Development and Validation of a Microplate Bioassay for Nystatin Using Analytical Quality by Design (QbD) Approach

Nystatin is an antifungal agent widely used in the treatment of candidiasis and other fungal infections. Agar diffusion official method has been used in the last decades to assess the relative potency of nystatin, however this method is laborious and time-spending. The aim of this work was to develop, optimize and validate a microplate bioassay for nystatin using an analytical Quality by Design (QbD) approach. Candida albicans (ATCC 10231) was used in microplate bioassay, due to its clinical importance. A design of experiments (DoE) was performed to establish inoculum proportion and nystatin concentration. Optimized conditions were achieved using inoculum proportion of 5%, incubation at 30 degrees C for 480 min, and nystatin concentration from 0.2 to 0.8 IU/mL. Microplate bioassay was specific, linear (R-Sq = 0.9978), accurate (mean recovery = 97%) and precise (RSD for repeatability = 6%, RSD for intermediate precision = 7%). Therefore, microplate bioassay is a useful alternative for routine analysis of pharmaceutical products containing nystatin.