Quantitative evaluation of besifloxacin ophthalmic suspension by HPLC, application to bioassay method and cytotoxicity studies.

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.

Phenolic composition in different genotypes of Guabiju fruits (Myrcianthes pungens) and their potential as antioxidant and antichemotactic agents.

Fruits of Myrcianthes pungens Berg. Legr. (Myrtaceae), known as guabiju, are widely consumed fresh as well as dried, processed into jam, marmalade, and juices. In this study, chemical composition and antichemotactic and antioxidant activities of fruits from a wild type (GB) and 2 genotypes, PL2 and PL1, of guabiju were investigated. Total anthocyanins for the genotypes ranged from 334 to 531 mg/100 g dry weight (dwt). Total flavonoids and polyphenols ranged from 79.8 to 154 mg/100 g and 2438 to 4613 mg/100 g (dwt), respectively. A reversed phase liquid chromatography method with photodiode array detection was used to determine chemical profiles of the main anthocyanins found in the extracts. An HPLC method for the quantification of flavonoids is proposed, providing a simple procedure with rapid sample preparation. All samples contained 5 identical anthocyanidins, distributed differently, with cyanidin as the main compound. Identified flavonoids were quercitrin, hyperoside, and isoquercitrin; their relative amounts varied among the extracts. The antioxidant activity of guabiju methanolic extract was comparable to that of Trolox, and at a test concentration of 0.25 mg/mL, GB and PL2 activities were higher than those exhibited by Trolox. Total dry extracts of guabiju exhibited greater inhibition of chemotaxis at a concentration of 4 µg/mL, except for GBH (wild-type hydrolyzed extract) which already presented high values at a concentration of 2 µg/mL. These results suggest that the consumption of this fruit, rich in polyphenols, may be beneficial to human health. Practical Application: The paper is the first attempt on the improvement of this native fruit, since it is widely consumed regularly as part of the South American diet. The content of phenolic compounds demonstrates that consumption of guabiju would be beneficial to human health. Differences among samples, originating from open pollination of plants growing on the same site, lead to the conclusion that improvements can be made in the chemical composition and beneficial activity of guabiju fruits by simply selecting genotypes for these characteristics among open-pollinated seedlings.