, tenofovir, and dolutegravir in bulk and their tablet dosage form. Future J Pharm Sci. 2015;1(2):73-7. doi: 10.1016/j. fjps.2015.11.002

Kumar P, Dwivedi SC, Kushnoor A. A validated stability indicating RP-HPLC method for the determination of lamivudine in bulk and tablet dosage form. Res J Pharm Technol. 2012;5(2):1396-8.

Gomes NA, Vaidya VV, Pudage A, Joshi SS, Parekh SA. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of tenofovir and emtricitabine in human plasma and its application to a bioequivalence study. J Pharm Biomed Anal. 2008 Nov;48(3):918-26. doi: 10.1016/j.jpba.2008.07.022, PMID 18783908

Venkatesh M, Sinha VR, Goel H, Yang Y, Karwa M. Development and validation of an UPLC-MS/MS method for determination of dolutegravir in human plasma. Drug Dev Ind Pharm. 2019 Sep;45(9):1451-8.

ICH Harmonised Tripartite Guideline. Validation of Analytical Procedures: Text and Methodology. In: International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. Vol. Q2. Geneva: ICH, ICH Harmonised Tripartite Guideline; 2005. p. R1.

Pawar AK, Unade TT. A new stability indicating UPLC method for the determination of two antidiabetic drugs in combination: Applications to bulk and tablet formulation. Int J Appl Pharm. 2022;14(4):192-9. doi: 10.22159/ijap.2022v14i4.44774

Noviza D, Julianto T, Abdul Majeed AB, Hamid KA. Bioanalytical of UPLC method development and validation of xanthorrizol and its application to pharmacokinetic study. Int J Appl Pharm. 2024;16(1):186-93. doi: 10.22159/ijap.2024v16i1.49340

Ponnekanti K, Sundararajan R. Development and validation of new RP-UPLC method for the determination of cefdinir in bulk and dosage form. Int J Pharm Pharm Sci. 2018;10(1):178-84. doi: 10.22159/ ijpps.2018v10i1.23256

Mule KL. Rapid analytical method for assay determination for prochlorperazine edisylate drug substances by ultra-performance liquid chromatography. Int J Curr Pharm Res. 2017;9(4):118-22. doi: 10.22159/ijcpr.2017v9i4.20973

Bhaskararao P, Sarika M, Madhavi CL, Kollipara MG, Prashanthi G. The industrial importance of technology transfer for analytical method development and validation-application to vilazodone hydrochloride dosage form. Int J Chem Res. 2024;8(4):12-7. doi: 10.22159/ ijcr.2024v8i4.235

Balaji N, Sayeeda SU. Stability indicating UPLC method development and validation for simultaneous estimation of emtricitabine, tenofovirdisoproxil fumarate and rilpivirine hydrochloride in pharmaceutical dosage forms. Int J Pharm Pharm Sci. 2017;9(9):61-6.

Anandakumar K, Abirami G, Murugan S, Ashok B. RP-HPLC method for simultaneous estimation of lamivudine, tenofovir disoproxil fumarate and efavirenz in tablet formulation. J Anal Chem. 2013 Sep;68(9):815-21. doi: 10.1134/S1061934813090025

Raju NA, Begum S. Simultaneous RP-HPLC method for the estimation of the emtricitabine, tenofovir disoproxil fumerate and efavirenz in tablet dosage forms. Res J Pharm Technol. 2008;1(4):522-5.

Venkatesh P, Daggumati M. Development and validation of HPLC method for the determination of tenofovir disoproxil fumarate and lamivudine in combined dosage form. Int J Pharm Res Scholars. 2012;1(2):33-8.

Chiluka R, Boddu S, Kovvasu SP. Development and validation of RP-UPLC method for simultaneous estimation of dolutegravir and lamivudine in bulk and tablet dosage form. Int J Pharm Sci Res. 2019 Apr;10(4):1744-9.

Patel SB, Bareja M, Munde M, Vyas AJ. Development and validation of stability-indicating RP-UPLC method for the determination of dolutegravir sodium. Int J Pharm Pharm Sci. 2017;9(11):60-6.

Kumar S, Rao PS, Krishna SR, Kumar KS. A novel validated RP-UPLC method development for the estimation of dolutegravir in bulk and its tablet dosage form. J Pharm Res. 2017;11(1):25-33.

Badgujar BP, Mahajan MP, Sawant SD. Development and validation of RP-UPLC method for simultaneous estimation of dolutegravir and lamivudine. Int J Chem Tech Res. 2019;12(3):80-90.

Mallikarjuna Rao N, Gowri Sankar D. Development and validation of UPLC method for simultaneous estimation of lamivudine, tenofovir and dolutegravir in combined dosage form. J Glob Trends Pharm Sci. 2016;7(1):2990-7.