QbD-based RP-HPLC method development for quantitative computation of phase III composition comprising apixaban and clopidogrel

Rashmi Shukla Ankit Chaudhari Pinak Patel Krunal Detholia   

Open Access   

Published:  Jun 07, 2024

DOI: 10.7324/JAPS.2024.181311

The presented work highlights the use of the quality by design methodology in accordance with International Council on Harmonization (ICH) recommendations for developing an reverse phase high performance liquid chromatographic (RP-HPLC) method for apixaban (APX) and clopidogrel (CLP). An efficient approach based on a factorial design incorporating the essential method parameters of the RP-HPLC method such as flow rate, pH, and proportion of organic phase, is presented. The optimum conditions for analysis were derived by using Design Expert software 10.0 Version, i.e., Hypersil ODS C18 column (5.0 μ, 25 cm × 4.6 mm), methanol and 0.05M potassium dihydrogen phosphate buffer (63.5:36.5%v/v, pH 3) as mobile phase, and 0.8 ml/minute as the flow rate. The derived condition gave excellent resolution between APX and CLP with Rt of 4.89 and 14.35 minutes, respectively, with the best possible system suitability parameters. The developed method presented excellent linearity in the range of 1.25–3.75 μg/ml for APX and 37.5–112.5 μg/ml for CLP at 245 nm. The optimized method was further validated in accordance with ICH guidelines on analytical method validation. Finally, the approach was successful in determining APX and CLP from a binary combination.

Keyword:     Quality by design (QbD) analytical method validation RP-HPLC apixaban clopidogrel


Shukla RR, Chaudhary A, Patel P, Detholia K. QbD-based RP-HPLC method development for quantitative computation of phase III composition comprising apixaban and clopidogrel. J Appl Pharm Sci. 2024. Online First. http://doi.org/10.7324/JAPS.2024.181311

Copyright: © The Author(s). This is an open-access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

HTML Full Text


1. Monks K, Molnar I, Rieger HJ, Bogati B, Szabo E, Quality by design: multidimensional exploration of the design space in high performance liquid chromatography method development for better robustness before validation. J Chromatogr A. 2011;1232:218-30. https://doi.org/10.1016/j.chroma.2011.12.041

2. Orlandini S, Pinzauti S, Furlanetto S. Application of quality by design to the development of analytical separation methods. Anal Bioanal Chem. 2013;405:443-50. https://doi.org/10.1007/s00216-012-6302-2

3. Mehta B, Joshi H. Shah U, Patel P. Implementation of QbD Principles for Simultaneous Quantitative Expression of Olmesartan Medoxomil, Telmisartan and Hydrochlorothiazide by RP-HPLC. J Pharm Res Int. 2021;33(41A):50-61. https://doi.org/10.9734/jpri/2021/v33i41A32301

4. Budovich A, Zargarova O, Nogid A. Role of apixaban (eliquis) in the treatment and prevention of thromboembolic disease. P T. 2013;38(4):206-31.

5. Wi?niewski A, Filipska K, The Phenomenon of Clopidogrel high on-treatment platelet reactivity in ischemic stroke subjects: a comprehensive review. Int J Mol Sci. 2020;21(17):6408. https://doi.org/10.3390/ijms21176408

6. Lopes RD, Heizer G, Aronson R, Vora AN, Massaro T, Mehran R, et al. Antithrombotic therapy after acute coronary syndrome or PCI in atrial fibrillation. N Engl J Med. 2019;380(16):1509-24. https://doi.org/10.1056/NEJMoa1817083

7. Mahendra B, Harika Sundari K, Vimalakkannan T. Method developed for the determination of apixaban by using U.V. spectrophotometric. Int J Res Pharm Chem Anal. 2019;1(3):83-7. https://doi.org/10.33974/ijrpca.v1i3.115

8. Landge SB, Jadhav SA, Dahale SB, Solanki PV, Bembalkar SR, Mathad VT, Development and validation of stability indicating RP-HPLC method on core shell column for determination of degradation and process related impurities of apixaban-an anticoagulant drug. Am J Anal Chem. 2015;6(6):539-50. https://doi.org/10.4236/ajac.2015.66052

9. Haque A, Soundharya R, Venu J, Monika ML, Bakshi V. Method development and validation of Apixaban using RP-HPLC method and its stress stability studies. Int J Chem Pharm Anal. 2017;5(1):1- 11.

10. Subramanian VB, Katari NK, Dongala T, Jonnalagadda SB. Stability-indicating RP-HPLC method development and validation for determination of nine impurities in apixaban tablet dosage forms. Robustness study by quality by design approach. Biomed Chromatogr. 2020;34(1):e4719. https://doi.org/10.1002/bmc.4719

11. Gouveia F, Bicker J, Santos J, Rocha M, Alves G, Falcão A, et al. Development, validation, and application of a new HPLC-DAD method for simultaneous quantification of apixaban, dabigatran, edoxaban and rivaroxaban in human plasma. J Pharm Biomed Anal. 2020;181:113109. https://doi.org/10.1016/j.jpba.2020.113109

12. Lagoutte-Renosi J, Le Poupon J, Girard A, Montange D, Davani S. A simple and fast HPLC-MS/MS method for simultaneous determination of direct oral anticoagulants apixaban, dabigatran, rivaroxaban in human plasma. J Chromatogr B Analyt Technol Biomed Life Sci. 2018;1100:43-9. https://doi.org/10.1016/j.jchromb.2018.09.026

13. Damle MC, Waghmare SS, Sinha PU. Development and validation of stability-indicating HPTLC method for determination of apixaban as bulk drug. Int J Pharm Pharm Sci. 2019;11:37-42. https://doi.org/10.22159/ijpps.2019v11i5.28071

14. Hamdy MM, Korany MA, Ebied SA, Haggag RS. ICH and US-FDA validated HPTLC methods with greenness assessments for the assay of mixtures prescribed in stroke prophylaxis: application to pharmaceutical preparations and human plasma. J Planar Chromatogr Modern TLC. 2022;35(5):519-32. https://doi.org/10.1007/s00764-022-00201-4

15. Zaazaa HE, Abbas SS, Abdelkawy M, Abdelrahman MM. Spectrophotometric and spectrodensitometric determination of Clopidogrel Bisulfate with kinetic study of its alkaline degradation. Talanta. 2009;78(3):874-84. https://doi.org/10.1016/j.talanta.2008.12.064

16. Antypenko L, Gladysheva S, Vasyuk S, Development and validation of clopidogrel Bisulphate determination in bulk by UV spectrophotometric method. Scr Sci Pharm. 2016;3(2):17-22. https://doi.org/10.14748/ssp.v3i2.1704

17. Mitakos A, Panderi I. A validated LC method for the determination of clopidogrel in pharmaceutical preparations. J Pharm Biomed Anal. 2002;28(3-4):431-8. https://doi.org/10.1016/S0731-7085(01)00620-3

18. Vocilkova L, Opatrilova R, Sramek V. Determination of clopidogrel by chromatography. Curr Pharm Anal. 2009;5(4):424-31. https://doi.org/10.2174/157341209789649131

19. Javed MK, Iqbal Z, Khan A, Khan A, Shah Y, Ahmad L, Development and validation of HPLC-UV method for the determination of clopidogrel in pharmaceutical dosage form and human plasma. J Liq Chromatogr Relat Technol. 2011;34(18):2118-29. https://doi.org/10.1080/10826076.2011.585482

20. Chaudhari PB, Pawar PD, Narkhede KP. Stability indicating spectrophotometric method for determination and validation of Clopidogrel bisulfate in tablet dosage form. Int J Res Ayur Pharm. 2010;1(2):418-23.

21. Srinivas A, Jayapaul B, Madhukar A, Kumar RV. Simple and sensitive analytical method development and validation of clopidogrel by RP-HPLC. Pharm Sci. 2012;1:5-7.

22. Silvestro L, Gheorghe M, Iordachescu A, Ciuca V, Tudoroniu A, Rizea Savu S, et al. Development and validation of an HPLC-MS/ MS method to quantify clopidogrel acyl glucuronide, clopidogrel acid metabolite, and clopidogrel in plasma samples avoiding analyte back-conversion. Anal Bioanal Chem. 2011;401:1023-34. https://doi.org/10.1007/s00216-011-5147-4

23. Liu G, Dong C, Shen W, Lu X, Zhang M, Gui Y, et al. Development and validation of an HPLC-MS/MS method to determine clopidogrel in human plasma. Acta Pharm Sin B. 2016;6(1):55-63. https://doi.org/10.1016/j.apsb.2015.11.001

24. Agrawal H, Kaul N, Paradkar AR, Mahadik KR. Stability indicating HPTLC determination of clopidogrel bisulphate as bulk drug and in pharmaceutical dosage form. Talanta. 2003;61(5):581-9. https://doi.org/10.1016/S0039-9140(03)00364-3

25. Patel RB, Shankar MB, Patel MR, Bhatt KK. Simultaneous estimation of acetylsalicylic acid and clopidogrel bisulfate in pure powder and tablet formulations by high-performance column liquid chromatography and high-performance thin-layer chromatography. J AOAC Int. 2008;91(4):750-5. https://doi.org/10.1093/jaoac/91.4.750

26. Sinha PK, Damle MC, Bothara KG. A validated stability indicating HPTLC method for determination of aspirin and clopidogrel bisulphate in combined dosage form. Eur J Anal Chem. 2009;4(2):152-60.

27. Tiwari PK, Jain A, Dubey BK, Pandey GK, Dhakad S, Analytical method development and validation for the simultaneous estimation of aspirin, clopidogrel and rosuvastatin in pharmaceutical dosage form. J Drug Deliv Ther. 2019;9(4-s):432-8. https://doi.org/10.22270/jddt.v9i4-s.3351

Article Metrics
140 Views 21 Downloads 161 Total



Related Search

By author names