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Method development in liquid chromatography with a charged cyclodextrin additive for chiral resolution ofrac-amlodipine utilising a central composite design

1996; Wiley; Volume: 8; Issue: 7 Linguagem: Inglês

10.1002/(sici)1520-636x(1996)8

1520-636X

Paul K. Owens, Anthony F. Fell, Michael W. Coleman, John C. Berridge,

Microfluidic and Capillary Electrophoresis Applications

A negatively charged derivative of β-cyclodextrin, sulphobutyl ether-β-cyclodextrin (SBE-β-CD), was examined as a chiral mobile phase additive in reversed-phase high-performance liquid chromatography for the enantiomeric resolution of the calcium channel blocker rac-amlodipine. 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 orthogonality within the design and the augmentation of centrepoint experiments to allow a larger factor space by increasing the distance of axial star points. Optimised separation was achieved using a reverse-phase column with eluent comprising: acetonitrile (ACN)—potassium dihydrogen phosphate (pH 3.93) containing 2.66 mM SBE-bgr;-CD (26.5:73.5% v/v) at a flow rate of 1.0 ml/min. This yielded a Kaiser peak separation index, Pi = 0.96, at tR2 = 52 min with satisfactory reproducibility, relative standard deviation values: tR1, 0.39%; tR2, 0.47% (n = 5). These experimental results were in excellent agreement with those predicted by the SAS software package for a chromatographic response function model. Multiple regression analysis in four dimensions, with three response models based on Rs, Pi, and a function of Pi, produced response surfaces which revealed zones of optimum robustness and illustrated 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 cyclodextrin are also discussed. These examine the possibility of ion-pairing and inclusion phenomena to account for the excellent resolution observed. © 1996 Wiley-Liss, Inc.