Validation of an HPLC method for the detection of Erythromycin and In-Vitro study into the effect of Sodium Butyrate on the metabolism of Erythromycin in chicken hepatocyte cell cultures

Abstract

The aim of this project was to validate a HPLC method for the detection of erythromycin and to then use this validated method to show the effect of sodium butyrate on erythromycin metabolism in in-vitro chicken hepatocyte cells. The method described is a high performance liquid chromatography method for the determination of the concentration of erythromycin in tissue samples with UV detection of 260-350nm. The method involves a liquid-liquid phase extraction procedure followed by a simple phase separation step. All calibration curves had correlation coefficients >0.98. The method has a limit of detection of 0.031μM at an erythromycin concentration of 100μM, 0.035μM at 50μM and 0.033μM at 10μM erythromycin concentration. This shows that the method is sufficiently sensitive. Accuracy should be as close as possible to zero and aall samples have an accuracy of ≤4.99. At 100μM erythromycin concentration the sample recovery was within the acceptable range of +/- 20%. The validated method was used to determine if there is any correlation between the concentration of sodium butyrate utilized by the in-vitro chicken hepatocytes and the hepatocytes ability to metabolise the erythromycin. The in-vitro study showed that erythromycin degradation has occurred in all samples. Significant differences were seen between samples (P<0.05) when comparing the samples to the zero standard (no cells and no sodium butyrate). When comparing samples with cells to the same samples without cells (both had the same concentration of sodium butyrate) a significant difference only occurred in samples with an erythromycin concentration of 50μM. Samples were also compared to the sample containing cells but no sodium butyrate and P<0.05 shows a significant difference in all samples at 50μM of erythromycin. There is a significant difference in some of the samples at 100μM erythromycin but there was no significant difference in samples at 10μM of erythromycin until the concentration of sodium butyrate was increased to 10mM. Erythromycin metabolism occurred in all samples at all concentrations of sodium butyrate but it increased as the concentration of sodium butyrate increased in the samples. The results show that the HPLC method for detection of erythromycin from cell culture medium is a new and appropriate method that has been successfully validated. The chicken hepatocytes are able to metabolise the erythromycin after treatment with sodium butyrate with the highest percentage of metabolism occurring at an erythromycin concentration of 50μM. It also shows that the metabolism of erythromycin increases with increased concentration of sodium butyrate up to 10mM.