DEVELOPMENT OF HPLC PACKING MATERIALS FOR DRUG ANALYSIS AND THEIR APPLICATIONS

High-performance liquid chromatography (HPLC) has been used for the as says of pharmaceuticals and their impurities, and drugs and their meta bolites in biological fluids. However, HPLC packing materials so far d eveloped are not necessarily suitable for the above mentioned purposes . During the past decade, we developed of new HPLC packing materials f or direct serum injection analysis of drugs, and for chiral resolution of drug enantiomers. A sample preparation step such as deproteinizati on and/or extraction is required for HPLC assays of drugs and their me tabolites in biological fluids. Direct serum injection analysis of dru gs can be achieved by the use of the packing materials having properti es 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 sep arated. Such a material is called restricted access stationary phase ( RASP). We developed two RASPs, an improved internal-surface reversed-p hase (ISRP) material and a mixed functional phase (MFP) material. The preparation methods of the ISRP and MFP materials and their applicatio ns to the assays of drugs in the serum are described. HPLC chiral stat ionary phases based on chiral small. molecules, and macromolecules suc h as polysaccharides and proteins have been used for separations of dr ug enantiomers. Disadvantages of protein-bonded stationary phases incl ude low capacity,,lack of column ruggedness and limited understanding of the chiral recognition mechanism. We tried to overcome the disadvan tages of protein-bonded stationary phases by modification of side-chai n amino acid and preparation of protein-domain or -fragment column. We isolated each ovomucoid domain and examined chiral recognition abilit y of each domain. The ovomucoid third domain exhibited chiral recognit ion for only a few benzodiazepines and 2-arylpropionic acid derivative s. 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, wa s isolated from crude ovomucoid preparations, characterized and bound to silica gels. It was found that the chiral recognition ability of th e ovomucoid reported previously came from the ovoglycoprotein, which i s present in crude ovomucoid as an impurity.