title: Development, optimization and validation of a rapid colorimetric microplate bioassay for neomycin sulfate in pharmaceutical drug products.
abstract: Microbiological assays have been used to evaluate antimicrobial activity since the discovery of the first antibiotics. Despite their limitations, microbiological assays are widely employed to determine antibiotic potency of pharmaceutical dosage forms, since they provide a measure of biological activity. The aim of this work is to develop, optimize and validate a rapid colorimetric microplate bioassay for the potency of neomycin in pharmaceutical drug products. Factorial and response surface methodologies were used in the development and optimization of the choice of microorganism, culture medium composition, amount of inoculum, triphenyltetrazolium chloride (TTC) concentration and neomycin concentration. The optimized bioassay method was validated by the assessment of linearity (range 3.0 to 5.0μg/mL, r=0.998 and 0.994 for standard and sample curves, respectively), precision (relative standard deviation (RSD) of 2.8% and 4.0 for repeatability and intermediate precision, respectively), accuracy (mean recovery=100.2%) and robustness. Statistical analysis showed equivalency between agar diffusion microbiological assay and rapid colorimetric microplate bioassay. In addition, microplate bioassay had advantages concerning the sensitivity of response, time of incubation, and amount of culture medium and solutions required.
authors.affiliation: Departamento de Farmácia, Faculdade de Ciências Farmacêutica, Universidade de São Paulo, Av. Prof. Lineu Prestes, 580-B13, Cidade Universitária, CEP 05508-000, São Paulo/SP, Brazil.
authors.forename: Fabiane Lacerda
authors.initials: FL
authors.lastname: Francisco
authors.affiliation: Departamento de Farmácia, Faculdade de Ciências Farmacêutica, Universidade de São Paulo, Av. Prof. Lineu Prestes, 580-B13, Cidade Universitária, CEP 05508-000, São Paulo/SP, Brazil.
authors.forename: Alessandro Morais
authors.initials: AM
authors.lastname: Saviano
authors.affiliation: Departamento de Farmácia, Faculdade de Ciências Farmacêutica, Universidade de São Paulo, Av. Prof. Lineu Prestes, 580-B13, Cidade Universitária, CEP 05508-000, São Paulo/SP, Brazil.
authors.forename: Terezinha de Jesus Andreoli
authors.initials: Tde J
authors.lastname: Pinto
authors.affiliation: Departamento de Farmácia, Faculdade de Ciências Farmacêutica, Universidade de São Paulo, Av. Prof. Lineu Prestes, 580-B13, Cidade Universitária, CEP 05508-000, São Paulo/SP, Brazil. Electronic address: feliperl@usp.br.
authors.forename: Felipe Rebello
authors.initials: FR
authors.lastname: Lourenço
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D003124:Colorimetry / Q000379:methods* / Q000592:standards; D057166:High-Throughput Screening Assays; D008826:Microbial Sensitivity Tests / Q000379:methods* / Q000592:standards; D009355:Neomycin / Q000737:chemistry / Q000494:pharmacology*; D011500:Protein Synthesis Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013778:Tetrazolium Salts
chemical_list: D000900:Anti-Bacterial Agents; D011500:Protein Synthesis Inhibitors; D013778:Tetrazolium Salts; C009591:triphenyltetrazolium; D009355:Neomycin
keywords: Aminoglycosides; Bioassay; Factorial design; Response surface methodology; Tetrazolium salt