METHOD DEVELOPMENT IN LIQUID-CHROMATOGRAPHY WITH A CHARGED CYCLODEXTRIN ADDITIVE FOR CHIRAL RESOLUTION OF RAC-AMLODIPINE UTILIZING A CENTRAL COMPOSITE DESIGN

A negatively charged derivative of beta-cyclodextrin, sulphobutyl ethe r-beta-cyclodextrin (SBE-beta-CD), was examined as a chiral mobile pha se additive in reversed-phase highperformance liquid chromatography fo r the enantiomeric resolution of the calcium channel blocker rac-amlod ipine. Theoretical and practical aspects are discussed for setting up a central composite design applicable to any analytical method. These include the correct location of factor points for maintaining orthogon ality within the design and the augmentation of centre-point experimen ts to allow a larger factor space by increasing the distance of axial star points. Optimised separation was achieved using a reverse-phase c olumn with eluent comprising: acetonitrile (ACN)-potassium dihydrogen phosphate (pH 3.93) containing 2.66 mM SBE-beta-CD (26.5:73.5% v/v) at a now rate of 1.0 ml/min. This yielded a Kaiser peak separation index , P-i = 0.96, at t(R2) = 52 min with satisfactory reproducibility, rel ative standard deviation values: t(R1), 0.39%; t(R2), 0.47% (n = 5). T hese experimental results were in excellent agreement with those predi cted by the SAS software package for a chromatographic response functi on model. Multiple regression analysis in four dimensions, with three response models based on R(s), P-i, and a function of P-i, produced re sponse surfaces which revealed zones of optimum robustness and illustr ated the interactions involved between the key chromatographic factors . Putative proposals for a mechanism involving the interaction of each of the positively charged enantiomers with the negatively charged cyc lodextrin are also discussed. These examine the possibility of ion-pai ring and inclusion phenomena to account for the excellent resolution o bserved. (C) 1996 Wiley-Liss, Inc.