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.ApparatusThe 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 conditionsBEH 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 preparationsPreparation of 0.002M of dipotassium hydrogen phosphate bufferThe buffer solution was prepared by dissolving 0.348 gm of dipotassium hydrogen phosphate in DI water. Preparation of Mobile Phase530 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 solutionExactly 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 mixPreparation of standard solutionPrecisely 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 makeup to volume with diluent and mix.Preparation of Sample solution: 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 make up the volume with diluent and mix.
Diluent: 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