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

Abstract

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.

Besifloxacin: A Critical Review of Its Characteristics, Properties, and Analytical Methods

“…[19] Besifloxacin has been approved in the United States by the Food and Drug Administration in May 2009 for the treatment of bacterial conjunctivitis in adults and children over one year of age. [3,[20][21][22] This fluoroquinolone started to be used in Brazil in 2011, when it was approved by the National Health Surveillance Agency, for the same purpose. [21] This drug is marketed as besifloxacin hydrochloride (Chemical Abstract Service [CAS]: 405165-61-9) by the US pharmaceutical company Bausch & Lomb, under the trade name Besivance TM , in the pharmaceutical form of 0.6% ophthalmic suspension.…”

“…[3,[20][21][22] This fluoroquinolone started to be used in Brazil in 2011, when it was approved by the National Health Surveillance Agency, for the same purpose. [21] This drug is marketed as besifloxacin hydrochloride (Chemical Abstract Service [CAS]: 405165-61-9) by the US pharmaceutical company Bausch & Lomb, under the trade name Besivance TM , in the pharmaceutical form of 0.6% ophthalmic suspension. This pharmaceutical form was developed with drug delivery technology known as DuraSite TM (InSite Vision, Inc., Alameda, USA), which makes the formulation more viscous than other suspensions and ophthalmic solutions, allowing the antimicrobial agent to remain for longer time in contact with the ocular surface.…”

“…This pharmaceutical form was developed with drug delivery technology known as DuraSite TM (InSite Vision, Inc., Alameda, USA), which makes the formulation more viscous than other suspensions and ophthalmic solutions, allowing the antimicrobial agent to remain for longer time in contact with the ocular surface. [21,23] Pharmaceutical and clinical aspects Chemical structure [24] Its chemical formula is C 19 H 21 ClFN 3 O 3 and the molecular weight is 430.40 g/mol. [22,25] It is a synthetic chiral fluoroquinolone marketed in a pure enantiomeric form.…”

Quantitative fingerprinting based on the limited‐ratio quantified fingerprint method for an overall quality consistency assessment and antioxidant activity determination of Lianqiao Baidu pills using HPLC with a diode array detector combined with chemometric methods

“…Considering the content and the distribution ratio of each component in the sample, the projection of ⃖⃖ ⃗ on ⃖⃖ ⃗ is defined as the projection content similarity (C), according to Eq. (7). In theory, S F , C and P can be utilized to compute the macro-quantitative similarity (P m ) based on Eq.…”

Uv-HPLC Method Development and Validation for Quantification of Besifloxacin Hydrochloride

“…The purpose of this proposed work was to develop and validate the method using ultraviolet-high performance liquid chromatographic (UV-HPLC) for quantification of besifloxacin hydrochloride. In literature, other analytical methods described for the quantitative analysis of besifloxacin hydrochloride but no report found using UV-HPLC method [3][4][5][6][7][8][9]. The newly developed method was a unique advantage over the other available methods, as it is simple, accurate, precise, and specific for quantitative determination of besifloxacin hydrochloride in pharmaceutical dosage form.…”