Bioassay Applied to Quantitative Determination of Doripenem in Powder for Injection – Method Validation and Degradation Kinetics Study

Fernanda      Fuhr; Patricia Klitzke      Paliosa; Marcia C. N.      Costa; Cassia Virginia      Garcia; Elfrides E. S.      Schapoval; Martin      Steppe; Andreas S. L.      Mendez

Abstract

The present study proposes an agar diffusion microbiological assay for quantitation of the antibiotic doripenem in powder for injection. The method was developed according to official guidelines and its stability-indicating performance was studied by stress testing in pre-defined conditions of heat and UV-C radiation. Based on the response obtained from growth inhibition of a strain of Staphylococcus epidermidis ATCC 12228, the quantitation was carried out in the range of 1.0 - 4.0 µg ml-1. The percentage of drug estimated was 99.42% (RSD=3.12%), with satisfactory intra-day and inter-day precision. In the recovery test, the method was found to be reliable and accurate in quantitation of doripenem (mean recovery = 100.30%). Through alkaline hydrolysis experiments and by comparison to HPLC, bioassay was found to be specific. In kinetics degradation experiments, where the influence of temperature (45 ºC) and UV-C radiation were evaluated, higher rate of degradation was observed for the first, with a drug remaining content of 55.74% after 12h of storage. The reactions could be described by zero-order kinetics. The proposed bioassay was statistically compared to HPLC and CE methods concerning quantitative purposes and the results indicated they are equivalent, which demonstrates that the bioassay can be routinely used for determination of antibiotic potency, besides of being a complementary tool in stability studies of drugs.

Keywords: Agar diffusion bioassay, degradation kinetics, doripenem, method validation, photostability, thermal stability.

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