Box-Behnken guided development of an ecofriendly RP-HPLC analytical method for simultaneous quantification of pantoprazole sodium and piperine co-loaded mucoadhesive GRDDS formulation for H. pylori eradication

Ashutosh Gupta Shiran Shetty Srinivas Mutalik Prerana D. Navti Moumita Saha Sudheer Moorkoth   

Open Access   

Published:  Jun 14, 2024

DOI: 10.7324/JAPS.2024.179147

Helicobacter pylori (H. pylori) infection is the leading cause of chronic peptic ulcer disease worldwide. Many treatment options are available to treat H. pylori infection. However, the eradication is still a challenge due to the poor bioavailability of the currently available formulations. To improve the efficacy of therapy, novel formulations are necessary. Proton pump inhibitors (PPIs) are already a part of the treatment regimen with antibiotics. P-glycoprotein (P-gp) inhibitor was reported to have increased the efficacy of antibiotic treatment in H. pylori infections. To make available the P-gp inhibitor and PPI on the intestinal mucosal surface we have formulated a gastroretentive drug delivery system (GRDDS) as an adjuvant therapy with antibiotics. The objective of this study is to simultaneously estimate pantoprazole (PAN) and piperine (PIP) by reverse-phase (RP) high-performance liquid chromatography (HPLC) method from the chitosan-based sodium alginate mucoadhesive beads utilizing the design of experiments (DOEs) methodology. The HPLC settings were optimized using DOEs software. The final optimized HPLC method used a hyperclone Octadecylsilane C18 column as the stationary phase and methanol: ammonium acetate at pH 4.5 (70:30 v/v) as the mobile phase. The flow rate was 0.9 ml/minute. The validation of the developed RP-HPLC method was done as per the International Conference on Harmonization (ICH) Q2(R1) guideline. The method was linear from 0.5 to 20 μg/ml for both PAN and PIP with an R2 value of 0.999 and 0.999, respectively. The validated RP-HPLC method showed specificity for both drugs despite interference from degradation products and other GRDDS excipients. The entrapment efficiency of the final formulation was determined to be 80%–85% for PAN and 60%–67% for PIP. The novelty and merit of the DOE-based method development are that it reduces the number of trials, thereby reducing reagent wastage, and is environmentally friendly suggested by the Green Analytical Procedure Index (GAPI) tool scoring six green, six yellow, and three red.

Keyword:     Box–Behnken design GRDDS pantoprazole piperine HPLC H. pylori


Gupta A, Shetty S, Mutalik S, Navti PD, Saha M, Moorkoth S. Box-Behnken guided development of an ecofriendly RP-HPLC analytical method for simultaneous quantification of pantoprazole sodium and piperine co-loaded mucoadhesive GRDDS formulation for H. pylori eradication. J Appl Pharm Sci. 2024. Online First.

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. Chmiela M, Kupcinskas J. Review: pathogenesis of helicobacter pylori infection. Helicobacter. 2019;24(S1):1-5.

2. Ahn HJ, Lee DS. Helicobacter pylori in gastric carcinogenesis. World J Gastrointest Oncol. 2015 Dec 15;7(12):455-65.

3. Gupta A, Shetty S, Mutalik S, Chandrashekar HR, Nandakumar K, Mathew EM, et al. Treatment of H. pylori infection and gastric ulcer: need for novel pharmaceutical formulation. Heliyon. 2023 Oct 1;9(10):e20406.

4. Ullah G, Nawaz A, Latif MS, Shah KU, Ahmad S, Javed F, et al. Clarithromycin and pantoprazole gastro-retentive floating bilayer tablet for the treatment of Helicobacter Pylori: formulation and characterization. Gels. 2023 Jan;9(1):43.

5. Damanhuri NS, Kumolosasi E, Omar MS, Razak AFA, Mansor AH. The influence of P-glycoprotein expression in the standard treatment of Helicobacter pylori infection in Sprague Dawley rats. Daru. 2021 Jun 1 [cited 2023 May 23];29(1):13-22.

6. Lomovskaya O, Bostian KA. Practical applications and feasibility of efflux pump inhibitors in the clinic-A vision for applied use. Biochem Pharmacol [Internet]. 2006 Mar 30 [cited 2023 May 23];71(7):910-8.

7. Tharmalingam N, Kim SH, Park M, Woo HJ, Kim HW, Yang JY, et al. Inhibitory effect of piperine on Helicobacter pylori growth and adhesion to gastric adenocarcinoma cells. Infectious Agents Cancer. 2014;9(1):1-15.

8. Mohamed D, ELbalkiny HT. Application of solidified floating organic droplet dispersive liquid-liquid microextraction for determination of veterinary antibiotic residues in milk samples with greenness assessment. Microchem J. 2023 Oct 1;193:109153.

9. Chiriac U, Rau H, Frey OR, Röhr AC, Klein S, Meyer AL, et al. Validation and application of an HPLC-UV method for routine therapeutic drug monitoring of dalbavancin. Antibiotics. 2022 May;11(5):541.

10. Benito-Peña E, Partal-Rodera AI, León-González ME, Moreno-Bondi MC. Evaluation of mixed mode solid phase extraction cartridges for the preconcentration of beta-lactam antibiotics in wastewater using liquid chromatography with UV-DAD detection. Anal Chim Acta. 2006 Jan 25;556(2):415-22.

11. Zimmer J, Röhr AC, Kluge S, Faller J, Frey OR, Wichmann D, et al. Validation and application of an HPLC-UV method for routine therapeutic drug monitoring of cefiderocol. Antibiotics. 2021 Mar;10(3):242.

12. Mutalik SP, Mullick P, Pandey A, Kulkarni SS, Mutalik S. Box-Behnken design aided optimization and validation of developed reverse phase HPLC analytical method for simultaneous quantification of dolutegravir sodium and lamivudine co-loaded in nano-liposomes. J Sep Sci. 2021 Aug;44(15):2917-31.

13. Naik S, Mullick P, Mutalik SP, Hegde AR, Lewis SA, Bhat K, et al. Full factorial design for development and validation of a stability-indicating RP-HPLC method for the estimation of timolol maleate in surfactant-based elastic nano-vesicular systems. J Chromatogr Sci. 2022 Jul 12;60(6):584-94.

14. Chaudhari BB, Devadiga BH, Matcha S, Lewis LE, Mallayasamy S, Moorkoth S. Validated HPLC method for ceftriaxone from dried blood spots for pharmacokinetic studies and therapeutic drug monitoring in neonatal population. Bioanalysis. 2023 Apr;15(8):449-63.

15. Ganorkar SB, Shirkhedkar AA. Design of experiments in liquid chromatography (HPLC) analysis of pharmaceuticals: analytics, applications, implications and future prospects. Rev Anal Chem. 2017 Sep 1 [cited 2023 Jul 9];36(3):20160025.

16. Hamid MHM, Elsaman T. A stability-indicating RP-HPLC-UV method for determination and chemical hydrolysis study of a novel naproxen prodrug. J Chem. 2017 Oct 25;2017:e5285671.

17. Gupta A, Navti PD, Mutalik S, Saha M, Moorkoth S. DoE guided development of an HPLC method for evaluation of amoxicillin and metronidazole co-loaded mucoadhesive GRDDS formulation for H. pylori eradication. Chromatographia. 2023 Oct 28;86:729-42. [cited 2023 Oct 29].

18. Zaman B, Hassan W, Khan A, Mushtaq A, Ali N, Bilal M, et al. Forced degradation studies and development and validation of HPLC-UV method for the analysis of velpatasvir copovidone solid dispersion. Antibiotics (Basel). 2022 Jul 5;11(7):897.

19. ?uromska-Witek B, ?mudzki P, Szlósarczyk M, Ma?lanka A, Hubicka U. Development and validation of stability-indicating HPLC methods for the estimation of lomefloxacin and balofloxacin oxidation process under ACVA, H2O2, or KMnO4 treatment. Kinetic evaluation and identification of degradation products by mass spectrometry. Molecules. 2020 Nov 11;25(22):5251.

20. Goyal PK, Jaimini M. Development and validation of stability indicating Rp-Hplc method for quantitative estimation of metoprolol succinate and azelnidipine from synthetic mixture. J Pharm Negat Results. 2023 Feb 6;772-9.

21. Emanuelli J, Eva Scherman Schapoval E. Stability-indicating HPLC method for estimation of omarigliptin in tablets - Oxidative and photolytic kinetics and degradation products formed under oxidative conditions. Microchem J. 2020 Sep 1;157:105084.

22. Peraman R, Lalitha KV, Raja NM, Routhu HB. Identification of degradation products and a stability-indicating RP-HPLC method for the determination of flupirtine maleate in pharmaceutical dosage forms. Sci Pharm. 2014;82(2):281-93.

23. Gadzi?ski P, Froelich A, Jadach B, Wojty?ko M, Tatarek A, Bia?ek A, et al. Ionotropic gelation and chemical crosslinking as methods for fabrication of modified-release gellan gum-based drug delivery systems. Pharmaceutics. 2023 Jan;15(1):108.

24. Noreen S, Hasan S, Ghumman SA, Anwar S, Gondal HY, Batool F, et al. Formulation, statistical optimization, and In Vivo pharmacodynamics of cydonia oblonga mucilage/alginate mucoadhesive microspheres for the delivery of metformin HCl. ACS Omega. 2023 Feb 14;8(6):5925-38.

25. Hoizey G, Lamiable D, Frances C, Trenque T, Kaltenbach M, Denis J, et al. simultaneous determination of amoxicillin and clavulanic acid in human plasma by HPLC with UV detection. J Pharm Biomed Anal. 2002 Oct 15;30(3):661-6.

26. Batrawi N, Wahdan S, Al-Rimawi F. A validated stability-indicating HPLC method for simultaneous determination of amoxicillin and enrofloxacin combination in an injectable suspension. Sci Pharm. 2017;85(1):6.

27. Bellur Atici E, Yazar Y, A?ta? Ç, Ridvano?lu N, Karl??a B. Development and validation of stability indicating HPLC methods for related substances and assay analyses of amoxicillin and potassium clavulanate mixtures. J Pharm Biomed Anal. 2017 Mar 20;136:1-9.

28. Dorn C, Kratzer A, Schießer S, Kees F, Wrigge H, Simon P. Determination of total or free cefazolin and metronidazole in human plasma or interstitial fluid by HPLC-UV for pharmacokinetic studies in man. J Chromatogr B Analyt Technol Biomed Life Sci. 2019 Jun 15;1118-1119:51-4.

29. Maher HM, Youssef RM, Khalil RH, El-Bahr SM. Simultaneous multiresidue determination of metronidazole and spiramycin in fish muscle using high performance liquid chromatography with UV detection. J Chromatogr B Analyt Technol Biomed Life Sci. 2008 Dec 15;876(2):175-81.

30. Wang Y, Zhang P, Jiang N, Gong X, Meng L, Wang D, et al. Simultaneous quantification of metronidazole, tinidazole, ornidazole and morinidazole in human saliva. J Chromatogr B. 2012 Jun 15;899:27-30.

31. Yang DZ, An YQ, Jiang XL, Tang DQ, Gao YY, Zhao HT, et al. Development of a novel method combining HPLC fingerprint and multi-ingredients quantitative analysis for quality evaluation of traditional Chinese medicine preparation. Talanta. 2011 Aug 15;85(2):885-90.

32. P?otka-Wasylka J. A new tool for the evaluation of the analytical procedure: green analytical procedure index. Talanta. 2018 May 1;181:204-9.

33. Gupta A, Akhtar J, Rastogi KC, Badruddeen, Khan MI, Ahmad M. Stability indicating HPLC method for In-vitro determination of pantoprazole sodium and its degradation products in simulated gastric and intestinal fluids. Curr Pharm Anal. 2023 Dec 1;19(10):767-75.

34. Sanam S, Halder S, Rahman SMA. Simultaneous pharmacokinetic evaluation of pantoprazole and vitamin B complex for assessing drug-drug interactions in healthy bangladeshi adults by a newly developed and validated HPLC method. Separations. 2023 Mar;10(3):170.

35. Todorovi? N, ?anji Pani? J, Zaviši? M, Krtolica J, Ratajac R, Petrovi? J, et al. Compounding of liquid and solid dose adjustable formulations with pantoprazole: comparison of stability, applicability and suitability. Pharmaceutics. 2023 Mar;15(3):717.

36. Panda SS, Panigrahi S, Mohanty S, Bera RKVV, Mekap SK. Liquid chromatographic methods for simultaneous quantification of fixed-dose combinations of pantoprazole with itopride and ondansetron: application to method greenness assessment. Sep Sci Plus. 2023;6(5):2300007.

37. Lin J, Liu Y, Zhang J, Lu Z, Guo J, Huang Y, et al. Development and application of a supercritical fluid chromatography-tandem mass spectrometry method for the simultaneous determination of pantoprazole enantiomers in rat plasma. J Pharm Biomed Anal Open. 2023 Dec 1;2:100016.

38. Setyaningsih D, Santoso YA, Hartini YS, Murti YB, Hinrichs WLJ, Patramurti C. Isocratic high-performance liquid chromatography (HPLC) for simultaneous quantification of curcumin and piperine in a microparticle formulation containing Curcuma longa and Piper nigrum. Heliyon. 2021 Mar;7(3):e06541.

39. Gupta I, Adin SN, Aqil M, Mujeeb M, Sultana Y. Quality by design-based development and validation of an HPLC method for simultaneous estimation of pregabalin and piperine in dual drug-loaded liposomes. Biomed Chromatogr. 2023 Jan;37(1):e5510.

40. Kurangi B, Jalalpure S, Jagwani S. A validated stability-indicating HPLC method for simultaneous estimation of resveratrol and piperine in cubosome and human plasma. J Chromatogr B Analyt Technol Biomed Life Sci. 2019 Aug 1;1122-1123:39-48.

41. Kamal YT, Mohammed Musthaba S, Singh M, Parveen R, Ahmad S, Baboota S, et al. Development and validation of HPLC method for simultaneous estimation of piperine and guggulsterones in compound Unani formulation (tablets) and a nanoreservoir system. Biomed Chromatogr. 2012;26(10):1183-90.

42. Sethi P, Dua VK, Mohanty S, Mishra SK, Jain R, Edwards G. Development and validation of a reversed phase HPLC method for simultaneous determination of curcumin and piperine in human plasma for application in clinical pharmacological studies. J Liq Chromatogr Relat Technol. 2009 Nov 18;32(20):2961-74.

Article Metrics
153 Views 26 Downloads 179 Total



Related Search

By author names