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

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 (R2 > 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.