QBD-Enabled HPLC Method Development and Validation for the Simultaneous Estimation of Benserazide and Levodopa in Pharmaceutical Dosage Forms

This study presents the development and validation of a high-performance liquid chromatography (HPLC) method for the simultaneous estimation of Benserazide and Levodopa in pharmaceutical formulations. A Box-Behnken response surface design was employed to systematically optimize key chromatographic parameters, including mobile phase composition, buffer pH, and flow rate. Statistical analysis revealed that flow rate significantly influenced resolution, while buffer pH and organic phase ratio affected peak tailing. The optimized method utilized a SPURCIL C18 column (250×4.6 mm, 5 µm) with a mobile phase of ammonium acetate buffer (pH 5.0) and methanol in a 70:30 ratio, at a flow rate of 1.0 ml/min and UV detection at 220 nm. The system suitability parameters resolution >2, tailing factor 2000 confirmed the method’s efficiency and reliability. Validation was conducted in accordance with ICH Q2(R1) guidelines, demonstrating excellent accuracy, precision, linearity, sensitivity, and robustness. Recovery rates for Benserazide and Levodopa were within the acceptable range of 98–102%, with %RSD values below 2% for both repeatability and intermediate precision. Linearity was observed over the ranges of 10–50 µg/ml for Benserazide and 40–200 µg/ml for Levodopa, with correlation coefficients of 0.9997 and 0.9998, respectively. The method exhibited low limits of detection and quantification, confirming its sensitivity. Robustness testing showed consistent performance under minor variations in chromatographic conditions. Overall, the developed HPLC method is reliable, reproducible, and suitable for routine quality control of Benserazide and Levodopa in combined pharmaceutical dosage forms.