QBD-based RP-HPLC Method Development and Validation for Thioacetazone and Isoniazide in Pharmaceutical Dosage Forms as per ICH Guidelines

Abstract

A novel and comprehensive High-Performance Liquid Chromatography (HPLC) method was developed and optimized for the simultaneous estimation of Isoniazid and Thioacetazone in pharmaceutical formulations. Method optimization was achieved using a Box-Behnken design coupled with response surface methodology, evaluating critical chromatographic parameters such as flow rate, buffer ratio, and buffer pH. The optimized conditions employed a Platisil C18 column (150×3.0 mm, 3μm) with a mobile phase of 0.1% orthophosphoric acid buffer (pH 3.5) and methanol (40:60 v/v), at a flow rate of 0.8 mL/min and detection at 258 nm. System suitability tests confirmed acceptable resolution (≥2), tailing factor (≤2), and theoretical plate count (≥2000), ensuring reliable separation and peak symmetry for both analytes. The method was rigorously validated in accordance with ICH guidelines, demonstrating excellent linearity for Isoniazid (10–50 µg/mL, R² = 0.9998) and Thioacetazone (5–25 µg/mL, R² = 0.9995). Precision studies yielded %RSD values below 2%, confirming reproducibility under varied conditions. Sensitivity was established through low LOD and LOQ values, while recovery studies at multiple concentration levels (50%, 100%, 150%) showed accuracy within 98–102%. Robustness was maintained despite deliberate variations in chromatographic parameters. Overall, the validated method is accurate, precise, sensitive, and robust, making it highly suitable for routine quality control of Isoniazid and Thioacetazone in pharmaceutical dosage forms.