[Development of HPLC packing materials for drug analysis and their applications]

Abstract

High-performance liquid chromatography (HPLC) has been used for the assays of pharmaceuticals and their impurities, and drugs and their metabolites in biological fluids. However, HPLC packing materials so far developed are not necessarily suitable for the above mentioned purposes. During the past decade, we developed of new HPLC packing materials for direct serum injection analysis of drugs, and for chiral resolution of drug enantiomers. A sample preparation step such as deproteinization and/or extraction is required for HPLC assays of drugs and their metabolites in biological fluids. Direct serum injection analysis of drugs can be achieved by the use of the packing materials having properties as follows: large molecules such as proteins are eluted in the void volume without destructive accumulation, but small molecules such as drugs and their metabolites can reach the hydrophobic sites and be separated. Such a material is called restricted access stationary phase (RASP). We developed two RASPs, an improved internal-surface reversed-phase (ISRP) material and a mixed functional phase (MFP) material. The preparation methods of the ISRP and MFP materials and their applications to the assays of drugs in the serum are described. HPLC chiral stationary phases based on chiral small molecules, and macromolecules such as polysaccharides and proteins have been used for separations of drug enantiomers. Disadvantages of protein-bonded stationary phases include low capacity, lack of column ruggedness and limited understanding of the chiral recognition mechanism. We tried to overcome the disadvantages of protein-bonded stationary phases by modification of side-chain amino acid and preparation of protein-domain or -fragment column. We isolated each ovomucoid domain and examined chiral recognition ability of each domain. The ovomucoid third domain exhibited chiral recognition for only a few benzodiazepines and 2-arylpropionic acid derivatives. The chiral binding site and mechanism on the ovomucoid third domain was investigated by proton NMR measurements and molecular modeling of the ligand-protein complex. Further, new protein, ovoglycoprotein, was isolated from crude ovomucoid preparations, characterized and bound to silica gels. It was found that the chiral recognition ability of the ovomucoid reported previously came from the ovoglycoprotein, which is present in crude ovomucoid as an impurity.