Validation of Symmetrical Two-Dose Parallel Line Assay Model for Nystatin Potency Determination in Pharmaceutical Product

Objectives: The microbiological antibiotic assay is an important quality control test for the determination of the potency of the antimicrobials whose activity cannot be estimated by the conventional analysis methods (e.g., chemical and HPLC) as raw materials and in the final medicinal dosage form such as tablets, capsules, gels, ointments, creams, powders for reconstitution or any other pharmaceutical preparation. However, biological assay tests must be validated to ensure that the determined potency of the sample is statistically valid. The present study aimed to validate the potency of nystatin in topical pharmaceutical preparations using agar diffusion methodology. Methods: The adopted assay module was 2 x 2 Parallel Line Model (PLM) in 8 x 8 large rectangular antibiotic assay plates. The assay model comprises a Latin square design with two treatment (high and low) doses for standard and test preparations. The investigated module is symmetrical with an equal number of replicates in each dose level for both standard and test. The validation methodology parameters cover the evaluation of selectivity, linearity, precision and accuracy (at 50%, 100% and 150%) parameters, in addition to a robustness that includes variation in pH of the antibiotic medium, incubation time and temperature outside the normal range of the regular parameters for the nystatin assay. The selected two-dose concentrations were determined based on the containment within the linear range from the calibration curve. The acceptance criteria for the validation study were established for linearity (r2 ≥ 0.98), precision (intraassay variation Relative Standard Deviation (RSD) ≤ 11%; inter-assay variation RSD ≤ 10%), accuracy, and specificity tests. Antibiotic plates are Incubated at temperatures ranging between 32 and 35oC for a period of 24 hours. Results: The assays were calculated statistically by the linear parallel model and regression analysis and verified using analysis of variance (ANOVA). The calibration curve showed r2 ≈ 0.99 for the individual and the pooled linearity determination. For accuracy, precision and robustness, none of the RSDs for the individual tests within groups exceeded 7% and the pooled or combined values did not exceed 4%. All validation criteria were met. Conclusions: The validation of PLM for nystatin in large 8 x 8 rectangular plates showed an acceptable level of the evaluated parameters and the assay design can be used for the routine quality control (QC) analysis of nystatin activity in medicinal products. Due to the relatively lower number of treatments per preparation on average, an optimum throughput could be achieved with minimal efforts and resources using a 2 x 2 PLM scheme - if compared with 3 x 3 and 5 + 1 designs - with satisfactorily accepted validity for antifungal potency determination of nystatin. Another interesting design that could be investigated in the future is of 3 + 1 type.