INTRODUCTION:

Bifonazole is also chemically known as 1-(biphenyl-4-yl-(phenyl)-methyl)-1H-imidazole which is substituted imidazole analogue. It has broad-spectrum activity against dermatophytes, moulds, yeasts, dimorphic fungi, and some gram-positive bacteria and it is broad-spectrum imidazole antimycotic.¹ Bifonazole, a strong cytochrome P-450 Aromatase inhibitor, works well to stop the production of oestrogens from androgens, which helps treat seborrhoeic dermatitis and tinea infections.^2,3,4 Bifonazole breaks down the fungal cell wall and creates holes in the cell membranes, which allows the vital components of the fungal cells to escape and ultimately kills the fungus.⁽⁵⁾ This medication's selective interference makes it an excellent treatment option for invasive mucosal infections by blocking the fungal ergo sterol production pathway and terpenoids biosynthesis.^6,7

According to the European Pharmacopoeia, bifonazole is an official medication. Bifonazole can be found in pharmaceutical formulations as lotions, powders, or creams.⁸When compared to other topical antifungal medicines, it will yield higher patient compliance with regard to topical treatment once a day.⁹

The N-substituted imidazole class of azoles includes bifonazole, which shares structural similarities with other medications in this class, including miconazole, econazole and clotrimazole. It comes in a variety of topical formulations, including cream, gel, and powder, and is recommended to be taken once daily, which is advantageous because it increases compliance.¹⁰

Figure 1: - Bifonazole structure

Physiochemical Properties

Bifonazole's antifungal action is effective against various pathogenic yeasts, dimorphic pathogens, dermatophytes, tinea versicolour, and filamentous fungi due to its low water solubility and lipophilic nature.¹¹

Bifonazole's mole fraction solubility in 14 pure solvents, including ethanol, n-propanol, iso-propanol, n-butanol, iso-butanol, sec-butanol, n-pentanol, iso-pentanol, methyl acetate, ethyl acetate, n-propyl acetate, n-butyl acetate, acetonitrile, and DMF, ranges from 283.15 to 323.15 K under 101.3 kPa^a.¹²

Bifonazole has a molar mass (g/mol) of 310.39 and a molar volume of 288.1c.¹² It has a pKa ³⁰⁻³⁶ of 6.29, a glass transition temperature Tg of 14.1±0.3, and a melting point Tm of 149.8±0.2.¹³ It was discovered that the Bifonazole gel's pH was 5.6 ± 0.07 and its viscosity was 593 ± 0.98 cps. This gel formulation has a spreadability of 6.5 ± 0.05 cm.¹⁴

Pharmacokinetic Properties

The pharmacokinetic characteristics of antifungals are important when choosing a prescription because of possible adverse effects like decreased medication freedom or impaired gastrointestinal function. Some antifungal medications, such as echinocandins and amphotericin B, must be administered intravenously because of their poor gastrointestinal absorption.¹⁵

Various bifonazole formulations have been administered orally and intravenously. After dermal application, bifonazole is mostly kept on the skin's surface, with a high capacity for skin penetration and a mean half-life of 19–32 hours. It has a 95% metabolism, so that unmodified bifonazole is eliminated faster. Its renal clearance is less than 0.006 L/h and its total body clearance is 50 L/h, underscoring the significance of metabolic pathways in its efficacy.¹⁶

Pharmacodynamic Properties

Antifungal experts demonstrate a variety of in vivo movement examples; such as focus autonomous or fixation hungry, depending on the fraction reaction bend state at clinically achieved concentrations.¹⁷Medication and pathogen MIC can be connected to in vivo activity to determine the best dosage strategies that maximize antifungal viability while minimizing toxicity. Predicting infection areas where antifungal drugs are less effective is possible with pharmacodynamic data.¹⁸

According to in vitro research, bifonazole causes degenerative alterations in hyphal structure at sub-inhibitory concentrations and prevents T. mentagrophytes' hyphal tips from growing normally.^{19, 20} It has been demonstrated that bifonazole works well to treat experimental dermatophytic and Candida infections. Topical use of bifonazole at 0.05 and 1.0% dosages resulted in a high percentage of mycological cures in guinea pigs with experimentally generated trichophytosis.^{20, 21}

Mechanism of action

It inhibits the production of ergo-sterol, a crucial component of the fungal cell membrane. It functions by the destabilisation of the fungal cytochrome P-450-51 enzyme (lanosterol 14-alpha demethlase). The structure of the fungal cell membrane depends on this. When it is suppressed, cells are lysed. ²²

Figure 2: Mechanism of action

Need for analytical method development and validation

Analytical technique validation is essential for quality control, sound science, and regulatory requirements. The accuracy, sensitivity, specificity, and repeatability of test procedures are required by CFR 311.165c. To prove the method's accuracy, sensitivity, specificity, and repeatability, scientists should use excellent science.²³ Despite its initial expense and time-consuming nature, analytical method validation improves time management, removes annoying repeats, and gives developers and consumers more confidence.²⁴

HPLC based analytical method development

The Russian botanist M.S. Tswett created HPLC in 1903, and it is now a commonly used analytical method in more than 85% of general pharmaceuticals. It includes streamlining optimization, assessing performance, and choosing the appropriate phase and factors. A solid approach needs advanced tools like computer modeling and few experimental runs.²⁵One effective analytical technique for separating, identifying, and quantifying chemicals in samples is High Performance Liquid Chromatography (HPLC). It entails pushing a liquid through a porous column at high pressure after injecting a sample solution into it. The rates of migration between the stationary and mobile phases determine how the sample is separated.²⁶

Table 1: HPLC based analytical method development for bifonazole

Title	Method	Sample	Stationary phase	Mobile phase	Wavelength (nm)	Reference
Determination of bifonazole and identification of its photo-catalytic degradation products using UPLC-MS/MS	UPLC-MS/MS	Solid /Cream	Zorbax Eclipse XDB-C18 column (250×4.6 mm)	Methanol: Ammonium acetate in water	252 nm	29
Analytical method development and validation of bifonazole and its stability study by using sophisticated RP-HPLC Method.	RP-HPLC	Solid /cream	Hemochrom C18 (150 mm× 4.5 mm × 5µm)	Acetonitrile: 0.05% TFA in water (20:80)	256 nm	30
Simultaneous determination of bifonazole and benzyl alcohol in pharmaceutical formulation by reverse-phase HPLC.	RP-HPLC	Cream /Solid form	Merck C18 Column	Buffer solution: Acetonitrile (50:50)	210 nm	31
An RP-HPLC developed for determination of bifonazole in pharmaceutical formulation.	RP-HPLC	Cream	Eurosphere C18 Column (25cm× 4.6mm × 5µm )	Methanol: Sodium acetate (70:30)	252 nm	32
AQbD approach applied to NIR in a complex topical formulation: Bifonazole as case study	HPLC	Cream	Merck C18 Column	Buffer solution at pH 3.2 :acetonitrile (50:50)	210	33

UV-spectroscopy-based analytical method development

UV spectroscopy is a technique that employs visible and ultraviolet light to examine the dynamics, composition, and chemical structure of molecules. It allows for the sensitive and quick analysis of ecosystem activities as well as the identification of pollutants and organic waste. ²⁷ The use of nanotechnology improves the sensitivity, specificity, and real-time process management of UV spectroscopy in online monitoring systems, biomedical applications, environmental monitoring, and pharmaceutical analysis. ²⁸

Table 2: UV- spectroscopy based analytical method development for bifonazole

Title	Sample	Method/Instrument	Solvent/Solution	Wavelength (nm)	Reference
Determination of bifonazole in creams containing methyl- and propyl p-hydroxybenzoate by derivative spectrophotometric method	Cream	Derivative Spectrophotometer	0.1 M HCL	241.5 nm	34
Determination and validation of UV spectrophotometric procedure for estimation of bifonazole in bulk	Bulk	UV-spectrophotometer/ Analytik jena Specord 200	0.1M HCL	255-256 nm	35
Validated spectrophotometric methods for the determination of bifonazole in pharmaceuticals by charge transfer complexation	Gel	Double beam spectrophotometer/ Shimadzu Uv-2550	0.1% CAA /0.1% DDQ	517 nm /457 nm	36
Validated UV spectrophotometric method for estimation of bifonazole in their bulk drug and cream pharmaceutical formulation	Bulk / Cream	Shimadzu spectrophotometer	0.1 M HCL	255 nm	37

NIR METHOD (Near infrared method)

For the quantification of bifonazole (BFZ) in cream formulations, the study used near-infrared spectroscopy (NIR), a non-destructive, non-invasive, and chemically specific technology to supplement conventional analytical techniques like RP-HPLC. A Spectrum 400 FT-IR/FT-NIR spectrometer was used for the NIR study, which projected that the formulation's bifonazole concentration would be 8.48 mg/g. The precision and dependability of the NIR approach were confirmed by the results, which displayed a 1.25% relative standard deviation when compared to RP-HPLC.³³

DSC AND PXRD METHOD

(Differential scanning calorimetric and Powder X-ray diffraction)

The PerkinElmer DSC model 8500 was used for DSC measurements, and it was calibrated using indium for temperature and enthalpy scales. For PXRD measurements, a STOE Stadi P diffractometer was used for the samples that were received. The PXRD and calorimetric results show that instead of creating distinct polymorphic structures, bifonazole displays a complicated partial crystallization behavior that is heavily influenced by its scanning rates and thermal history. ³⁸

CONCLUSION:

Drug development is an analytical technique that enables the study and development of a drug's analytical procedure. This process includes important characteristics including sensitivity, robustness, specificity, accuracy, and precision. The purpose of reviewing the bifonazole analytical method is to provide a comprehensive overview of the complete process, including its characteristics, mode of action, pharmacokinetics, and pharmacodynamic investigations. In this article, the various analytical techniques for calculating bifonazole in bulk and various dosage forms were covered. In addition to helping you discover new analytical methods, this article lets you study all of the more traditional bifonazole methods at once.

REFERENCE:

1. Rieth H. Bifonazole. Basel: Karger Publishers; 1985. doi:10.1159/isbn.978-3-318-01573-7

2. Wheatley D, Richardson MD, Scott EM. Tinea Infections Treated with Bifonazole Gel/Dermatophytosen‐Behandlung mit Bifonazol‐Gel. Mycoses. 1988 Sep; 31(9): 471-5..

3. Egbuta C, Lo J, Ghosh D. Mechanism of inhibition of estrogen biosynthesis by azole fungicides. Endocrinology. 2014 Dec 1; 155(12): 4622-8.

4. Faergemann J. Treatment of seborrhoeic dermatitis with bifonazole. Mycoses. 1989 Jun; 32(6): 309-11..

5. Ahmad K, Khan MK, Baig MH, Imran M, Gupta GK. Role of azoles in cancer prevention and treatment: present and future perspectives. Anti-Cancer Agents in Medicinal Chemistry-Anti-Cancer Agents). 2018 Jan 1; 18(1):46-56.

6. Wächtler B, Wilson D, Hube B. Candida albicans adhesion to and invasion and damage of vaginal epithelial cells: stage-specific inhibition by clotrimazole and bifonazole. Antimicrobial Agents and Chemotherapy. 2011 Sep;55(9):4436-9.

7. Adriana PT. Havard’s Nursing Guide to Drugs.

8. Čakar M, Popović G. Spectrophotometric determination of Bifonazole and Clotrimazole in lotions Bicutrin® and Canesten®. Arhiv za Farmaciju. 2004; 54(1-2):1-3.

9. Mushik OV, Tkach VI, Karandeeva NI, Glukhova OI, Tsyganok LP. Electrochemical and analytical properties of solid-contact ion-selective electrodes reversible to the imidazole derivatives clotrimazole and bifonazole. Journal of Analytical Chemistry. 1998; 53(12): 1110-2.

10. Drabińska B, Dettlaff K, Ratajczak T, Kossakowski K, Chmielewski MK, Cielecka-Piontek J, Kujawski J. Structural and spectroscopic properties of isoconazole and bifonazole—experimental and theoretical studies. International Journal of Molecular Sciences. 2022 Dec 28; 24(1): 520..

11. Fromtling RA. Overview of medically important antifungal azole derivatives. Clinical Microbiology Reviews. 1988 Apr; 1(2): 187-217..

12. Ma, Miao, Jidong Wang, Xiaotong Yang, and Zhong Zhao. Determination and Simulation of the Solubility of Bifonazole in 14 Pure Solvents at 283.15–323.15 K. Journal of Chemical and Engineering Data. 2023: 68(7): 1748-1762.

13. Yarlagadda DL, Kawakami K, Samavedi S. Leveraging Molecular Interactions to Develop a Generalized Design Framework for Coamorphous Drug–Drug Mixtures Exhibiting Elevated Glass Transition Temperatures. Molecular Pharmaceutics. 2025 May 16; 22(6): 3084-96.

14. Deep, Aman, Manish Kumar, Rakesh Pahwa, Sumeet Gupta, Shailendra Bhatt, Beena Kumari, Prabhat Kumar Upadhyay, and A. Pandurangan. Formulation and in vivo pharmacodynamics studies of nanostructured lipid carriers for topical delivery of bifonazole. ACTA Pharmaceutica Sciencia. 2021; 59(1).

15. Rajendiran A, Rawat S, Singh A, Parveen A, Goutham S. Role of azole antifungal agents in seborrheic dermatitis and dandruff: a review. World Journal of Pharmaceutical Research. 2021 July;12(10):367-386.

16. Lackner TE, Clissold SP. Bifonazole: a review of its antimicrobial activity and therapeutic use in superficial mycoses. Drugs. 1989 Aug; 38(2): 204-225.

17. Drusano GL. Antimicrobial pharmacodynamics: critical interactions of'bug and drug. Nature Reviews Microbiology. 2004 Apr 1; 2(4): 289-300.

18. Andes D, Van Ogtrop M. In vivo characterization of the pharmacodynamics of flucytosine in a neutropenic murine disseminated candidiasis model. Antimicrobial Agents and Chemotherapy. 2000 Apr 1; 44(4): 938-42.

19. Osumi M, Yamada N, Okada J, Yamaguchi H, Hiratani T, Plempel M. The effect of bifonazole on the structure of Trichophyton. Arzneimittel-Forschung. 1983 Jan 1;33(10):1484-91.

20. Plempel M, Berg D, Abbink J. Antimycotic characteristics of bifonazole. 1987

21. Plempel M, Regel E, Büchel KH. Antimycotic efficacy of bifonazole in vitro and in vivo. Arzneimittel-Forschung. 1983 Jan 1; 33(4): 517-24.

22. Gujjarappa R, Kabi AK, Sravani S, Garg A, Vodnala N, Tyagi U, Kaldhi D, Velayutham R, Singh V, Gupta S, Malakar CC. Overview on biological activities of imidazole derivatives. Nanostructured Biomaterials: Basic Structures and Applications. 2022 Mar 8: 135-227.

23. Lavanya G, Sunil MM, Eswarudu MM, Eswaraiah MC, Harisudha K, Spandana BN. Analytical method validation: an updated review. International Journal of Pharmaceutical Sciences and Research. 2013 Apr 1; 4(4):1280..

24. Pranshu T, Singh RP, Vikash J. Validation: A critical parameter for quality control of pharmaceuticals. Journal of Drug Delivery and Therapeutics. 2012; 2(3): 34-40.

25. Ravisankar, P., S. Gowthami, and G. Devlala Rao. A review on analytical method development. Indian Journal of Research in Pharmacy and Biotechnology. 2014; 2(3): 1183.

26. Vidushi Y, Meenakshi B, Bharkatiya M. A review on HPLC method development and validation. Res J Life Sci, Bioinform, Pharm Chem Sci. 2017; 2(6): 178.

27. Nnachi AB. Advances in UV spectroscopy for monitoring the environment. RIJBAS. 2024; 3(1): 82.

28. Maurya RK, Kumar GP, Singh G, Malik J, Singh SP, Tiwari A. Recent developments in UV-Spectroscopy techniques for quantitative analysis in pharmaceutical dosage forms. International Journal of Pharmaceutical Science and Medicine 2024; 2(1): 21-27

29. Kryczyk-Poprawa A, Żmudzki P, Hubicka U. Determination of bifonazole and identification of its photocatalytic degradation products using UPLC-MS/MS. Biomed Chromatogr. 2017 Sep;31(9). doi: 10.1002/bmc.3955.

30. Prajapati SR, Nagar AR, Hamrapurkar PD. Analytical method development and validation of Bifonazole and It’s stability study by using sophisticated RP-HPLC method. World Journal of Pharmaceutical Research. 2023; 12(6):643-652.

31. Čudina OA, Čomor MI, Janković IA. Simultaneous determination of bifonazole and benzyl alcohol in pharmaceutical formulations by reverse-phase HPLC. Chromatographia. 2005 Apr; 61(7): 415-8.

32. Wahab SI, Zaheer Z. An RP-HPLC method developed for determination of bifonazole in pharmaceutical formulation. Bulletin of Pharmaceutical and Medical Sciences. 2013; 1(1).

33. Chiarentin L, Moura V, Pais AA, Vitorino C. AQbD Approach Applied to NIR in a Complex Topical Formulation: Bifonazole as Case Study. Pharmaceutics. 2025 Jun 26; 17(7): 835.

34. Popović G, Čakar M, Agbaba D. Determination of bifonazole in creams containing methyl-and propyl p-hydroxybenzoate by derivative spectrophotometric method. Journal of pharmaceutical and biomedical analysis. 2003 Sep 15;33(1):131-6.

35. Antypenko LM, Gladysheva SA, Korzhova AS. Development and validation of UV spectrophotometric procedure for estimation of bifonazole in bulk.2016; 4(4): 111-115.

36. Ismail NB, Narayana B, Divya K. Validated spectrophotometric methods for the determination of bifonazole in pharmaceuticals by charge transfer complexation. Journal of the Association of Arab Universities for Basic and Applied Sciences. 2016 Feb 1;19:8-14.

37. Wahab SI, Zaheer Z, Ali SA. Validated UV spectrophotometric method for estimation of bifonazole in their bulk drug and cream pharmaceutical formulation. Inventi Rapid-Pharm Analysis and Quality Assurance. 2013 Apr 19;3:1-3

38. Sonaglioni D, Capaccioli S, Tombari E. Bifonazole: a missing case of polymorphism. Journal of Thermal Analysis and Calorimetry. 2025 May; 150(9):6777-83.

Received on 17.08.2025 Revised on 24.10.2025

Accepted on 28.11.2025 Published on 10.12.2025

Available online from December 26, 2025

International Journal of Technology. 2025; 15(2):51-55.

DOI: 10.52711/2231-3915.2025.00010

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