Green Ecofriendly RP-HPLC Method for Estimating Bifonazole in Transethosomes: Application of Box Behnken Design

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

The analysis was carried out using a C18 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.

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.

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.

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