Development and Validation of Ultra High Performance Liquid Chromatographic (UHPLC) Method for the Determination of Roxithromycin in the Broiler Plasma

Abstract

Aims: The present study was designed to develop and validate the UHPLC method for quantitative determination of roxithromycin, a macrolide antimicrobial drug, in broiler plasma for the application of pharmacokinetic studies. Methodology: UHPLC apparatus comprised of ultraviolet (UV) detector was used in the present study. Chromatographic separation was performed by using reverse phase C18 column. Mobile phase was combination of buffer and 55 acetonitrile in the ratio of 55: 45. Buffer part used was 0.1% trifluoroacetic acid (v/v) having pH of 2.1. Erythromycin was used as an internal standard. Isocratic elution mode was employed with flow rate of 1 ml/min and effluents were monitored at wavelength of 220 nm. Liquid-liquid extraction using ice-cold acetonitrile was performed to extract roxithromycin from plasma samples. The data integration was performed using Chromeleon™ version 6.8 software. Results: The linear calibration curve with a mean correlation coefficient (R2) value of 0.9999 was observed for concentrations ranging from 0.20 to 12.80 µg/ml. At any concentration, accuracy was not found to be less than 90%. The mean extraction recovery (n=5) for concentrations of 0.40 µg/ml was 81.36%. The calculated intraday and interday C.V. % was not more than 7.70% and 9.42%, respectively, at any concentration studied. The specificity of the analysis was reflected by the narrow range of retention time ranging between 6.983 to 7.178 minutes. LOD and LOQ of the method under investigation were calculated as 0.131 and 0.398 µg/ml, respectively. Conclusion: A reliable, reproducible, accurate, precise, specific and sensitive method for analysis of roxithromycin in broiler plasma was developed and validated for application in the pharmacokinetic study of the roxithromycin.