Quality by Design Approach for the Development of a Rapid Stability-Indicating Spectrophotometric Method for the Estimation of Amphotericin B in Bulk and Formulations

This study presents the first application of an analytical quality by design approach using face-centered central composite design to optimize an ultraviolet-visible spectrophotometric method for the estimation of amphotericin B (Amp B). The optimal critical method parameters established using the desirability approach indicated a detection wavelength of 385 nm in a solvent of 0.1 N HCl (pH 1.2), which significantly improved (p < 0.05) the extinction coefficient while minimizing aggregation. The method, when validated as per ICH Q2(R2), displayed high linearity (R² = 0.997) in the concentration range from 6–36 µg/mL. The method was found to display high accuracy, with recoveries ranging from 95.41 ± 0.02% to 97.77 ± 0.13%, and high precision (relative standard deviation < 2%) for intra- and inter-day studies. The limit of detection and limit of quantification were found to be 4.75 and 14.40 µg/mL, respectively. The method was found to be highly specific for Amp B as it ruled out the possibility of any interference by excipients, and was quite robust and reproducible across analysts and conditions. The method was able to successfully assess the improved thermal stability, reduced aggregation, and therefore the decreased irritation potential of the nano-vesicular formulation compared to the marketed product. The validated method is simple, rapid, cost-effective, and accurate for routine quantification of Amp B, and it offers a valuable in vitro alternative tool for assessing ocular irritancy potential.