Box–Behnken Design–Based Development and Validation of RP‐HPLC Method for Determination of Related Substances in Nitrofurantoin Formulations

ABSTRACT A stable reversed‐phase high‐performance liquid chromatography method was developed and validated to quantify related substances in nitrofurantoin capsule formulations. Based on response surface methodology and experimental design, the Box–Behnken design technique improved method performance and dependability. The mobile phase‐A was formulated by mixing 0.2% formic acid solution and tetrahydrofuran in the ratio of 95:5% ( v / v ), and the mobile phase‐B was formulated by mixing methanol, acetonitrile, and tetrahydrofuran in the ratio of 50:40:10 ( v / v / v ) using gradient elution with a flow rate of 0.8 mL/min. Chromatographic separation was achieved using an Inertsil ODS‐3 C18 column (250 mm × 4.6 mm, 5 μm), with detection at 285 nm and column temperature maintained at 30°C. The method was validated according to ICH and USP <1225> guidelines. This is demonstrated by high‐level accuracy, covering an entire range from 92% to 111%. Precision (resistance ≤ 1.0%) and linearity ( R 2 > 0.999). Box–Behnken design–based RP‐HPLC technique for nitrofurantoin formulations is significant because it uses a systematic, statistically optimized approach to method development to assure robustness, accuracy, and dependability. This work provides a proven stability‐indicating approach for routine quality control and regulatory compliance to address safety concerns about nitrofurantoin degradation products and impurities.