Costa Márcia C N, Barden Amanda T, Andrade Juliana M M, Oppe Tércio P, Schapoval Elfrides E S
Programa de Pós-Graduação em Ciências Farmacêuticas, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul., Av. Ipiranga 2752, Lab. 402, F: 55-51-3308-5214 Porto Alegre/RS, CEP 90610-000, Brazil.
Programa de Pós-Graduação em Ciências Farmacêuticas, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul., Av. Ipiranga 2752, Lab. 402, F: 55-51-3308-5214 Porto Alegre/RS, CEP 90610-000, Brazil.
Talanta. 2014 Feb;119:367-74. doi: 10.1016/j.talanta.2013.10.051. Epub 2013 Nov 20.
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.
贝西沙星（BSF）是一种合成的手性氟喹诺酮类药物，用于眼部感染的局部治疗。本研究报告了一种采用圆筒平板法测定眼用混悬液中BSF的微生物学测定方法的建立与验证。为评估该方法，采用高效液相色谱法（HPLC）对BSF进行定量分析，建立并验证了该方法。HPLC法具有特异性、在20 - 80 µg mL(-1)范围内呈线性（r = 0.9998）、精密度、准确度和稳健性。微生物学方法基于BSF对用作测试微生物的表皮葡萄球菌ATCC 12228菌株的抑制作用。生物测定验证方法取得了优异的结果，包括线性、精密度、准确度、稳健性和选择性。通过方差分析（ANOVA）对测定结果进行统计学处理，发现在0.5 - 2.0 µg mL(-1)范围内呈线性（r = 0.9974）、精密度良好（批间精密度：RSD = 0.84）、准确度为101.4%、具有特异性和稳健性。使用学生t检验比较了生物测定法和先前验证的高效液相色谱（HPLC）法，结果表明这两种方法之间没有统计学上的显著差异。这些结果证实，所提出的微生物学方法可用于眼用混悬液中BSF定量测定的常规分析。在HPLC验证期间进行了初步稳定性研究，结果表明BSF在紫外条件下不稳定。在UVA光下，BSF在水中的光降解动力学对药品（眼用混悬液）呈一级反应，对参考标准品（RS）呈二级反应。还对UVA降解的BSF样品进行了研究，以确定其对单核细胞的初步体外细胞毒性。结果表明，在测试的实验条件下，BSF不会改变细胞膜，被认为对人单核细胞无毒。
