Analytical Quality by Design Approach for a Stability-Indicating Method to Determine Apixaban and Its Related Impurities

  • Jéssica B. EllwangerEmail author
  • Nathalie Ribeiro Wingert
  • Nadia Maria Volpato
  • Cássia Virginia Garcia
  • Elfrides E. S. Schapoval
  • Martin Steppe


Quality by Design (QbD) is a systematic approach that takes predefined objectives and emphasizes process understanding and control. Analytical QbD (AQbD) is widely used when it comes to the development and optimization of analytical methods. Since impurities and contaminants may be present in the pharmaceutical product, current regulatory guidelines recommend monitoring these substances. Thus, the aim of this work was to develop and validate a fast and sensitive stability indicating method by high-performance liquid chromatography (HPLC) for the simultaneous determination of apixaban and three synthesis impurities using AQbD approach. Experiments were designed and assessed on MODDE® 11 (Umetrics, Sweden) software and carried out in a Shimadzu® LC-20A Prominence HPLC–DAD at 220 nm. Design of experiments was applied to achieve the optimum conditions through response surface methodology with four quantitative critical method parameters (CMPs): column temperature, flow rate, mobile phase pH and organic percentage. Besides, the chromatographic column was the fifth CMP taken as a qualitative variable. The HPLC method was established using an Inertsil® CN-3 column at the temperature of 30 °C. The mobile phase consisted of methanol and water (50.2:49.8) at a flow rate of 1.015 mL/min with no pH adjustment. In order to confirm method selectivity excipients mixture was evaluated, as well as hydrolytic, photolytic, thermolytic, and oxidative conditions. The method was also validated for sensitivity (LOQ 0.5 μg/mL, LOD 0.01–0.05 μg/mL for impurities), linearity (concentration range 1–35 μg/mL for APX and 0.5–10 μg/mL for impurities, r > 0.999), precision (RSD ≤ 10%) and accuracy between levels (RSD < 3.0 for APX and < 4.0% for impurities). The application of AQbD led to a well-understood and validated analytical method that provided assured separation of APX and all significant impurities, turning it into a powerful decision-making tool for the analytical methodologies development.


Apixaban Quality-by-design AQbD Experimental design Drug impurities 



Authors are thankful to CAPES and CNPq (Brazil) for financial support.

Compliance with Ethical Standards

Conflict of Interest

The authors declare the absence of conflicts of interest.

Ethical Approval

This article does not contain any studies with human participants performed by any of the authors.

Supplementary material

10337_2019_3815_MOESM1_ESM.tiff (111 kb)
Supplementary material 1 (TIFF 111 kb)


  1. 1.
    Turpie AGG (2007) Oral, direct factor Xa inhibitors in development for the prevention and treatment of thromboembolic diseases. Arterioscler Thromb Vasc Biol 27:1238–1247. CrossRefPubMedGoogle Scholar
  2. 2.
    Lassen MR, Davidson BL, Gallus A et al (2007) The efficacy and safety of apixaban, an oral, direct factor Xa inhibitor, as thromboprophylaxis in patients following total knee replacement. J Thromb Haemost 5:2368–2375. CrossRefPubMedGoogle Scholar
  3. 3.
    Frost C, Wang J, Nepal S et al (2013) Apixaban, an oral, direct factor Xa inhibitor: single dose safety, pharmacokinetics, pharmacodynamics and food effect in healthy subjects. Br J Clin Pharmacol 75:476–487. CrossRefPubMedGoogle Scholar
  4. 4.
    Huynh K (2017) Milestone 10: Era of the NOACs. Nat Rev Cardiol. CrossRefPubMedGoogle Scholar
  5. 5.
    International Conference on Harmonisation (2006) Impurities N Drug Subst Q3a(R2):15Google Scholar
  6. 6.
    Furlanetto S, Orlandini S, Pasquini B et al (2013) Quality by design approach in the development of a solvent-modified micellar electrokinetic chromatography method: finding the design space for the determination of amitriptyline and its impurities. Anal Chim Acta 802:113–124. CrossRefPubMedGoogle Scholar
  7. 7.
    Bueno LM, Manoel JW, Giordani CFA et al (2017) HPLC method for simultaneous analysis of ticagrelor and its organic impurities and identification of two major photodegradation products. Eur J Pharm Sci 97:22–29. CrossRefPubMedGoogle Scholar
  8. 8.
    Karmarkar S, Yang X, Garber R et al (2014) Quality by design (QbD) based development and validation of an HPLC method for amiodarone hydrochloride and its impurities in the drug substance. J Pharm Biomed Anal 100:167–174. CrossRefPubMedGoogle Scholar
  9. 9.
    Kumar N, Devineni SR, Dubey SK, Kumar P (2017) Potential impurities of anxiolytic drug, clobazam: identification, synthesis and characterization using HPLC, LC-ESI/MSnand NMR. J Pharm Biomed Anal 137:268–278. CrossRefPubMedGoogle Scholar
  10. 10.
    Holm R, Elder DP (2016) Analytical advances in pharmaceutical impurity profiling. Eur J Pharm Sci 87:118–135. CrossRefPubMedGoogle Scholar
  11. 11.
    Secrétan P-H, Sadou-Yayé H, Aymes-Chodur C et al (2015) A comprehensive study of apixaban’s degradation pathways under stress conditions using liquid chromatography coupled to multistage mass spectrometry. RSC Adv. CrossRefGoogle Scholar
  12. 12.
    Tantawy MA, El-Ragehy NA, Hassan NY, Abdelkawy M (2016) Stability-indicating spectrophotometric methods for determination of the anticoagulant drug apixaban in the presence of its hydrolytic degradation product. Spectrochim Acta Part A Mol Biomol Spectrosc. CrossRefGoogle Scholar
  13. 13.
    Argentine MD, Owens PK, Olsen BA (2007) Strategies for the investigation and control of process-related impurities in drug substances. Adv Drug Deliv Rev 59:12–28. CrossRefPubMedGoogle Scholar
  14. 14.
    Roy J (2002) Pharmaceutical impurities–a mini-review. AAPS Pharm Sci Tech 3:E6. CrossRefGoogle Scholar
  15. 15.
    Agrawal R, Jain P, Dikshit SN (2012) Apixaban: a new player in the anticoagulant class. Curr Drug Targets 13:863–875. CrossRefPubMedGoogle Scholar
  16. 16.
    Nevuluri NR, Rapolu RK, Iqbal J et al (2017) A morpholine-free process amenable convergent synthesis of apixaban: a potent factor Xa inhibitor. Monatshefte fur Chemie 148:1477–1482. CrossRefGoogle Scholar
  17. 17.
    Maxwell BD, Tran SB, Chen SY et al (2011) The syntheses and in vitro biotransformation studies of [14C]apixaban, a highly potent, selective, efficacious and orally bioavailable inhibitor of blood coagulation Factor Xa. J Label Compd Radiopharm 54:418–425. CrossRefGoogle Scholar
  18. 18.
    Pinto DJP, Orwat MJ, Quan ML et al (2006) 1-[3-Aminobenzisoxazol-5′-yl]-3-trifluoromethyl-6-[2′-(3-(R)-hydroxy-N-pyrrolidinyl)methyl-[1,1′]-biphen-4-yl]-1,4,5,6-tetrahydropyrazolo-[3,4-c]-pyridin-7-one (BMS-740808) a highly potent, selective, efficacious, and orally bioavailable inhibitor of bloo. Bioorganic Med Chem Lett 16:4141–4147. CrossRefGoogle Scholar
  19. 19.
    Sun X, Hong Z, Liu M et al (2017) Design, synthesis, and biological activity of novel tetrahydropyrazolopyridone derivatives as FXa inhibitors with potent anticoagulant activity. Bioorganic Med Chem 25:2800–2810. CrossRefGoogle Scholar
  20. 20.
    Christopher I, Ramsey P, Chant GR, et al (2015) (12) United States Patent (10) Patent NoGoogle Scholar
  21. 21.
    Xu Q, Shuang H, Weisi L et al (2015) Process for preparation od anti-thrombotic agent apixaban, China Patent CN 104892601 AGoogle Scholar
  22. 22.
    Orlandini S, Pinzauti S, Furlanetto S (2013) Application of quality by design to the development of analytical separation methods. Anal Bioanal Chem 405:443–450. CrossRefPubMedGoogle Scholar
  23. 23.
    International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (2009) ICH harmonised tripartite guideline, pharmaceutical development Q8(R2)Google Scholar
  24. 24.
    Yu LX, Amidon G, Khan MA et al (2014) Understanding pharmaceutical quality by design. AAPS J 16:771–783. CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Dispas A, Avohou HT, Lebrun P et al (2018) ‘Quality by Design’ approach for the analysis of impurities in pharmaceutical drug products and drug substances. TrAC Trends Anal Chem 101:24–33. CrossRefGoogle Scholar
  26. 26.
    Wingert NR, Ellwanger JB, Bueno LM et al (2018) Application of Quality by Design to optimize a stability-indicating LC method for the determination of ticagrelor and its impurities. Eur J Pharm Sci 118:208–215. CrossRefPubMedGoogle Scholar
  27. 27.
    Debrus B, Guillarme D, Rudaz S (2013) Improved quality-by-design compliant methodology for method development in reversed-phase liquid chromatography. J Pharm Biomed Anal 84:215–223. CrossRefPubMedGoogle Scholar
  28. 28.
    Cavazzuti M (2013) Optimization methods: from theory to design scientific and technological aspects in mechanics. Google Scholar
  29. 29.
    Boukouvala F, Muzzio FJ, Ierapetritou MG (2010) Design space of pharmaceutical processes using data-driven-based methods. J Pharm Innov 5:119–137. CrossRefGoogle Scholar
  30. 30.
    ICH (2005) Validation of a analytical Procedures : text and methodology Q2(R1). Guidance 1994:17. Accessed 1 July 2017
  31. 31.
    Vogt FG, Kord AS (2011) Development of quality-by-design analytical methods. J Pharm Sci 100:797–812CrossRefGoogle Scholar
  32. 32.
    Djuris J, Djuric Z (2017) Modeling in the quality by design environment: regulatory requirements and recommendations for design space and control strategy appointment. Int J Pharm. CrossRefPubMedGoogle Scholar
  33. 33.
    International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (2005) ICH harmonised tripartite guideline. Quality risk management Q9Google Scholar
  34. 34.
    Hanai T (1999) HPLC: a practical guide. HPLC A Pract Guid. CrossRefGoogle Scholar
  35. 35.
    Montgomery DC (2012) Design and analysis of experiments Livro montgomerry, 8th ednGoogle Scholar
  36. 36.
    Miller JN, Miller JC (2005) Statistics and chemometrics for analytical chemistry, 4th edn. Prentice HallGoogle Scholar
  37. 37.
    Kazakevich Y, LoBrutto R (2006) HPLC for pharmaceutical scientists. Wiley. Google Scholar
  38. 38.
    Ribani M, Grespan Bottoli CB, Collins CH et al (2004) Validação em métodos cromatográficos e eletroforéticos. Quim Nova 27:771–780CrossRefGoogle Scholar
  39. 39.
    Zhang X, Hu C (2017) Application of quality by design concept to develop a dual gradient elution stability-indicating method for cloxacillin forced degradation studies using combined mixture-process variable models. J Chromatogr A 1514:44–53. CrossRefPubMedGoogle Scholar
  40. 40.
    Landge SB, Jadhav SA, Dahale SB et al (2015) 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 6:539–550. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Jéssica B. Ellwanger
    • 1
    Email author
  • Nathalie Ribeiro Wingert
    • 1
  • Nadia Maria Volpato
    • 1
  • Cássia Virginia Garcia
    • 1
  • Elfrides E. S. Schapoval
    • 1
  • Martin Steppe
    • 1
  1. 1.Laboratory of Pharmaceutical Quality ControlFederal University of Rio Grande do SulPorto AlegreBrazil

Personalised recommendations