Supercritical fluid chromatography and liquid chromatography for isomeric separation of a multiple chiral centers analyte.

The development of a chiral separation strategy has always been a challenge of crucial importance, particularly in the pharmaceutical field. Chromatographic methods have become popular, particularly High Performance Liquid Chromatography and Supercritical Fluid Chromatography from a preparative scale point of view. A bioactive compound bearing three stereogenic centers was entrusted in our laboratory and the aim of this work was to obtain the complete resolution of the eight stereoisomers. Nine different polysaccharide-based columns were tested in SFC under various carbon dioxide-based mobile phases. The use of a single chiral column Lux Cellulose-2 under 30% 2-PrOH in carbon dioxide, at a flow-rate of 1 mL/min, column temperature of 40°C, 120 bar outlet pressure allowed the obtention of eight peaks. To further improve the resolution of the two last isomers, two columns were serially coupled . The results obtained with the six different combinations are discussed. The tandem column supercritical fluid chromatography has demonstrated to be a useful technique to resolve the eight stereoisomers on Lux Cellulose-2//Cellulose-2 tandem of coupled columns with 30% 2-PrOH in carbon dioxide, at a flow-rate of 1 mL/min, column temperature of 40°C and 120 bar outlet pressure, despite a long analysis time. In order to compare the two methods (i.e supercritical and liquid), chiral liquid chromatography under polar aqueous-organic mode, polar organic mode and normal-phase mode, was implemented. The last mode allowed the full baseline resolution of the eight isomers on Cellulose-5 CSP, with 20% 2-PrOH in n-heptane at a flow-rate of 0.8 mL/min, at 25°C, λ = 220 nm. The limits of detection and of quantification were determined for this method and the best values obtained for isomer 8 were equal to 2.84 and 9.37 nM respectively. Finally, a small-scale preparative separation of the multiple chiral centers compound was implemented on Cellulose-5 CSP within 10% 2-PrOH in n-heptane in order to study the stereoisomer elution order on Cellulose-2, Cellulose-5 and Chiralpak AD-H, under EtOH or 2-PrOH in n-heptane mobile phases, and partial reversal elution orders were observed.

Electrophoretic separation of multiple chiral center analyte with a three cyclodextrins mixture.

A capillary electrokinetic chromatography method (CEKC) was developed for complete stereoisomeric separation of a neutral, hydrophobic, multiple chiral center dihydropyridone analogue, a drug candidate proposed in type 2 diabetes treatment. A background electrolyte comprising three cyclodextrins was found to successfully separate the eight isomers. First an anionic cyclodextrin, the SBE-ß-CD, was selected to allow the chiral separation of our neutral compound and partial resolutions of the eight isomers were obtained. Then, the effects of different parameters such as the nature and concentration of the other cyclodextrins added and pH of the buffer were examined. Finally, a triple CD-system consisted of 15 mM SBE-ß-CD plus 15 mM Î³-CD and 40 mM HP-γ-CD in a 50 mM borate background electrolyte at pH 10, was found to successfully separate the eight isomers. Last, the selectivity and limits of detection and quantification were evaluated for this optimized method.