EXPERIMENTAL DESIGN-BASED OPTIMIZATION APPROACHES IN UPLC METHOD DEVELOPMENT: SIMULTANEOUS QUANTITATIVE ESTIMATION OF SULFADIAZINE AND TRIMETHOPRIM IN A COMBINED VETERINARY FORMULATION

Abstract

This study reports a comparative application of three different experimental designs to develop ultra-performance liquid chromatography (UPLC) methods for simultaneous quantification of sulfadiazine and trimethoprim in veterinary tablets. The effects of column temperature, buffer ratio, and flow rate on chromatographic response were systematically evaluated by response surface methodology using full factorial design (FFD), Box–Behnken design (BBD), and central composite design (CCD). Full quadratic models were constructed and evaluated for each design, using statistical parameters and studying the response surface plots. Then, optimal conditions providing efficient peak resolution in shortest runtime were calculated, and experimentally confirmed. FFD and BBD produced nearly identical optimal conditions, while CCD yielded conditions with shorter run time and sufficient resolution. The optimal conditions were determined from two experimental design methods: the first set from FFD and BBD (collectively referred to as FFD/BBD-UPLC), and the second set from CCD (referred to as CCD-UPLC). Both sets of conditions underwent system suitability tests and were validated for linearity, limits of detection and quantification, precision, accuracy, and specificity. Recoveries for sulfadiazine and trimethoprim were within 99.3-100.5% with relative standard deviations less than 1.8%. The developed methods were successfully applied to commercial veterinary tablet analysis, with statistically comparable assay results in accordance with the label claims for both drugs.