A stability-indicating high-performance liquid chromatographic (HPLC) method was developed and verified by conducting forced degradation studies on BDQ that included exposure to light, heat, acid, base and oxidation. The method was validated for reproducibility, system stability, linearity, specificity, ruggedness, filter suitability and working standard stability, and used to determine potency of BDQ in the two formulations on Days 0, 15 and 30. Three aliquots were withdrawn from each of the three bottles for a total of n = 9 at each time point.
The diluent was prepared with water and acetonitrile (1:1), and allowed to equilibrate at room temperature. The stock standard solution was prepared by dissolving 60 mg of BDQ fumarate bulk powder in the diluent in a 100-mL volumetric flask for a 0.5 mg/mL (as BDQ). The working standard solution was prepared by diluting the stock standard solution with diluent to a concentration of 0.02 mg/mL. The stock sample solution was prepared by volumetrically diluting 5 mL of BDQ (20 mg/mL) suspension to a concentration of 0.5 mg/mL in diluent. A 4-mL aliquot of this mixture was further diluted with the diluent to 100 mL for a concentration of 0.02 mg/mL. An aliquot of this mixture was filtered through a 0.45 μm polypropylene membrane syringe filter and the filtrate was analyzed by HPLC. Details of the HPLC method are provided inTable 3 .
Ten μL of working standard solution was injected into the chromatographic system and the area under the BDQ peak eluting at 4.5 to 5.5 min was recorded. Ten μL of the diluent was injected as a blank to ensure no interference occurred with the BDQ peak. Five consecutive injections of the working standard were made to show the reproducibility of BDQ peak areas. Ten μL of the sample solution were injected into the chromatographic system, and the area under the BDQ peak was recorded as a percentage of the theoretical concentration. The working standard solution was injected periodically, throughout the run and at the end of the run to demonstrate HPLC system stability. The BDQ peak area response agreed with the system suitability average within 2%.
The diluent was prepared with water and acetonitrile (1:1), and allowed to equilibrate at room temperature. The stock standard solution was prepared by dissolving 60 mg of BDQ fumarate bulk powder in the diluent in a 100-mL volumetric flask for a 0.5 mg/mL (as BDQ). The working standard solution was prepared by diluting the stock standard solution with diluent to a concentration of 0.02 mg/mL. The stock sample solution was prepared by volumetrically diluting 5 mL of BDQ (20 mg/mL) suspension to a concentration of 0.5 mg/mL in diluent. A 4-mL aliquot of this mixture was further diluted with the diluent to 100 mL for a concentration of 0.02 mg/mL. An aliquot of this mixture was filtered through a 0.45 μm polypropylene membrane syringe filter and the filtrate was analyzed by HPLC. Details of the HPLC method are provided in
Ten μL of working standard solution was injected into the chromatographic system and the area under the BDQ peak eluting at 4.5 to 5.5 min was recorded. Ten μL of the diluent was injected as a blank to ensure no interference occurred with the BDQ peak. Five consecutive injections of the working standard were made to show the reproducibility of BDQ peak areas. Ten μL of the sample solution were injected into the chromatographic system, and the area under the BDQ peak was recorded as a percentage of the theoretical concentration. The working standard solution was injected periodically, throughout the run and at the end of the run to demonstrate HPLC system stability. The BDQ peak area response agreed with the system suitability average within 2%.
Free full text: Click here
