Development and Validation of Quality by Design‐Based HPLC Method for Impurity Profiling of Alprazolam

ABSTRACT A quality by design (QbD)‐aided stability‐indicating method was developed for the simultaneous quantification of alprazolam and its four impurities using HPLC. The method was systematically optimized, followed by the establishment of a “method operable design region (MODR).” Critical parameters and ICH (Q14) emphasize the adherence to a “low‐risk approach” as per “USP 1210” and compliance with it. Chromatographic separation was achieved using a Poroshell 120 phenyl hexyl column (100 mm × 4.6 mm, 2.7 µm) and measured at 254 nm. The composition of Mobile Phase A was 44:56 vol/vol buffer (pH 3.5) and methanol, whereas Mobile Phase B had 5:95 vol/vol buffer (pH 3.5) and methanol in gradient elution mode (Time/%A—0/98, 6/98, 14/1, 16/1, 18/98, 22/98) at a flow rate of 1.0 mL/min and an injection volume of 10 µL. Forced degradation studies were conducted, where no significant degradation product peaks were observed under any degradation conditions. The developed method was validated as per ICH (Q2(R2)) guidelines. The method exhibited good linearity, as correlation coefficient ( r 2 ) values for all analytes range from 0.976 to 0.986. %RSD values in precision studies were within the acceptance criteria (<15%). The %RSD values for six replicate injections at the LOQ level for these impurities range from 0.9% to 4.3%. In the combined study of validation of accuracy and precision as per USP 〈1210〉 guidelines, the computed interval bounds for all impurities were within the specified limits of 95%–105%, ensuring the method's robustness. The recovery values in the accuracy study ranged from 98.9% to 101.8% across all impurities. The tolerance interval analysis, performed at a 95% confidence level with 5% alpha, confirmed that the computed intervals for all impurities fell within the specification limits. Collectively, these results demonstrate that the method is a low‐risk approach, and the developed method is suitable for routine quality control in pharmaceutical manufacturing.