Nariyasu Mano

Development of a flavoprotein column for chiral separation by high-performance liquid chromatography

Abstract A new chiral stationary phase using flavoprotein, a glycoprotein present in chicken egg-white, was developed for high-performance liquid chromatography. This column could achieve baseline separations of acidic (ketoprofen), weakly acidic (warfarin) and neutral (benzoin) compounds. In the flavoprotein-conjugated silica gel column, the capacity factor and enantioselectivity for several model drugs were greatly influenced by the pH and the concentration of organic solvents and salts in the mobile phase, and the optimum conditions for chiral separation varied from compound. However, this column shows good stability to pH variation and to organic solvents, and it should be applicable for the chiral separation of many compounds in the reversed-phase mode. This column can directly separate optical isomers with an aqueous mobile phase, so it should be very useful in the fields of pharmacokinetics and clinical chemistry.

Studies of ovomucoid-, avidin-, conalbumin- and flavoprotein-conjugated chiral stationary phases for separation of enantiomers by high-performance liquid chromatography

Abstract The effects of organic modifiers, buffer salts and pH on the retention and chiral separation of four protein-immobilized chiral stationary phases (CSPs), an ovomucoid-CSP, an avidin-CSP, a conalbumin-CSP and a flavoprotein-CSP, were investigated. Both retention and enantioselectivity were affected by alteration of the mobile phase conditions, and it was elucidated that the hydrophobic and ionic interactions between enantiomers and chiral recognition moieties in immobilized protein molecules were important to the chiral separation of each CSP. The protein bindings of enantiomers for four native proteins were also examined with chiral separation chromatography using a commercial ovomucoid-CSP (Ultron ES-OVM). The racemate which showed significant differences in protein binding abilities among its enantiomers was excellently resolved by chromatography.

Conalbumin-conjugated silica gel, a new chiral stationary phase for high-performance liquid chromatogaphy

A new chiral stationary phase using conalbumin (from chicken egg white) was developed for high-performance liquid chromatography. Chiral resolution of racemic azelastine, an antiallergic drug, was achieved on a conalbumin-conjugated silica gel column. The effects of the pH, the concentration of organic solvents and salts in the mobile phase, and the temperature on the capacity factor and resolution of racemic azelastine were examined. This column shows good stability and can separate optical isomers with an aqueous mobile phase. It should be very useful in studies on pharmacokinetics and in clinical chemistry.

Enantioselective high-performance liquid chromatographic assay for determination of the enantiomers of a new anti-ulcer agent, E3810, in Beagle dog plasma and rat plasma

An enantioselective high-performance liquid chromatographic method for the determination of E3810, a new anti-ulcer agent, in Beagle dog plasma and rat plasma has been developed. After extraction from plasma with ethyl acetate, E3810 enantiomers were measured by reversed-phase high-performance liquid chromatography on a Chiralcel OD-R column. The enantiomers were detected by ultraviolet absorbance detection at 290 nm. The recoveries of E3810 enantiomers and internal standard were greater than 91%. The calibration curves were linear from 0.03 to 20 micrograms/ml for Beagle dog plasma and from 0.1 to 100 micrograms/ml for rat plasma. The limits of quantification of both enantiomers were 0.03 micrograms/ml for Beagle dog plasma and 0.1 micrograms/ml for rat plasma. The intra- and inter-day accuracy and precision data showed good reproducibility of the method. The assay was applied for the analysis of E3810 enantiomers in plasma after intravenous administration of racemic E3810 to Beagle dogs and rats. This method should be very useful for enantioselective pharmacokinetic studies of E3810.

Investigation of Interactions Between Drug Enantiomers and Flavoprotein as a Chiral Selector by Affinity Capillary Electrophoresis

Abstract Interactions between drug enantiomers and flavoprotein as a pseudo chiral stationary phase were investigated by using affinity capillary electrophoresis (CE) in order to avoid the effects of nonspecific interactions that occur in chiral HPLC. Circular dichroism (CD) measurement was used to monitor changes of the secondary structure of flavoprotein under various analysis conditions in affinity CE. The chiral discrimination region for ketoprofen on the flavoprotein surface was concluded to consist of α-helix structure, and the decrease of chiral separation ability with increase of methanol content in the electrophoretic buffer was directly related to conformational change of the α-helix. Studies with chemically modified flavoprotein indicated that two types of interaction at the chiral discrimination region are required for chiral separation: π-π interaction of a tryptophan residue with the aromatic ring of ketoprofen, and ionic interaction of the carboxyl group of ketoprofen with an amino group an...

Resolution of 4-(4-chlorobenzyl)-2-(hexahydro-1-methyl-1H-azepin-4yl)-1(2H)-phthalazinone enantiomers in plasma with frit-FAB LC—MS using a conalbumin column

A new column-switching method for analysis of drug enantiomers in plasma has been developed with liquid chromatography-frit fast atom bombardment mass spectrometry in combination with a chiral resolution column, which consists of conalbumin (egg-white glycoprotein) immobilized on silica gel and can be used in the reversed-phase separation mode. This method makes it possible to inject a large volume of deproteinized plasma and obtain resolution of drug enantiomers with high sensitivity. The optimum mobile phase, including a non-volatile buffer such as phosphate buffer, for separation of drugs from a large amount of endogenous compounds can be used, because of inclusion of a trapping column with a desalting function. This method is very simple and rapid, and should be very powerful in studies requiring high-sensitivity analysis with chiral separation of drugs from biological samples such as plasma.

Investigation of retention behavior for racemate drugs on avidin- and modified avidin-column

Abstract The binding properties of a native avidin were investigated by recording the fluorescence spectra of the native avidin, and by measuring the avidin-drug binding and the hydrophobicty of drugs. Some emission intensities were diminished by mixing avidin and drugs solutions compared with the native avidin solution. Also, the Scatchard plot analysis were performed to evaluate the avidin-drug binding. Then, modified avidin columns were prepared by acylation of amino groups and carboxyl groups to examine the retention and enantioselective properties of drugs. As a result, these functional groups contributed to chiral recognition of the avidin column for some drugs. Based on the results in this study, the avidin column have multiple binding sites for chiral separation.