The Erythromycin is one of the most frequently used macrolid type antibiotic in the veterinary practice, which metabolises intensively in the liver. In our in vitro experiments we investigated the changes of Erythromycin concentrations in different liver cell culture samples using different amounts of Sodium butyrate. In our in vivo experiments we determined the Erythromycin concentrations in chicken plasma samples to define the pharmacokinetic parameters.
The aim of these investigations was to develop a selective, sensitive and routinely used HPLC method, wich is usable to determine the Erythromycin concentrations in liver cell culture and chicken plasma samples.
We had to use a derivatization procedure – the reagent was fluorenylmethyl chloroformiate – with fluorescence detection for these investigations. During the sample preparation we used liquid-liquid extraction for recovering Erythromycin from liver cell culture and chicken plasma samples. We applied a reversed phase liquid chromatographic system for the measurment of Erythromycin using RP18 type stationary phase and fluorescence detection.
During the development of the HPLC method we optimized the sample preparation – the ratio of the organic and the water solution volume – , the derivatization procedure – the reaction time and temperature -, and the parameters of the HPLC measurment – type of the RP18 column, wavelength of detection, composition of the mobile phase.
Before using this HPLC method routinely, we validated it on the basis of some parameters: specificity, linearity, recovery, reproducibilty and limit of quatification.