Separation of Beraprost sodium isomers using different cyclodextrin stationary phases

Abstract

The separation of isomers present in a new prostacyclin analogue (Beraprost sodium) on several different yclodextrin stationary phases was investigated. Beraprost sodium, which contains six chiral centers, is a mixture of four isomers; two diastereomers each containing a pair of enantiomers. The cyclodextrin stationary phases used included: α-, β- and γ-cyclodextrins as well as several derivatized β-cyclodextrins. The mobile phase variables that were found to affect the chiral separation were: type and concentration of organic modifier, type and concentration of buffer, the cation and anion associated with the buffer, mobile phase pH, preparation of the mobile phase, and column temperature. The cyclodextrin stationary phases that were found to separate all four isomers were: the acetylated β-cyclodextrin (Cyclobond I AC) and the para-toluoyl ester-derivatized β-cyclodextrin (Cyclobond I PT). The selectivities of the isomers were better on the Cyclobond I AC column than on the Cyclobond I PT column. Therefore the Cyclobond I AC column was used to determine what effect each mobile phase variable had on isomer retention and resolution. Calibration curves were done for the four isomers and the correlation coefficients determined using UV detection. Plots of log peak area versus concentration of each isomer provided correlation coefficients of 0.998 or better. Detection limits of 75 ng/ml and quantitation limits of 500 ng/ml of each isomer were found.