title: Development, Optimization, and Validation of a Microplate Bioassay for Relative Potency Determination of Linezolid Using a Design Space Concept, and its Measurement Uncertainty.
abstract: The aim of this study was to develop, optimize, and validate a microplate bioassay for relative potency determination of linezolid in pharmaceutical samples using quality-by-design and design space approaches. In addition, a procedure is described for estimating relative potency uncertainty based on microbiological response variability. The influence of culture media composition was studied using a factorial design and a central composite design was adopted to study the influence of inoculum proportion and triphenyltetrazolium chloride in microbial growth. The microplate bioassay was optimized regarding the responses of low, medium, and high doses of linezolid, negative and positive controls, and the slope, intercept, and correlation coefficient of dose-response curves. According to optimization results, design space ranges were established using: (a) low (1.0 μg/mL), medium (2.0 μg/mL), and high (4.0 μg/mL) doses of pharmaceutical samples and linezolid chemical reference substance; (b) Staphylococcus aureus ATCC 653 in an inoculum proportion of 10%; (c) antibiotic No. 3 culture medium pH 7.0±0.1; (d) 6 h incubation at 37.0±0.1ºC; and (e) addition of 50 μL of 0.5% (w/v) triphenyltetrazolium chloride solution. The microplate bioassay was linear (r2=0.992), specific, precise (repeatability RSD=2.3% and intermediate precision RSD=4.3%), accurate (mean recovery=101.4%), and robust. The overall measurement uncertainty was reasonable considering the increased variability inherent in microbiological response. Final uncertainty was comparable with those obtained with other microbiological assays, as well as chemical methods.
authors.affiliation: Universidade de São Paulo, Faculdade de Ciências Farmacêuticas, Departamento de Farmácia, Av. Prof. Lineu Prestes, 580-Bloco 13-Cidade Universitária, São Paulo, SP, Brazil.
authors.forename: Alessandro Morais
authors.initials: AM
authors.lastname: Saviano
authors.forename: Fabiane Lacerda
authors.initials: FL
authors.lastname: Francisco
authors.forename: Celina Silva
authors.initials: CS
authors.lastname: Ostronoff
authors.forename: Felipe Rebello
authors.initials: FR
authors.lastname: Lourenço
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001681:Biological Assay; D003470:Culture Media / Q000737:chemistry; D005163:Factor Analysis, Statistical; D000069349:Linezolid / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests; D015203:Reproducibility of Results; D012107:Research Design; D013211:Staphylococcus aureus / Q000187:drug effects* / Q000254:growth & development; D013778:Tetrazolium Salts / Q000737:chemistry; D035501:Uncertainty
chemical_list: D000900:Anti-Bacterial Agents; D003470:Culture Media; D013778:Tetrazolium Salts; C009591:triphenyltetrazolium; D000069349:Linezolid