Applications Of Chiral HPLC To Separation Of Pharmaceutical Active Compounds

A pair of enantiomers of a chiral drug often show different pharmacological behaviors and undergo different metabolic pathways in vivo. To study the pharmacological and metabolic properties of chiral drugs, appropriate methods for separation and preparation of optically pure enantiomers from racemates must be developed. In this thesis, we report the development of high-performance liquid chromatography (HPLC) methods in chiral stationary phase (CSP) and chiral mobile phase additive (CMPA) modes and their applications to chiral separation of several important pharmaceutically active compounds. The thesis work can be devided into three parts as follows:Optically pureβ-amino acids can be used as building blocks for a number of therapeutic drugs. Two methods were developed that allow the separation ofβ-amino acid enantiomers from their synthetic racemates. Firstly, chiral separation ofβ-amino acids was achieved in a reverse phase liquid chromatography (RPLC) using optically pure L-phenylalanine as a chiral mobile phase additive. The chiral ligand in the mobile phase can form diastereomers with racemicβ-amino acids in the presence of metal ions such as Cu²⁺ and the diastereomers can be readily resolved on a RPLC system. Secondly, chiral separation ofβ-amino acids was achieved using chiral stationary phases (CSP). Three types of ligand exchange CSPs were prepared by coating alkylated amino acids derivatives such as N-hexadecyl-L-hydroxyproline (C₁₆-L-Hyp), N-hexadecyl-L-proline (C₁₆-L-Pro) and N-hexadecyl-alanine (C₁₆-L-Ala) onto the octadecyl-bonded silica (ODS) support. With an aqueous solution of cupper sulfate as the mobile phase, five differentβ-phenylalanine racemates were all baseline separated on these ligand exchange CSPs. Chiral separation method based on ligand exchange CSPs offers the merits of easy operation and low operating cost and can be scaled up for preparative scale separations ofβ-amino acids.Equol is a chiral metabolite of daidzein found in both human and animal urines.Currently equol is extensively studied with regard to its pharmaceutical potentials. A reversed-phase chromatography with a chiral mobile phase additive (CMPA) was adapted for separation of equol enantiomers from its synthetic racemates. The column used was a BaseLine C18 (250 mm×4.6 mm, 5μm), and the mobile phases were composed of aqueous solution ofβ-cyclodextrin and its derivatives with some proportion of organic modifiers. Within twenty-five minutes, a baseline separation of equol enantiomers could be achieved. After optimization of the chromatographic parameters, a separation factor up to 1.21 was obtained for the paired enantiomers of eqoul reaches, which is higher than reported separation factor of 1.12 obtained on a hydroxypropyl-β-cyclodextrin bonded silica.To overcome the limited supply of the equol isolated from animal urines, we developed a method for preparation of equol with potential for large scale production. In this method, a mixture of soybean isoflavone glycosides were used as starting materials. After the glycosides were treated with hydrochloric acid, three aglycons (daidzein,genistein and glycitein) were obtained. Then, the aglycons were subjected to hydrogenation in an autoclave with carbon palladium as a catalyst, giving rise to reduction products which contain eqoul and other two new chiral compounds. The HPLC method developed for equol was applied to simultaneous separation of the hydrogenation products. To the best of our knowledge, the preparation and chrial resolution of racemic hydrogenated derivatives of genistein and glycitein were not reported before. Furthermore, we brifely examined the methods of chiral resolution for the composite racemic compounds in CSP includingβ-cyclodextrin and polysaccharide CSP in order to overcome the deficit of CMPA mode that needs complicated post-separation purifications to get rid of CMPA from desired products.