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All the reagents were of LC grade except stated or else Milli-Q-water was used throughout the research. operational standard of Erythromycin estolate was procured from M/S ADCOCK INGRAM, RD&I, Sabax Road, Aero ton, Johannesburg, 2013, South Africa. Dipotassium hydrogen phosphate and Acetonitrile were procured from Merck, Mumbai
Apparatus
The UPLC system consisted of high pressure pump, Photodiode array detector and 10 ?L capacity injector loops. The column used was BEH C18; 50 x 2,1mm; 1.7µm column. The output signal was monitored and processed using Empower software.
Chromatographic conditions
BEH C18; 50×2.1 mm, 1.7?m column was used for separation. Chromatographic separation was attained using a mobile phase comprising of 0.002M di-potassium hydrogen phosphate buffer and Acetonitrile 53:47v/v. The flow rate of the mobile phase was 0.6 ml/min with detection at 210 NM. The column temperature was kept at 40 0C and the injection volume was 2 ?L.Solution preparations
Preparation of 0.002M of di-potassium hydrogen phosphate buffer
Buffer solution was prepared by dissolving 0.348 gm of di-potassium hydrogen phosphate in DID water.
Preparation of Mobile Phase
530 mL of 0.002M of di-potassium hydrogen phosphate was mixed with 470 mL of acetonitrile. The solution was degassed in an ultrasonic water bath for 5 minutes and filtered through 0.45 ?m filter under vacuum.
Preparation of stock solution
Exactely weighed 400mg of Erythromycin estolate running standard into a 50mL volumetric flask; 35 ml of diluent was added and sonicate for 5 min to dissolve completely. Cool to room temperature; make up the volume with diluent and mix
Preparation of standard solution
Precisely weighed 80mg of Erythromycin estolate operational standard into a 20ml volumetric flask. approximately 15 ml of diluent was added and sonicate for 5 min to dissolve completely; cool to room temperature, make up to volume with diluent and mix.
Preparation of Sample solution:

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

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Diluent
0.002M of di-potassium 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
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.
Stress degradation study
To determine the developed analytical method was stability indicating, Erythromycin estolate standard solution was stressed under various conditions includes
Oxidative 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 Solution was prepared in 0.01 N sodium hydroxide and kept at room temperature for 1hour
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
Photo stability studies:
Erythromycin 250mg capsules were exposed to light to reach greater than 1.2 million Lux Hours. The drug solution was prepared and subjected to analysis
.
Thermal/Humidity studies:
Placebo, Erythromycin 250mg capsules is subjected directly at 50°C/75% RH for 7days. The samples were analyzed.
Method validation
The optimized method was validated as per International Conference on Harmonization (ICH) guidelines. The validated parameters were system suitability, specificity, and linearity, accuracy, precision, and robustness

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