Development of an HPLC Method for the Chemical Quality and Cleaning Validation of Cephalosporin Products

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A new cost effective HPLC method of analysis has been developed to assay separately and simultaneously these compounds; cefuroxime (injection), ceftriaxone (injection) and cefepime (injection). 80% Phosphate buffer and 20% methanol as mobile phase with C18 reverse phase column; Zorbax ODS (C18) - 4.6mm x 25cm was used. The detector used was UV-Vis with a wavelength of 260nm. The flow rate and the injector volume were 1.5ml/min and 20μl respectively. Diluents was methanol ( 80% phosphate buffer in the ratio 1:4). The results were analyzed statistically using ANOVA, T-test and F-Test. At 95% confidence level, the method is robust. The average retention time for Cefepime, Ceftriaxone and Cefepime were 4.78min ± 0.016, 5.87 ± 0.039 and 15.39 ± 0.026 respectively. The percentage recovery for respective cephalosporin injections in the assay performed at the three different concentration levels were greater than 98% and their relative standard deviation were less than 2% and within acceptable limits of accuracy. The linear regression analysis results showed a correlation coefficient of about 0.99 for each calibration plot. This gave an indication of good linear relationship between instrument response and analytes concentration in the range 30μg/ml to 120μg/ml for each cephalosporin analytes. The limit of Quantification (LOQ) and limit of Detection (LOD) based on instrument response were as follows Cefepime: LOQ – 0.003933mg/ml and LOD – 0.001180mg/ml; Ceftriaxone: LOQ - 0.001595mg/ml and LOD - 0.0004786mg/ml. and Cefuroxime: LOQ – 0.0007239mg/ml and LOD – 0.0002172mg/ml. The HPLC method of analysis can effectively assay separately and simultaneously cefuroxime, ceftriaxone and cefepime products and hence had performance of the developed method as a validation procedure for cleaning of equipments after manufacturing the cephalosporin products assured at confidence level of 95%.
A thesis submitted to the Department of Pharmaceutical Chemistry Faculty of Pharmacy and Pharmaceutical sciences College of Health sciences Kwame Nkrumah University of science and Technology, Kumasi in Partial Fulfillment of the requirements for the Degree of Master of science: Pharmaceutical Analysis and Quality Control