Bioassays for quantitating ciprofloxacin and tobramycin in aqueous humor

Development of new therapies for bacterial keratitis requires assays of antibiotics in infected ocular tissues; however, methods for such drug quantitation can be expensive and can lack precision and sensitivity. To develop a more suitable assay for quantitating tobramycin and ciprofloxacin in the aqueous humor, sixteen bioassays were compared. Variables assessed included: the strain of bacteria used as indicator (Staphylococcus aureus ATCC 25923 versus Klebsiella pneumoniae ATCC 10031), the application of bacteria to the agar surface versus inclusion in molten agar, the volume of the agar, and the application of antibiotic on paper discs versus instillation into agar wells. The best results were obtained when the appropriate indicator organism (S. aureus for tobramycin and K. pneumoniae for ciprofloxacin) was applied to the surface of the agar, antibiotic solution was put into agar wells, and the volume of agar was 30 ml per plate. The best sensitivity for assays of tobramycin and ciprofloxacin diluted in water were 0.98 and 0.12 micrograms/ml, respectively. These sensitivities improved as much as fourfold when antibiotics were diluted in aqueous humor or normal saline. The precision of all assays tested was high as determined by regression coefficients (r > 0.90). The sensitivity and precision of these assays indicate their utility for quantitating fluoroquinolones and aminoglycosides in aqueous humor.