title: Method development in liquid chromatography with a charged cyclodextrin additive for chiral resolution of rac-amlodipine utilising a central composite design.
abstract: A negatively charged derivative of beta-cyclodextrin, sulphobutyl ether-beta-cyclodextrin (SBE-beta-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-beta-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.
authors.affiliation: Pharmaceutical Analysis Research Unit, Pharmaceutical Chemistry, School of Pharmacy, University of Bradford, United Kingdom.
authors.forename: P K
authors.initials: PK
authors.lastname: Owens
authors.forename: A F
authors.initials: AF
authors.lastname: Fell
authors.forename: M W
authors.initials: MW
authors.lastname: Coleman
authors.forename: J C
authors.initials: JC
authors.lastname: Berridge
mesh_terms: D017311:Amlodipine / Q000737:chemistry* / Q000302:isolation & purification*; D002121:Calcium Channel Blockers / Q000737:chemistry* / Q000302:isolation & purification*; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000706:statistics & numerical data; D003505:Cyclodextrins / Q000737:chemistry*; D003627:Data Interpretation, Statistical; D004563:Electrochemistry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D013237:Stereoisomerism; D047392:beta-Cyclodextrins
chemical_list: D002121:Calcium Channel Blockers; D003505:Cyclodextrins; C103513:SBE7-beta-cyclodextrin; D047392:beta-Cyclodextrins; D017311:Amlodipine