Skip to main content
SpringerLink
Log in

DoE Guided Development of an HPLC Method for Evaluation of Amoxicillin and Metronidazole Co-loaded Mucoadhesive GRDDS Formulation for H. pylori Eradication

  • Original
  • Published:
Chromatographia Aims and scope Submit manuscript

Abstract

H. Pylori infection is a major reason for chronic Peptic Ulcer Disease (PUD) and related complications worldwide. Traditional treatment options fail to eradicate the organism completely which leads to antibiotic resistance. Novel formulations are being developed with an aim for effective treatment. Mucoadhesive GRDDS system is an option to increase the antibiotic bioavailability. The objective of this work is to develop a RP-HPLC–PDA based analytical method to evaluate the entrapment efficiency of the GRDDS systems developed for H. pylori treatment containing amoxicillin and metronidazole. DoE was used for optimizing the HPLC conditions. The optimized method used a HyperClone ODS C18 column as stationary phase and methanol: phosphate buffer (15:85) at pH 6.4 as the mobile phase. The flow rate was 0.9 mL/min. Method was validated as per the ICH Q2(R1) guideline. Method was linear from 0.5 to 20 µg/ml for both amoxicillin and metronidazole with an R2 value of 0.9995 and 0.9996 respectively. The validated HPLC method showed specificity for both drugs in the presence of degradation products as well as from other excipients of the GRDDS system. The validated method was applied to determine the entrapment efficiency as a part of formulation optimization. The entrapment efficiency of the final formulation was determined to be 78–84% and 75–82% for amoxicillin and metronidazole respectively.

Graphical Abstract

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Data availability

The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.

Abbreviations

AMO:

Amoxicillin trihydrate

MTZ:

Metronidazole

H. pylori :

Helicobacter pylori

GRDDS:

Gastroretentive drug delivery system

ANOVA:

Analysis of variance

BBD:

Box-Behnken design

DoE:

Design of experiments

OFAT:

One-factor at a time

ICH:

International Conference on Harmonization

TR :

Retention time

References

  1. Chmiela M, Kupcinskas J (2019) Review: pathogenesis of Helicobacter pylori infection. Helicobacter 24:1–5. https://doi.org/10.1111/hel.12638

    Article  Google Scholar 

  2. Sarem M, Corti R (2016) Role of Helicobacter pylori coccoid forms in infection and recrudescence. Gastroenterología y Hepatología (English Edition) 39:28–35. https://doi.org/10.1016/j.gastre.2015.12.005

    Article  Google Scholar 

  3. Kakiuchi T, Nakayama A, Shimoda R, Matsuo M (2019) Atrophic gastritis and chronic diarrhea due to Helicobacter pylori infection in early infancy. Medicine (Baltimore) 98:e17986. https://doi.org/10.1097/MD.0000000000017986

    Article  PubMed  Google Scholar 

  4. Miller AK, Williams SM (2021) Helicobacter pylori infection causes both protective and deleterious effects in human health and disease. Genes Immun 22:218–226. https://doi.org/10.1038/s41435-021-00146-4

    Article  PubMed  PubMed Central  Google Scholar 

  5. Liu Z, Lu W, Qian L et al (2005) In vitro and in vivo studies on mucoadhesive microspheres of amoxicillin. J Control Release 102:135–144. https://doi.org/10.1016/j.jconrel.2004.06.022

    Article  CAS  PubMed  Google Scholar 

  6. Kim SY, Choi DJ, Chung J-W (2015) Antibiotic treatment for Helicobacter pylori: Is the end coming? World J Gastrointest Pharmacol Ther 6:183–198. https://doi.org/10.4292/wjgpt.v6.i4.183

    Article  PubMed  PubMed Central  Google Scholar 

  7. Hou J-Y, Gao L-N, Meng F-Y, Cui Y-L (2014) Mucoadhesive microparticles for gastroretentive delivery: preparation, biodistribution and targeting evaluation. Mar Drugs 12:5764–5787. https://doi.org/10.3390/md12125764

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Portero A, Remuñán-López C, Criado MT, Alonso MJ (2002) Reacetylated chitosan microspheres for controlled delivery of anti-microbial agents to the gastric mucosa. J Microencapsul 19:797–809. https://doi.org/10.1080/0265204021000022761

    Article  CAS  PubMed  Google Scholar 

  9. Vinchurkar K, Sainy J, Khan MA et al (2022) Features and facts of a gastroretentive drug delivery system-a review. Turk J Pharm Sci 19:476–487. https://doi.org/10.4274/tjps.galenos.2021.44959

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Gupta A, Shetty S, Mutalik S, et al (2023) Treatment of H. pylori infection and gastric ulcer: Need for novel Pharmaceutical formulation. Heliyon https://doi.org/10.1016/j.heliyon.2023.e20406

  11. Sahasathian T, Praphairaksit N, Muangsin N (2010) Mucoadhesive and floating chitosan-coated alginate beads for the controlled gastric release of amoxicillin. Arch Pharm Res 33:889–899. https://doi.org/10.1007/s12272-010-0612-8

    Article  CAS  PubMed  Google Scholar 

  12. Sen O, Manna S, Nandi G et al (2023) Recent advances in alginate based gastroretentive technologies for drug delivery applications. Med Novel Technol Devices 18:100236. https://doi.org/10.1016/j.medntd.2023.100236

    Article  Google Scholar 

  13. Kumar BR (2017) Application of HPLC and ESI-MS techniques in the analysis of phenolic acids and flavonoids from green leafy vegetables (GLVs). J Pharm Anal 7:349–364. https://doi.org/10.1016/j.jpha.2017.06.005

    Article  PubMed  PubMed Central  Google Scholar 

  14. Adigal S, Bhandary V, Hegde N et al (2023) Protein profile analysis of tear fluid with hyphenated HPLC-UV LED-induced fluorescence detection for the diagnosis of dry eye syndrome. RSC Advances 13:22559–22568. https://doi.org/10.1039/D3RA04389D

    Article  CAS  PubMed  Google Scholar 

  15. Nikolin B, Imamović B, Medanhodzić-Vuk S, Sober M (2004) High perfomance liquid chromatography in pharmaceutical analyses. Bosn J Basic Med Sci. https://doi.org/10.17305/bjbms.2004.3405

    Article  PubMed  PubMed Central  Google Scholar 

  16. Amarnath K, Amarnath V, Amarnath K et al (2003) A specific HPLC-UV method for the determination of cysteine and related aminothiols in biological samples. Talanta 60:1229–1238. https://doi.org/10.1016/S0039-9140(03)00232-7

    Article  CAS  PubMed  Google Scholar 

  17. Wu T, Annie Bligh SW, Gu L et al (2005) Simultaneous determination of six isoflavonoids in commercial Radix Astragali by HPLC-UV. Fitoterapia 76:157–165. https://doi.org/10.1016/j.fitote.2004.11.006

    Article  CAS  PubMed  Google Scholar 

  18. Porta V, Schramm SG, Kano EK et al (2008) HPLC-UV determination of metformin in human plasma for application in pharmacokinetics and bioequivalence studies. J Pharm Biomed Anal 46:143–147. https://doi.org/10.1016/j.jpba.2007.10.007

    Article  CAS  PubMed  Google Scholar 

  19. Sarısaltık Yaşın D, Arslantürk Bingül A, Karaküçük A, Teksin ZŞ (2021) Development and validation of an HPLC method using an experimental design for analysis of amlodipine Besylate and Enalapril maleate in a fixed-dose combination. Turk J Pharm Sci 18:306–318. https://doi.org/10.4274/tjps.galenos.2020.89725

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Tavares Luiz M, Santos Rosa Viegas J, Palma Abriata J et al (2021) Design of experiments (DoE) to develop and to optimize nanoparticles as drug delivery systems. Eur J Pharm Biopharm 165:127–148. https://doi.org/10.1016/j.ejpb.2021.05.011

    Article  CAS  PubMed  Google Scholar 

  21. Jankovic A, Chaudhary G, Goia F (2021) Designing the design of experiments (DOE) – An investigation on the influence of different factorial designs on the characterization of complex systems. Energ Build 250:111298. https://doi.org/10.1016/j.enbuild.2021.111298

    Article  Google Scholar 

  22. Tome T, Žigart N, Časar Z, Obreza A (2019) Development and optimization of liquid chromatography analytical methods by using AQbD principles: overview and recent advances. Org Process Res Dev 23:1784–1802. https://doi.org/10.1021/acs.oprd.9b00238

    Article  CAS  Google Scholar 

  23. Mutalik SP, Mullick P, Pandey A et al (2021) 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 44:2917–2931. https://doi.org/10.1002/jssc.202100152

    Article  CAS  PubMed  Google Scholar 

  24. Mohamed D, Elbalkiny HT (2023) Application of solidified floating organic droplet dispersive liquid–liquid microextraction for determination of veterinary antibiotic residues in milk samples with greenness assessment. Microchemical Journal 193:109153. https://doi.org/10.1016/j.microc.2023.109153

    Article  CAS  Google Scholar 

  25. Chiriac U, Rau H, Frey OR et al (2022) Validation and application of an HPLC-UV method for routine therapeutic drug Monitoring of Dalbavancin. Antibiotics 11:541. https://doi.org/10.3390/antibiotics11050541

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Benito-Peña E, Partal-Rodera AI, León-González ME, Moreno-Bondi MC (2006) 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 556:415–422. https://doi.org/10.1016/j.aca.2005.09.054

    Article  CAS  Google Scholar 

  27. Zimmer J, Röhr AC, Kluge S et al (2021) Validation and application of an HPLC-UV method for routine therapeutic drug Monitoring of cefiderocol. Antibiotics 10:242. https://doi.org/10.3390/antibiotics10030242

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Naik S, Mullick P, Mutalik SP et al (2022) 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 60:584–594. https://doi.org/10.1093/chromsci/bmab101

    Article  CAS  PubMed  Google Scholar 

  29. Chaudhari BB, Devadiga BH, Matcha S et al (2023) Validated HPLC method for ceftriaxone from dried blood spots for pharmacokinetic studies and therapeutic drug monitoring in neonatal population. Bioanalysis 15:449–463. https://doi.org/10.4155/bio-2023-0047

    Article  CAS  PubMed  Google Scholar 

  30. Ganorkar SB, Shirkhedkar AA (2017) Design of experiments in liquid chromatography (HPLC) analysis of pharmaceuticals: analytics, applications, implications and future prospects. Rev Analyt Chem. https://doi.org/10.1515/revac-2016-0025

    Article  Google Scholar 

  31. Hamid MHM, Elsaman T (2017) A stability-indicating RP-HPLC-UV method for determination and chemical hydrolysis study of a novel naproxen prodrug. J Chem 2017:e5285671. https://doi.org/10.1155/2017/5285671

    Article  Google Scholar 

  32. Zaman B, Hassan W, Khan A et al (2022) Forced degradation studies and development and validation of HPLC-UV method for the analysis of Velpatasvir Copovidone solid dispersion. Antibiotics (Basel) 11:897. https://doi.org/10.3390/antibiotics11070897

    Article  CAS  PubMed  Google Scholar 

  33. Żuromska-Witek B, Żmudzki P, Szlósarczyk M, et al (2020) 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 25:5251. https://doi.org/10.3390/molecules25225251

  34. Goyal PK, Jaimini M (2023) Development And Validation Of Stability Indicating Rp-Hplc Method For Quantitative Estimation Of Metoprolol Succinate And Azelnidipine From Synthetic Mixture. Journal of Pharmaceutical Negative Results 772–779. https://doi.org/10.47750/pnr.2023.14.03.101

  35. Emanuelli J, Eva Scherman Schapoval E (2020) Stability-Indicating HPLC method for estimation of omarigliptin in tablets – Oxidative and photolytic kinetics and degradation products formed under oxidative conditions. Microchem J 157:105084. https://doi.org/10.1016/j.microc.2020.105084

    Article  CAS  Google Scholar 

  36. Peraman R, Lalitha KV, Raja NM, Routhu HB (2014) Identification of degradation products and a stability-indicating RP-HPLC method for the determination of Flupirtine maleate in pharmaceutical dosage forms. Sci Pharm 82:281–293. https://doi.org/10.3797/scipharm.1310-01

    Article  CAS  PubMed  Google Scholar 

  37. Gadziński P, Froelich A, Jadach B et al (2023) Ionotropic Gelation and chemical crosslinking as methods for fabrication of modified-release Gellan gum-based drug delivery systems. Pharmaceutics 15:108. https://doi.org/10.3390/pharmaceutics15010108

    Article  CAS  Google Scholar 

  38. Noreen S, Hasan S, Ghumman SA et al (2023) Formulation, statistical optimization, and In Vivo Pharmacodynamics of Cydonia oblonga Mucilage/Alginate Mucoadhesive microspheres for the delivery of metformin HCl. ACS Omega 8:5925–5938. https://doi.org/10.1021/acsomega.2c07789

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Abdalla K, Kamoun E, Maghraby G (2015) Optimization of the entrapment efficiency and release of ambroxol hydrochloride alginate beads. J App Pharm Sci 013–019. https://doi.org/10.7324/JAPS.2015.50403

  40. Prezotti FG, Cury BSF, Evangelista RC (2014) Mucoadhesive beads of gellan gum/pectin intended to controlled delivery of drugs. Carbohyd Polym 113:286–295. https://doi.org/10.1016/j.carbpol.2014.07.021

    Article  CAS  Google Scholar 

  41. Deshmukh R, Harwansh RK, Prajapati M, Sharma B (2023) Formulation and Evaluation of Oral Mucoadhesive Microspheres of Ofloxacin for Peptic Ulcer Use. Trends in Sciences 20:5751–5751. https://doi.org/10.48048/tis.2023.5751

  42. Hoizey G, Lamiable D, Frances C et al (2002) Simultaneous determination of amoxicillin and clavulanic acid in human plasma by HPLC with UV detection. J Pharm Biomed Anal 30:661–666. https://doi.org/10.1016/S0731-7085(02)00289-3

    Article  CAS  PubMed  Google Scholar 

  43. Batrawi N, Wahdan S, Al-Rimawi F (2017) A validated stability-indicating HPLC method for simultaneous determination of amoxicillin and Enrofloxacin combination in an injectable suspension. Sci Pharm 85:6. https://doi.org/10.3390/scipharm85010006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Bellur Atici E, Yazar Y, Ağtaş Ç et al (2017) Development and validation of stability indicating HPLC methods for related substances and assay analyses of amoxicillin and potassium clavulanate mixtures. J Pharm Biomed Anal 136:1–9. https://doi.org/10.1016/j.jpba.2016.12.032

    Article  CAS  PubMed  Google Scholar 

  45. Dorn C, Kratzer A, Schießer S et al (2019) 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 1118–1119:51–54. https://doi.org/10.1016/j.jchromb.2019.04.025

    Article  CAS  Google Scholar 

  46. Maher HM, Youssef RM, Khalil RH, El-Bahr SM (2008) Simultaneous multiresidue determination of metronidazole and spiramycin in fish muscle using high performance liquid chromatography with UV detection. J Chromatogr B 876:175–181. https://doi.org/10.1016/j.jchromb.2008.10.033

    Article  CAS  Google Scholar 

  47. Wang Y, Zhang P, Jiang N et al (2012) Simultaneous quantification of metronidazole, tinidazole, ornidazole and morinidazole in human saliva. J Chromatogr B 899:27–30. https://doi.org/10.1016/j.jchromb.2012.04.032

    Article  CAS  Google Scholar 

  48. Yang D-Z, An Y-Q, Jiang X-L et al (2011) Development of a novel method combining HPLC fingerprint and multi-ingredients quantitative analysis for quality evaluation of traditional Chinese medicine preparation. Talanta 85:885–890. https://doi.org/10.1016/j.talanta.2011.04.059

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors are thankful to Indian Council of Medical Research (ICMR) for the funding provide under ICMR-SRF fellowship program. Authors are also grateful to Manipal academy of higher education, Manipal, Karnataka for providing the facilities. The authors are thankful for Biorender.com, a figure-making tool.

Funding

The work is funded by Indian Council of Medical Research (ICMR) File no. 3/2/2/16/2022-NCD-III

Author information

Authors and Affiliations

  1. Department of Pharmaceutical Quality Assurance, Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher Education, Manipal, Karnataka, 576104, India

    Ashutosh Gupta, Moumita Saha & Sudheer Moorkoth

  2. Department of Pharmaceutics, Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher Education, Manipal, Karnataka, 576104, India

    Prerana D. Navti & Srinivas Mutalik

Authors
  1. Ashutosh Gupta
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Prerana D. Navti
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Srinivas Mutalik
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Moumita Saha
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sudheer Moorkoth
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

All authors contributed to the study conception and design. Ashutosh Gupta carried out the experimental work, data analysis, and drafting the paper. Prerana D Navti carried out the the DoE analysis and data interpretation. Moumita Saha involved in the experimental work of HPLC method optimization. Sudheer Moorkoth and Srinivas Mutalik were involved in supervising the experimental work, data analysis, resources, and reviewing the paper. All authors have made a significant contribution to the research in the manuscript, approved its claims, agreed to be an author.

Corresponding author

Correspondence to Sudheer Moorkoth.

Ethics declarations

Conflict of interest

The authors state that the manuscript does not include any conflicts of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 1127 KB)

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Gupta, A., Navti, P.D., Mutalik, S. et al. DoE Guided Development of an HPLC Method for Evaluation of Amoxicillin and Metronidazole Co-loaded Mucoadhesive GRDDS Formulation for H. pylori Eradication. Chromatographia 86, 729–742 (2023). https://doi.org/10.1007/s10337-023-04290-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10337-023-04290-z

Keywords

Search

Navigation