Optimization of RPLC Method for Separation of Some Acetylcholinesterase Inhibitors by using Central Composite Design

Ramazan Uysal, Yaşar Doğan Daldal, Zehra Üstün, Ebru Çubuk Demiralay

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Acetylcholinesterase inhibitors, optimization, pKa estimation, central composite design (CCD), derringer’s desirability function


This study is described for reversed phase liquid chromatographic separation of acetylcholinesterase inhibitors (donepezil, galantamine and rivastigmine). In the first stage of method development, acetonitrile concentration, pH of mobile phase and column temperature were investigated using central composite design (CCD). Afterwards, the optimal conditions were found employing central composite design and Derringer's desirability function. Effect of these variables on the output responses such as retention factors, resolutions (Rs) and retention time (tR) were evaluated. The separation was applied by using X Terra C18 column (250 × 4.6 mm ID, 5 μm). The optimum assay conditions were: acetonitrile-water binary mixture (45:55, v/v) and pH 9.5 as the mobile phase and at column temperature 33 oC. Total chromatographic analysis time per sample was approximately 12.5 min. The method showed good agreement between the experimental data and predictive value throughout the studied parameter space. By using equations obtained CCD, protonation constant values (pKa) of donepezil, galantamine and rivastigmine were also predicted.