Skip to content
2000
image of Green Ecofriendly RP-HPLC Method for Estimating Bifonazole in Transethosomes: Application of Box Behnken Design

Abstract

Introduction

The antifungal drug bifonazole, a substituted imidazole, exhibits broad-spectrum action against Gram-positive bacteria, yeasts, moulds, dermatophytes, and dimorphic fungi.

Methods

The analysis was carried out using a C reversed-phase column (250 mm × 4.6 mm, 5 μm particle size) maintained at a constant temperature of 25°C. Methanol and 0.1% trifluoroacetic acid were utilized as the mobile phase. Detection was performed at 256 nm with a UV detector, and quantification was achieved using the standard method. The method validation was carried out for various parameters as per ICH guidelines. The developed eco-friendly HPLC method was employed to assess the vesicle size and entrapment efficiency of the transethosomes. The greenness assessment tool was applied to assess the environmental sustainability of the proposed HPLC method.

Results

The retention time was under 5 minutes, confirming the method's appropriateness for routine analysis. Linearity was observed in the concentration range of 5 to 25µg/ml, LOD and LOD were 2µg/ml and 8µg/ml respectively. Intraday and interday precision were within ICH limits (<2). The method was successfully used to analyse vesicle size (178nm) and entrapment efficiency (77.6±0.72%.) of the nanoformulation. The greenness assessment tool yielded a value of 0.71.

Discussion

The developed method is both analytically sound and eco-friendly, making a significant improvement in the estimation of bifonazole. The developed method's LOD and LOQ values were sufficiently low to facilitate the detection and measurement of bifonazole in pharmaceutical formulations frequently utilized in research applications, thereby supporting its potential utility in analytical and research settings.

Conclusion

Overall, this green, eco-friendly HPLC method is well-suited for routine analysis of bifonazole, demonstrating excellent cost-effectiveness, sensitivity, precision, and robustness.

© 2025 Bentham Science Publishers
Loading

Article metrics loading...

/content/journals/dmb/10.2174/0118723128396503250715215742
2025-07-31
2025-11-17

  • From This Site

    /content/journals/dmb/10.2174/0118723128396503250715215742
    dcterms_title,dcterms_subject,pub_keyword
    -contentType:Contributor -contentType:Concept -contentType:Institution
    10
    5
Loading full text...

Full text loading...

References

  1. Rang H.P. Dale M.M. Ritter J.M. Flower R.J. Henderson G. Rang and Dale’s Pharmacology. Elsevier Health Sciences 2011
    [Google Scholar]
  2. Reddy G.K.K. Padmavathi A.R. Nancharaiah Y.V. Fungal infections: Pathogenesis, antifungals and alternate treatment approaches. Curr. Res. Microb Sci. 2022 3 100137 10.1016/j.crmicr.2022.100137 35909631
    [Google Scholar]
  3. Hay R.J. Fungal infections. Clin. Dermatol. 2006 24 3 201 212 10.1016/j.clindermatol.2005.11.011 16714201
    [Google Scholar]
  4. Bseiso E. Nasr M. Abd El Gawad N.A. Sammour O. Recent advances in topical formulation carriers of antifungal agents. Indian J. Dermatol. Venereol. Leprol. 2015 81 5 457 463 10.4103/0378‑6323.162328 26261140
    [Google Scholar]
  5. Garber G. An overview of fungal infections. Drugs 2001 61 1 12.(Suppl. 1) 10.2165/00003495‑200161001‑00001 11219546
    [Google Scholar]
  6. Wang H. Sun F. Wang C. Ye J. Xia P. Wang W. Wu Y. A systematic analysis of the global, regional, and national burden of fungal skin diseases from 1990 to 2021. Front Epidemiol. 2024 4 1489148 10.3389/fepid.2024.1489148 39741899
    [Google Scholar]
  7. Lackner T.E. Clissold S.P. Bifonazole. A review of its antimicrobial activity and therapeutic use in superficial mycoses. Drugs 1989 38 2 204 225 10.2165/00003495‑198938020‑00004 2670516
    [Google Scholar]
  8. Plempel M. Berg D. Ritter W. Bifonazole, a new topical azole antimycotic with specific properties. Advances in Topical Antifungal Therapy. Springer 1986 4 20 10.1007/978‑3‑642‑71717‑8_2
    [Google Scholar]
  9. Sheehan D.J. Hitchcock C.A. Sibley C.M. Current and emerging azole antifungal agents. Clin. Microbiol. Rev. 1999 12 1 40 79 10.1128/CMR.12.1.40 9880474
    [Google Scholar]
  10. Carrillo-Muñoz A.J. Tur-Tur C. Comparative study of antifungal activity of sertaconazole, terbinafine, and bifonazole against clinical isolates of Candida spp., Cryptococcus neoformans and dermatophytes. Chemotherapy 1997 43 6 387 392 10.1159/000239596 9395851
    [Google Scholar]
  11. Finch J.J. Warshaw E.M. Toenail onychomycosis: Current and future treatment options. Dermatol. Ther. 2007 20 1 31 46 10.1111/j.1529‑8019.2007.00109.x 17403258
    [Google Scholar]
  12. Elewski B.E. Haley H.R. Robbins C.M. The use of 40% urea cream in the treatment of moccasin tinea pedis. Cutis 2004 73 5 355 357 15186053
    [Google Scholar]
  13. Chemello R.L. Castagna R.D. Cappelletti T. Stramari J.M. da Silva C.B. Comazzetto L.R. Comazzetto, Fungal Infections. Dermatology in Public. Health. Environments: A Comprehensive Textbook. Springer 2023 10.1007/978‑3‑031‑13505‑7_11
    [Google Scholar]
  14. Yuan Z. Yu X. Wu S. Wu X. Wang Q. Cheng W. Hu W. Kang C. Yang W. Li Y. Zhou X.Y. Instability mechanism of Osimertinib in plasma and a solving strategy in the pharmacokinetics study. Front. Pharmacol. 2022 13 928983 928983 10.3389/fphar.2022.928983 35935836
    [Google Scholar]
  15. Kadenatsi I.B. Agapitova I.V. Shustova L.V. Salib I. Dombrovskiĭ V.S. Gagaeva E.V. Kuleshova E.E. Arzamastsev A.P. Firsov A.A. Determination of bifonazole using HPLC in pharmacokinetic studies. Antibiot. Khimioter. 1996 41 5 19 24.May 9054312
    [Google Scholar]
  16. Guo X.J. Wei Z.Y. Li F.M. Determination of bifonazole in cream by high performance liquid chromatography. Se Pu 2001 19 3 279 280 12541819
    [Google Scholar]
  17. Atapattu S.N. Applications and retention properties of alternative organic mobile phase modifiers in reversed-phase liquid chromatography. J. Chromatogr Open 2024 5 100135 10.1016/j.jcoa.2024.100135
    [Google Scholar]
  18. Sharma U.D. Kumar L. Verma R. Selection of stationary phase and mobile phase in high performance liquid chromatography. Research J. Pharm. Technol 2022 15 4325 4332 10.52711/0974‑360X.2022.00726
    [Google Scholar]
  19. Patil S.D. Chalikwar S.S. A brief review on application of design of experiment for the analysis of pharmaceuticals using HPLC. Ann. Pharm. Fr. 2024 82 2 203 228 10.1016/j.pharma.2023.12.011 38159721
    [Google Scholar]
  20. Sahu P.K. Ramisetti N.R. Cecchi T. Swain S. Patro C.S. Panda J. An overview of experimental designs in HPLC method development and validation. J. Pharm. Biomed. Anal. 2018 147 590 611 10.1016/j.jpba.2017.05.006 28579052
    [Google Scholar]
  21. Haque S.K.M. Box–Behnken experimental design for optimizing the HPLC method to determine hydrochlorothiazide in pharmaceutical formulations and biological fluid. J. Mol. Liq. 2022 352 118708 10.1016/j.molliq.2022.118708
    [Google Scholar]
  22. Pena-Pereira F. Wojnowski W. Tobiszewski M. AGREE—Analytical GREEnness metric approach and software. Anal. Chem. 2020 92 14 10076 10082 10.1021/acs.analchem.0c01887 32538619
    [Google Scholar]
  23. Maliyakal J. Patel M. Green chemistry approaches in the analytical validation of fosravuconazole using UV spectrophotometry and HPLC. Green Analytical Chemistry 2025 12 100215 10.1016/j.greeac.2025.100215
    [Google Scholar]
  24. ICH Harmonised tripartite guideline. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human use. https://database.ich.org/sites/default/files/Q2%28R1%29%20Guideline.pdf
    [Google Scholar]
  25. Chiprikar P. Mastiholimath V. Development of RP-HPLC based analytical method for determination of cariprazine hydrochloride in bulk drug and pharmaceutical dosage form using box-behnken statistical design. Anal. Chem. Lett. 2023 13 3 297 312 10.1080/22297928.2023.2242857
    [Google Scholar]
  26. Kumbhar T. Chiprikar P. Ammanage A. Patel U. Mastiholimath V. Mastiholimath, formulation and analytical development of laxative polyherbal suspension. Int. J. Ayurvedic Med. 2024 15 383 10.47552/ijam.v15i2.4422
    [Google Scholar]
  27. Chaudhari M.V. Chaudhari U. Sahu J.K. Bagade S.B. Stability indicating method development and validation for the estimation of bempedoic acid by RP-HPLC. Drug Metab. Bioanal Lett 2024 17 1 23 33 10.2174/0118723128278080240404052506
    [Google Scholar]
  28. Chiprikar P. Mastiholimath V. Balekundri A. HP-TLC method development of cariprazine hydrochloride for applicative quantification of nanostructured lipid carriers. Chemistry Africa 2023 7 141 10.1007/s42250‑023‑00726‑z
    [Google Scholar]
  29. Raut R. Shaji J. HPLC method validation for quantification of tetrahydrocurcumin in bulk drug and formulation. Future J. Pharm. Sci. 2021 7 1 7 10.1186/s43094‑021‑00194‑7
    [Google Scholar]
  30. Wiggins D.E. System suitability in an optimized hplc system. J. Liq. Chromatogr. 1991 14 16-17 3045 3060 10.1080/01483919108049375
    [Google Scholar]
  31. Epshtein N.A. Validation of the specificity of chromatographic methods: Key points and practical recommendations. Pharm. Chem. J. 2022 56 5 702 711 10.1007/s11094‑022‑02698‑9
    [Google Scholar]
  32. Schill G. Approaches to improve specificity and sensitivity in chromatography. Drug Deliv. 1985 341 360 10.1007/978‑1‑4612‑5186‑6_19
    [Google Scholar]
  33. Abdulbaqi I.M. Darwis Y. Abou Assi R. Abdul Karim Khan N. Transethosomal gels as carriers for the transdermal delivery of colchicine: statistical optimization, characterization, and ex vivo evaluation. Drug Des. Devel. Ther. 2018 12 795 813 10.2147/DDDT.S158018 29670336
    [Google Scholar]
  34. Mishra K.K. Kaur C.D. Gupta A. Development of itraconazole loaded ultra-deformable transethosomes containing oleic-acid for effective treatment of dermatophytosis: Box-Behnken design, ex-vivo and in-vivo studies. J. Drug Deliv. Sci. Technol. 2022 67 102998 10.1016/j.jddst.2021.102998
    [Google Scholar]
  35. Asghar Z. Jamshaid T. Jamshaid U. Madni A. Akhtar N. Lashkar M.O. Gad H.A. In vivo evaluation of miconazole-nitrate-loaded transethosomal gel using a rat model infected with Candida albicans. Pharmaceuticals 2024 17 5 546 10.3390/ph17050546 38794118
    [Google Scholar]
  36. Qureshi M.I. Jamil Q.A. Usman F. Wani T.A. Farooq M. Shah H.S. Ahmad H. Khalil R. Sajjad M. Zargar S. Kausar S. Tioconazole-loaded transethosomal gel using Box–Behnken design for topical applications: In vitro, in vivo, and molecular docking approaches. Gels 2023 9 9 767 10.3390/GELS9090767
    [Google Scholar]
  37. Asghar Z. Jamshaid T. Sajid-ur-Rehman M. Jamshaid U. Gad H.A. Novel transethosomal gel containing miconazole nitrate; development, characterization, and enhanced antifungal activity. Pharmaceutics 2023 15 11 2537 10.3390/pharmaceutics15112537 38004517
    [Google Scholar]
  38. Mazhar D. Haq N.U. Zeeshan M. Ain Q.U. Ali H. Khan S. Khan S.A. Preparation, characterization, and pharmacokinetic assessment of metformin HCl loaded transfersomes co-equipped with permeation enhancer to improve drug bioavailability via transdermal route. J. Drug Deliv. Sci. Technol. 2023 84 104448 10.1016/j.jddst.2023.104448
    [Google Scholar]
  39. Joshi S.A. Jalalpure S.S. Kempwade A.A. Peram M.R. Development and validation of hplc method to determine colchicine in pharmaceutical formulations and its application for analysis of solid lipid nanoparticles. Curr. Pharm. Anal. 2017 14 1 76 83 10.2174/1573412912666161013124417
    [Google Scholar]
  40. Sartori T. Murakami F.S. Cruz A.P. de Campos A.M. Development and validation of a fast RP-HPLC method for determination of methotrexate entrapment efficiency in polymeric nanocapsules. J. Chromatogr. Sci. 2008 46 6 505 509 10.1093/chromsci/46.6.505 18647471
    [Google Scholar]
  41. Frkanec R. Travaš D. Krstanović M. Špoljar B.H. Ljevaković Đ. Vranešić B. Frkanec L. Tomašić J. Entrapment of peptidoglycans and adamantyltripeptides into liposomes: An HPLC assay for determination of encapsulation efficiency. J. Liposome Res. 2003 13 3-4 279 294 10.1081/LPR‑120026452 14670233
    [Google Scholar]
  42. Balekundri A. Hurkadale P. Hegde H. Greenness evaluation and simultaneous high-performance thin-layer chromatography method for determination of powder mixture and Ayurvedic formulation. Natl. Acad. Sci. Lett. 2024 47 3 233 237 10.1007/s40009‑023‑01342‑8
    [Google Scholar]
  43. Naguib I.A. Majed M. Albogami M. Alshehri M. Bukhari A. Alshabani H. Alsalahat I. Abd-ElSalam H.A.H. Greenness assessment of HPLC analytical methods with common detectors for assay of paracetamol and related materials in drug products and biological fluids. Separations 2023 10 283 10.3390/separations10050283
    [Google Scholar]
  44. Jonnalagadda R. Rathinam S. Nagappan K. Chandrasekar V. Green HPLC method for simultaneous analysis of three natural antioxidants by analytical quality by design. J. AOAC Int. 2024 107 1 14 21 10.1093/jaoacint/qsad105 37701979
    [Google Scholar]
  45. Vieira-Sellaï L. Quintana M. Diop O. Mercier O. Tarrit S. Raimi N. Ba A. Maunit B. Galmier M.J. Green HPLC quantification method of lamivudine, zidovudine and nevirapine with identification of related substances in tablets. Green Chem. Lett. Rev. 2022 15 3 695 704 10.1080/17518253.2022.2129463
    [Google Scholar]
  46. Bhujbal S. Rupenthal I.D. Agarwal P. Development and validation of a stability-indicating HPLC method for assay of tonabersat in pharmaceutical formulations. Methods 2024 231 178 185 10.1016/j.ymeth.2024.10.001 39368764
    [Google Scholar]
  47. Kokilambigai K.S. Lakshmi K.S. Analytical quality by design assisted RP-HPLC method for quantifying atorvastatin with green analytical chemistry perspective. J. Chromatogr Open 2022 2 100052 10.1016/j.jcoa.2022.100052
    [Google Scholar]
  48. Abdelgawad M.A. Abdelaleem E.A. Gamal M. Abourehab M.A.S. Abdelhamid N.S. A new green approach for the reduction of consumed solvents and simultaneous quality control analysis of several pharmaceuticals using a fast and economic RP-HPLC method; a case study for a mixture of piracetam, ketoprofen and omeprazole drugs. RSC Advances 2022 12 25 16301 16309 10.1039/D2RA02395D 35733689
    [Google Scholar]
/content/journals/dmb/10.2174/0118723128396503250715215742
Loading
Green Ecofriendly RP-HPLC Method for Estimating Bifonazole in Transethosomes: Application of Box Behnken Design
Bentham Science Publishers ; https://doi.org/10.2174/0118723128396503250715215742
/content/journals/dmb/10.2174/0118723128396503250715215742
/content/journals/dmb/10.2174/0118723128396503250715215742
Loading

Data & Media loading...


  • Article Type:     Research Article
Keywords: BBD ; HPLC ; bifonazole ; green method ; Gram-positive bacteria ; transethosomes ; pharmaceutical formulations
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error
Please enter a valid_number test