Besifloxacin (BSF) is a synthetic chiral fluoroquinolone developed for the topical treatment of ophthalmic infections. The present study reports the development and validation of a microbiological assay, applying the cylinder-plate method, for determination of BSF in ophthalmic suspension. To assess this methodology, the development and validation of the method was performed for the quantification of BSF by high performance liquid chromatography (HPLC). The HPLC method showed specificity, linearity in the range of 20-80 µg mL(-1) (r=0.9998), precision, accuracy and robustness. The microbiological method is based on the inhibitory effect of BSF upon the strain of Staphylococcus epidermidis ATCC 12228 used as a test microorganism. The bioassay validation method yielded excellent results and included linearity, precision, accuracy, robustness and selectivity. The assay results were treated statistically by analysis of variance (ANOVA) and were found to be linear (r=0.9974) in the range of 0.5-2.0 µg mL(-1), precise (inter-assay: RSD=0.84), accurate (101.4%), specific and robust. The bioassay and the previously validated high performance liquid chromatographic (HPLC) method were compared using Student's t test, which indicated that there was no statistically significant difference between these two methods. These results confirm that the proposed microbiological method can be used as routine analysis for the quantitative determination of BSF in an ophthalmic suspension. A preliminary stability study during the HPLC validation was performed and demonstrated that BSF is unstable under UV conditions. The photodegradation kinetics of BSF in water showed a first-order reaction for the drug product (ophthalmic suspension) and a second-order reaction for the reference standard (RS) under UVA light. UVA degraded samples of BSF were also studied in order to determine the preliminary in vitro cytotoxicity against mononuclear cells. The results indicated that BSF does not alter the cell membrane and has been considered non-toxic to human mononuclear cells in the experimental conditions tested.

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Cefpirome is a fourth-generation cephalosporin active against a broad spectrum of gram-negative and gram-positive bacterial infections. The present work describe the development and validation of a simple, sensitive and specific agar diffusion bioassay applying cylinder-plate method for quantification of cefpirome in raw material and powder for injectable preparation. The validation method yielded good results and included linearity, precision, accuracy and specificity. The assay is based on the inhibitory effect of cefpirome upon the strain of Kocuria rizophila ATCC 9341 as the test microorganism. The result of assay were treated statistically by ANOVA and the response graphs for standard and sample solutions were linear (r = 0.9948) in the range of 0.3 – 1.2 µg mL-1, precise (intra-assay: RSD = 0.11; inter-assay: RSD = 0.18) and accurate (mean recovery value = 99.41%). A preliminary stability study of cefpirome showed that the microbiological assay is specific for the determination cefpirome in the presence of its degradation products. The proposed microbiological method allows the quantitation of cefpirome in pharmaceutical dosage form and raw material and can be used for the drug analysis in routine quality control.

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