All the reagents were of LC grade except stated or else Milli-Q-water was used throughout the research. Working standard of Erythromycin estolate was obtained 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.
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.
BEH C18; 50×2.1 mm, 1.7?m column was used for separation. Chromatographic separation was achieved using a mobile phase consisting 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 0 C and the injection volume was 2 ?L.Solution preparations
Preparation of 0.002M of dipotassium hydrogen phosphate buffer
The buffer solution was prepared by dissolving 0.348 gm of dipotassium hydrogen phosphate in DID water.
Preparation of Mobile Phase
530 ml of 0.002M of dipotassium 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 filters under vacuum.
Preparation of stock solution
Exactly weighed 400mg of Erythromycin estolate operational standard into a 50mL volumetric flask, 35 ml of diluent was added and sonicate for 5 min to dissolve completely. Cool to room temperature and make up the volume with diluent and mix
Preparation of standard solution
Precisely weighed 80mg of Erythromycin estolate running 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 and make up to volume with diluent and mix.