0.002M of dipotassium hydrogen phosphate in water and the organic modifier acetonitrile was preferably chosen as appropriate mobile phase for ideal separation as no interference was found with the solvent. Several isocratic and gradient elution were tried to separate Erythromycin B and Erythromycin. Finally, the mobile phase composition of 53:47%v/v of 0.002M of dipotassium hydrogen phosphate and acetonitrile was found to be most appropriate for separation of Erythromycin B and Erythromycin with a resolution of greater than 2.
0. The sample was injected with an injection volume of 2 µl and the injector port temperature was maintained at 40°C± 2°C and the flow rate of 0.6ml/min. The column BEH C18; 50×2.
1mm; 1.7µm column was selected. The column was equilibrated by pumping the mobile phase through the column for at least 30 min prior to the injection of the drug solution. 2 ?L of the standard, sample solutions were injected into the chromatography system and measure the area of the erythromycin estolate peak. The detection of the drug peak was monitored at 210 NM. The runtime was set at 12 min. Under these optimized chromatographic conditions, the retention time obtained from the drug was 2.
69min. A typical chromatogram showing the separation of the drug is given in Figure 2. Stress degradation studyTo determine whether the developed analytical method was stability indicating, Erythromycin estolate standard solution was stressed under various conditions includesOxidative degradation:Erythromycin estolate solution was prepared in 3% hydrogen peroxide and kept in a mechanical shaker at at50°C for 1 hour to facilitate the oxidation of the drug. Acid hydrolysis:Erythromycin estolate solution was prepared in 0.01N hydrochloric acid and kept in a mechanical shaker at 50°C for 15 min Alkaline hydrolysis:Erythromycin estolate Solutions were prepared in 0.01 N sodium hydroxide and kept at roomThe temperature for I hour.
Temperature stress studies:Erythromycin 250mg capsules were exposed to dry heat (105°C) in a hot air oven for 2 h, 42min. The drug solution was prepared and subjected to analysis. Photostability studies: Erythromycin 250mg capsules were exposed to light to reach greater than 1.2 million LuxHours. The drug solution was prepared and subjected to analysisThermal/Humidity studies:Placebo, Erythromycin 250mg capsules is subjected directly at 50°C/75% RH for 7days.
The samples were analyzed.The optimized method was validated as per International Conference on Harmonization (ICH) guidelines for validation of analytical procedures. The validated parameters were system suitability, specificity, and linearity, accuracy, precision, and robustness.