DEVELOPMENT AND VALIDATION OF AN LC-MS/MS ANALYTICAL METHOD FOR THE CONCURRENT QUANTIFICATION OF CABOTEGRAVIR AND RILPIVIRINE IN RAT PLASMA
Keywords:
LC-MS/MS, Cabotegravir, Rilpivirine, Pharmacokinetics, Bioanalytical ValidationAbstract
Objective:
The aim of this study was to establish and authenticate a precise and dependable LC-MS/MS technique for the concurrent quantification of Cabotegravir (CAB) and Rilpivirine (RIL) in rat plasma, specifically for pharmacokinetic studies.
Methods:
A protein precipitation extraction procedure was employed, followed by chromatographic separation on a Kromasil C18 column using combination of ethanol and 0.1% triethylamine buffer (30:70, v/v, pH 2.5) as solvent system. Quantification was performed using an electrospray ionization (ESI) interface coupled with a SCIEX QTRAP 5500 mass spectrometer in multiple reaction monitoring (MRM) mode. The mass transitions for CAB (m/z 406.1240 → 218.3257), RIL (m/z 367.2103 → 174.1127), and the internal standard (IS) Raltegravir (m/z 445.1147 → 389.5261) were optimized for maximum sensitivity.
Results:
The method demonstrated excellent linearity (r² > 0.999), precision (≤ 15% CV), and accuracy (85–115%) for both intra- and inter-day assays. Recovery rates for CAB and RIL ranged from 94% to 98%. The method exhibited negligible matrix effects, with lower limits of quantification (LLOQ) of 15 ng/mL for CAB and 10 ng/mL for RIL.
Conclusion:
This validated LC-MS/MS method provides a robust and sensitive approach for the simultaneous quantification of CAB and RIL in rat plasma, suitable for pharmacokinetic studies, offering a reliable tool for future clinical and preclinical research on these drugs.
Downloads
References
1. Steulet C. Clinical pharmacology considerations and drug–drug interactions with long‐acting cabotegravir and rilpivirine relevant to sub‐Saharan Africa. Br J Clin Pharmacol. Published 2024. Available at: https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bcp.16154. Accessed July 22, 2025.
2. Overton ET, Richmond G, Rizzardini G, et al. Long-Acting Cabotegravir and Rilpivirine Dosed Every 2 Months in Adults With Human Immunodeficiency Virus 1 Type 1 Infection: 152-Week Results From ATLAS-2M, a Randomized, Open-Label, Phase 3b, Noninferiority Study. Clin Infect Dis. 2023;76(9):1646–1654. doi:10.1093/cid/ciad020.
3. Patel P, Teichner P, Elliot E, et al. Practical dosing guidance for the management of clinician-administered injections of long-acting cabotegravir and rilpivirine. Ther Adv Infect Dis. 2023;10:20499361231214626. doi:10.1177/20499361231214626.
4. Serris A, Ferre VM, Le Hingrat Q, et al. Real-world data on long-acting intramuscular maintenance therapy with cabotegravir and rilpivirine mirror Phase 3 results. J Antimicrob Chemother. 2024;79(11):2932–2938. doi:10.1093/jac/dkae308.
5. Sutton KC, De Vente J, Leblanc R, et al. Long-Term Efficacy, Safety, and Durability of Cabotegravir and Rilpivirine as 2-Drug Oral Maintenance Therapy After 6 Years of Study. Open Forum Infect Dis. 2022;9(4):ofac067. doi:10.1093/ofid/ofac067.
6. Sri KV, Jain GV, Maduri M. UV spectrophotometric method for the estimation of rilpivirine in bulk and pharmaceutical formulations. Res J Pharm Technol. 2014;7(4):463-466.
7. Prasanna A, Rani SS. A validated stability indicating RP-HPLC method development for simultaneous estimation of Cabotegravir and Rilpivirine in pharmaceutical dosage form. World J Pharm Sci. 2022;10(10):22–29. doi:10.54037/WJPS.2022.101003.
8. Suneetha A, Lakshmi MV, Jyothi K, Priyanka BY. Development and validation of stability indicating RP-HPLC method for the simultaneous determination of Cabotegravir and Rilpivirine in bulk and injection dosage form. J Pharm Res. 2022;21(4):145.
9. Rizwan UM. Development and validation of stability indicating RP-HPLC method for the simultaneous estimation of Cabotegravir and Rilpivirine in bulk and tablet dosage form [dissertation]. JKK Nattraja College of Pharmacy, Kumarapalayam; 2021.
10. Pandya Y, Patel S. RP-HPLC stability method development & validation for anti-HIV drugs Cabotegravir & Rilpivirine in IM injection and human plasma. Int J Health Sci. 2023;1:9104-9117.
11. Patil JA, Shah RR. Analytical method development and validation of Rilpivirine by RP-HPLC method. Int J Pharm Res. 2021;13(3):323-330.
12. Veeraswami B, Naveen VM. Development and validation of RP-HPLC method for the estimation of Dolutegravir and Rilpivirine in bulk and pharmaceutical dosage form and its application to rat plasma. Dev. 2019;12(2):121-130.
13. Bhavani R, Reddy TR. Method development and validation for simultaneous estimation of Rilpivirine and Dolutegravir by using RP-HPLC method in bulk dosage form. World J Pharm Sci. 2020:167-175.
14. Kavitha KY, Geetha G, Hariprasad R, Venkatnarayana R, Subramanian G. Development and validation of RP-HPLC analytical method for simultaneous estimation of Emtricitabine, Rilpivirine, Tenofovir Disoproxil Fumarate and its pharmaceutical dosage forms. Pharm Globale. 2013;4(1):1.
15. Madhuri M, Anusha V, Siresha VR. Development, validation of RP-HPLC method for the simultaneous estimation of Tenofovir Disoproxil Fumarate, Emtricitabine, and Rilpivirine hydrochloride in bulk, formulation, and used in nanosuspension. Indian Drugs. 2019;56(9):611-618.
16. Mhaske DK, Kumbhar AS. Simultaneous quantification of (E) and (Z) isomers of Rilpivirine and four degradation products in bulk and tablets by reversed-phase ultra-high-performance liquid chromatography and confirmation of all by molecular weight. J Sep Sci. 2023;2201067.
17. Vemuluri PC, Dodda S. Stability indicating reverse phase-high performance liquid chromatography method for simultaneous estimation of Cabotegravir and Rilpivirine. Indian J Pharm Educ Res. 2023;57(3s):s766-s771.
18. Suresh CV, Raaga MS, SanthoshIllendula DK. Development of stability indicating RP-HPLC method and validation for the estimation of Cabotegravir and Rilpivirine in pure form and marketed pharmaceutical dosage form.
19. Pasha SI, Varanasi MB, Mohammed I. Stability indicating RP-UPLC-PDA method development, validation of multi-drug combination of Emtricitabine, Tenofovir Alafenamide, and Rilpivirine in bulk drug and its tablet formulation. Orient J Chem. 2017;33(2):925-929.
20. Kovac L, Časar Z, TrdanLušin T, Roškar R. Development of an analytical method for determination of related substances and degradation products of Cabotegravir using analytical quality by design principles. ACS Omega. 2022;7(10):8896-8905.
21. Ramöller IK, Abbate MT, Vora LK, Hutton AR, Peng K, Volpe-Zanutto F, et al. HPLC-MS method for simultaneous quantification of the antiretroviral agents Rilpivirine and Cabotegravir in rat plasma and tissues. J Pharm Biomed Anal. 2022;213:114698. doi:10.1016/j.jpba.2022.114698.
22. Vejendla A, Talari S, Moturu R, Boddapati SM, Kola AE. Method development and validation for Cabotegravir and Rilpivirine by using HPLC and its degradants characterized by LCMS and FTIR. Future J Pharm Sci. 2021;7:1-8.
23. Rajoli RK, Curley P, Chiong J, Back D, Flexner C, Owen A, Siccardi M. Predicting drug–drug interactions between rifampicin and long-acting Cabotegravir and Rilpivirine using physiologically based pharmacokinetic modeling. J Infect Dis. 2019;219(11):1735-1742.
24. Hodge D, Back DJ, Gibbons S, Khoo SH, Marzolini C. Pharmacokinetics and drug–drug interactions of long-acting intramuscular Cabotegravir and Rilpivirine. Clin Pharmacokinet. 2021;60:835-853.
25. Bevers L, van Ewijk-Beneken Kolmer N, te Brake L. Development, validation, and clinical implementation of a UPLC-MS/MS bioanalytical method for simultaneous quantification of Cabotegravir and Rilpivirine E-Isomer in human plasma. J Pharm Biomed Anal. 2024;238:115832.
26. Courlet P, Alves-Saldanha S, Cavassini M, Marzolini C, Choong E, Csajka C, et al. Development and validation of a multiplex UHPLC-MS/MS assay with stable isotopic internal standards for the monitoring of the plasma concentrations of the antiretroviral drugs Bictegravir, Cabotegravir, Doravirine, and Rilpivirine in people living with HIV. J Mass Spectrom. 2020;55(6):e4506.
27. Saminathan J, Vetrichelvan T. Development and validation of HPTLC method for simultaneous estimation of Emtricitabine, Rilpivirine, and Tenofovir Disoproxil Fumarate in combined dosage form. Bangladesh Pharm J. 2016;19(1):114-121.
28. Sudha T, Shanmugasundram P. Reverse phase high-performance and HPTLC methods for the determination of Rilpivirine bulk and in tablet dosage form. World J Pharm Res. 2012;1(4):1183-1196.
29. Adhao VS, Ambhore JP, Bhuskat AD, Talokar DV. A Review on Analytical Methodologies for the Estimation of Anti-Retroviral Drugs Cabotegravir and Rilpivirine. Mathews J Pharm Sci. 2024;8(1). doi:10.30654/mjps.10030.
30. U.S. Food and Drug Administration. Bioanalytical method validation: Guidance for industry. U.S. Department of Health and Human Services; 2018. Available at: https://www.fda.gov/media/70858/download.
31. Gandhi BM, Rao AL, Rao JV. UPLC-MS/MS method for determination of sofosbuvir in human plasma. Ann Pharm Fr. 2017;75(4):257-266. doi:10.1016/j.pharma.2017.04.003.
Published
How to Cite
Issue
Section
Copyright (c) 2025 Ramya Nagabathula, Dr Kamepalli Sujana
This work is licensed under a Creative Commons Attribution 4.0 International License.
The publication is licensed under CC By and is open access. Copyright is with author and allowed to retain publishing rights without restrictions.