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

Abstract

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.