AN LC-MS/MS METHOD DEVELOPMENT AND VALIDATION FOR THE QUANTIFICATION OF ANTIVIRAL DRUGS IN PLASMA SAMPLES
DOI:
https://doi.org/10.22159/ijap.2026v18i2.56739Keywords:
Cabotegravir, Rilpivirine, HIV/AIDS, LC-MS/MS, Method validation, StabilityAbstract
Objective: A reliable and specific LC-MS/MS method was developed and validated for the simultaneous quantification of cabotegravir (CBTG) and rilpivirine (RLPV) in plasma samples.
Methods: Chromatographic separation was achieved on a Hypersil BDS C18 (50 × 4.6 mm, 5 µm) column maintained at 35 °C. The mobile phase consisted of 0.1% HCOOH, acetonitrile, and methanol in the ratio 10:80:10 (v/v/v). The injection volume was 10 µL, and the autosampler temperature was maintained at 10 °C. The retention times for RLPV, internal standard, and CBTG were 2.00, 3.20, and 5.15 min, respectively.
Results: Accuracy ranged from 96.32% to 98.19% for CBTG and 95.57% to 97.00% for RLPV. The intra- and inter-day precision values were 0.39%–3.82% RSD for CBTG and 1.56%–4.55% RSD for RLPV. Mean recovery values at three QC levels were 96.14%–97.70% for CBTG and 96.06%–97.75% for RLPV. All stability assessments were within acceptance criteria.
Conclusion: The method is robust for the quantitative determination of CBTG and RLPV in biological samples and is suitable for application in forensic, quality-control, bioavailability, and bioequivalence studies.
References
1. Ferretti F, Boffito M. Rilpivirine long-acting for the prevention and treatment of HIV infection. Curr Opin HIV AIDS. 2018;13(4):300-7. doi: 10.1097/COH.0000000000000474.
2. Stellbrink HJ, Hoffmann C. Cabotegravir: its potential for antiretroviral therapy and pre-exposure prophylaxis. Curr Opin HIV AIDS. 2018;13(4):334-40. doi: 10.1097/COH.0000000000000480.
3. Cattaneo D, Gervasoni C. Pharmacokinetics and pharmacodynamics of cabotegravir, a long-acting HIV integrase strand transfer inhibitor. Eur J Drug Metab Pharmacokinet. 2019;44(3):319-27. doi: 10.1007/s13318-018-0526-2.
4. Swindells S, Andrade-Villanueva JF, Richmond GJ, et al. Long-acting cabotegravir + rilpivirine as maintenance therapy: ATLAS week 48 results. Presented at: Conference on Retroviruses and Opportunistic Infections (CROI); 2019 Mar 4-7; Seattle, WA.
5. Orkin C, Arasteh K, Hernandez-Mora MG, et al. Long-acting cabotegravir + rilpivirine for HIV maintenance: FLAIR week 48 results. Presented at: Conference on Retroviruses and Opportunistic Infections (CROI); 2019 Mar 4-7; Seattle, WA.
6. Janssen Pharmaceutical Companies of Johnson & Johnson. Janssen reports positive top-line phase 3 study results of investigational, long-acting injectable HIV treatment regimen administered every two months [Internet]. 2019 [cited 2025 Sep 3]. Available from: https://www.drugs.com/clinical_trials/janssen-reports-positive-top-line-phase-3-studyresults-investigational-long-acting-injectable-hiv-18243.html
7. Jackson AG, Else LJ, Mesquita PM, et al. A compartmental pharmacokinetic evaluation of long-acting rilpivirine in HIV-negative volunteers for pre-exposure prophylaxis. Clin Pharmacol Ther. 2014;96(3):314-23. doi: 10.1038/clpt.2014.112.
8. Landovitz RJ, Li S, Grinsztejn B, et al. Safety, tolerability, and pharmacokinetics of long-acting injectable cabotegravir in low-risk HIV-uninfected individuals: HPTN 077, a phase 2a randomized controlled trial. PLoS Med. 2018;15(11):e1002690. doi: 10.1371/journal.pmed.1002690.
9. Margolis DA, Gonzalez-Garcia J, Stellbrink HJ, et al. Long-acting intramuscular cabotegravir and rilpivirine in adults with HIV-1 infection (LATTE-2): 96-week results of a randomized, open-label, phase 2b, non-inferiority trial. Lancet. 2017;390(10101):1499-510. doi: 10.1016/S0140-6736(17)31917-7.
10. Anuradha V, Subrahmanyam T, Raju M, Murthy B, Emmanuel K. Method development and validation for cabotegravir and rilpivirine by using HPLC and its degradants characterized by LC-MS and FTIR. Futu J Pharm Sci. 2021;7:226. doi: 10.1186/s43094-021-00355-8.
11. Ramöller IK, Abbate MTA, Vora LK, 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.
12. Courlet P, Alves Saldanha S, Cavassini M, et al. Development and validation of a multiplex UHPLC-MS/MS assay with stable isotopic internal standards for monitoring plasma concentrations of bictegravir, cabotegravir, doravirine, and rilpivirine in people living with HIV. J Mass Spectrom. 2020;55(6):e4506. doi: 10.1002/jms.4506.
13. Bevers LAH, van Ewijk-Beneken Kolmer EWJ, Te Brake HML, Burger DM. 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 Pharmacol Toxicol Methods. 2024;238:115832. doi: 10.1016/j.jpba.2023.115832.
14. Weld ED, Parsons TL, Gollings R, et al. Development and validation of a liquid chromatographic-tandem mass spectrometric assay for quantification of cabotegravir and rilpivirine from dried blood spots. J Pharm Biomed Anal. 2023;228:115307. doi: 10.1016/j.jpba.2023.115307.
15. European Medicines Agency. Guideline on bioanalytical method validation [Internet]. London: EMA; 2011 [cited 2025 Sep 3]. Available from: http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2011/08/WC500109686.pdf
16. US Food and Drug Administration. Guidance for industry: bioanalytical method validation [Internet]. Silver Spring (MD): FDA; 2018 [cited 2025 Sep 3]. Available from: https://www.fda.gov/downloads/drugs/guidances/ucm368107.pdf
17. Gallant JE, Thompson M, DeJesus E, et al. Antiviral activity, safety, and pharmacokinetics of bictegravir as 10-day monotherapy in HIV-1-infected adults. J Acquir Immune Defic Syndr. 2017;75(1):61-6. doi: 10.1097/QAI.0000000000001321.
18. Yee KL, Sanchez RI, Auger P, et al. Evaluation of doravirine pharmacokinetics when switching from efavirenz to doravirine in healthy subjects. Antimicrob Agents Chemother. 2017;61(2):e01757-16. doi: 10.1128/AAC.01757-16.
19. Dadhaniya T, Chaudhary K, Mehta P. Development of LC-MS/MS method for determination of iloperidone in rabbit plasma: application to a pharmacokinetic study. Int J Pharm Pharm Sci. 2013;7(4):294-7. Available from: https://journals.innovareacademics.in/index.php/ijpps/article/download/5121/8609/0
20. Deepan T, Basaveswara Rao MV, Dhanaraju MD. Bioanalytical method development and validation of canagliflozin in human plasma by LC-MS/MS. Asian J Pharm Clin Res. 2019;12(8):46-51. doi: 10.22159/ajpcr.2019.v12i18.33228.
21. Gurav P, Damle M. Bioanalytical method for estimation of teriflunomide in human plasma. Int J Pharm Pharm Sci. 2022;14(9):19-23. doi: 10.22159/ijpps.2022v14i9.45151.
22. Nimmakayala MR, Kolli D, Durga Bhavani PN. Bioanalytical method development and validation of maralixibat in rat plasma by LC-MS/MS detection and its application to a pharmacokinetic study. Int J Appl Pharm. 2023;15(4):166-72. doi: 10.22159/ijap.2023v15i4.47768.
23. Parmar I, Patel YA. Recent method development by analytical techniques of new FDA-approved drugs in 2021. Int J Curr Pharm Res. 2022;14(3):17-21. doi: 10.22159/ijcpr.2022v14i3.1975.
24. Ravi Y, Bhikshapathi D, Shankar C, Rajkamal B. Development of a fast and simple LC-ESI-MS/MS technique for the quantification of regorafenib; application to pharmacokinetics in healthy rabbits. Curr Pharm Anal. 2021;17(4):554-63. doi: 10.2174/1573412916666191111144707.
25. Rozet E, Ceccato A, Hubert C, et al. Analysis of recent pharmaceutical regulatory documents on analytical method validation. J Chromatogr A. 2007;1158(1-2):111-25. doi: 10.1016/j.chroma.2007.03.111.
26. Hubert P, Nguyen-Huu JJ, Boulanger B, et al. Harmonization of strategies for validation of quantitative analytical procedures. A SFSTP proposal—part I. J Pharm Biomed Anal. 2004;36(3):579-86. doi: 10.1016/j.jpba.2004.07.027.
27. Lolla S, Gubbiyappa KS, Shankar C, Bhikshapathi DVRN. Validation of an LC-MS/MS method for quantitation of fostemsavir in plasma. J Pharmacol Toxicol Methods. 2023;120:107254. doi: 10.1016/j.vascn.2023.107254.
28. Chapuzet E, Mercier N, Bervoas-Martin S, et al. Méthodes chromatographiques de dosage dans les milieux biologiques: stratégie de validation - rapport d'une commission SFSTP. STP Pharma Sci. 1997;7:169-94.
29. Krishnan VS, Bhikshapathi D, Shankar C. Method development and validation for quantification of abametapir in biological matrices by LC-ESI-MS/MS. Indian J Pharm Educ Res. 2024;58(3 Suppl):S1028-33. doi: 10.5530/ijper.58.3s.102.
Published
How to Cite
Issue
Section
Copyright (c) 2025 Raghavamma STV, DIVYA G
This work is licensed under a Creative Commons Attribution 4.0 International License.
