Weighed and finely powdered not less than 20 capsules. An accurately weighed quantity of powder corresponding to about 250mg of Erythromycin base from Capsule powder (about 440mg) was transferred into the 100mL volumetric flask, around 75 ml of diluent was added and sonicate for 5 minutes to dissolve entirely,cool to room temperature and makeup the volume with diluent and mix.

0.002M of dipotassium hydrogen phosphate buffer and acetonitrile in the ratio of 53:47 v/v was selected as diluent Since the Erythromycin estolate is also soluble in Acetonitrile
Method Development and optimization
Any analytical method was not reported in the stability studies of Erythromycin estolate in a capsule formulation. Hence it was noteworthy to commence the method development using reverse phase liquid chromatography as it is commonly used and C-18 columns are also available. Different columns were used with different mobile phases during the development of UPLC method suitable for the analysis of Erythromycin estolate in a capsule formulation. 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 suitable 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.

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