Integrating Box-Behnken and Plackett-Burman designs for the robust impurity profiling of levodopa and carbidopa tablets.

Pharmacopeial methods are essential for pharmaceutical quality control, yet impurity profiling of levodopa/carbidopa fixed-dose combination tablets remains challenging because both actives are chemically labile and several structurally related impurities exhibit closely spaced retention behavior. During verification of the compendial procedure, a process-related impurity (1 R,3S)-6,7-Dihydroxy-1-methyl-1,2,3,4-tetrahydroisoquinoline-1,3-dicarboxylic acid (IP2) was found to co-elute with levodopa, compromising selective quantification and peak-purity assessment; moreover, small univariate adjustments (e.g., mobile-phase composition and pH) improved the levodopa/IP2 separation but created new critical overlaps among carbidopa, catechol, and levodopa related compound C, indicating limited robustness. To systematically resolve these deficiencies, a Design of Experiment (DoE) strategy was applied using a Box-Behnken response surface design to model and optimize three critical method parameters-ethanol content 11.5 - 31.5%, v/v, mobile-phase pH 2.20 - 2.50 and column temperature 26°C - 32°C with chromatographic resolutions between the most critical pairs as the primary responses; quadratic (second-order) regression models were generated and used with desirability-based numerical optimization to define operating conditions that maximize separation while maintaining suitable chromatographic performance. Under the optimized conditions mobile-phase pH, column (temperature 26°C, flow rate 0.9 mL/min, UV 280 nm, gradient runtime 75min), baseline separation of the critical impurity pairs was achieved, and system suitability confirmed adequate selectivity, including a resolution between levodopa related compound C and carbidopa in the System Suitability Test (SST) solution. Method robustness was further assessed using a Plackett-Burman design to identify parameters most sensitive to deliberate variation, supporting reliable performance under routine analytical conditions and across potential raw-material/formulation variability.