Toshinobu Miwa

Characteristics of an avidin-conjugated column in direct liquid chromatographic resolution of racemic compounds

Abstract An avidin-conjugated column exhibited chiral recognition of acidic compounds such as profen derivatives. Amine enantiomers were not resolved by this column, of which biotin acted as a potent modifier. Avidin showed hydrophobic interaction with the solutes. A differential scanning calorimetry study showed that thermodynamically stable avidin is inapplicable as a chiral recognition ligand.

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.

Characteristics of ovomucoid-conjugated columns in the direct liquid chromatographic resolution of racemic compounds

Abstract The chiral recognition properties of ovomucoid-conjugated columns were investigated. Trypsin slightly affected the chiral recognition characteristics of the column. Neuraminidase treatment of ovomucoid columns altered their acidic solute retention properties. Deglycosylated ovomucoid-conjugated columns dit not resolve racemic chlorpheniramine or ketoprofen. A sugra chain is essential for the exhibition of chiral recognition ability for ovomucoid.

Drug-plasma protein binding assay by electrokinetic chromatography-frontal analysis

We developed a rapid, microscale and reliable analytical method for binding of drugs to plasma proteins using capillary electrophoresis (CE) with ionic cyclodextrins (CD) combined with frontal analysis. These CDs were used as pseudostationary phases of electrokinetic chromatography (EKC). The CD‐modified EKC (CDEKC) approach allowed us to separate anionic drugs from plasma proteins, whereas CZE could not separate these drugs from plasma proteins because they had a similar mobility like plasma proteins. CDs uniquely interact with these drugs but not with plasma proteins. Therefore, CDEKC could be coupled with frontal analysis to measure the binding of anionic drugs to plasma proteins. The binding values obtained by CDEKC were highly consistent with those determined by the ultrafiltration method. Our CDEKC approach should expand the applicability of CE to protein binding analysis.

Chiral Separation of Lorazepam on Ovomucoid-Bonded Columns: Peak Coalescence Due to Racemization

Abstract Racemic lorazepam (LZ) was separated on an ovomucoid (OVM)-bonded column by changing the column temperature. Chromatographic peak coalescence, appearing as a plateau between the resolved peaks, was observed above column temperatures of 15°C. Each LZ enantiomer was isolated on another chiral column using a non-aqueous eluent. Racemization of each LZ enantiomer in two different solutions and peak coalescence on these chiral columns were investigated. These results revealed that the peak coalescence should be due to racemization of LZ on the OVM-bonded column. By reducing the column temperature to 7°C, the enantiomeric composition of LZ before chromatography could be determined on the OVM-bonded column.

Application of an ovomucoid-conjugated polymer column for the enantiospecific determination of chlorprenaline concentrations in plasma

An ovomucoid-conjugated polymer column was prepared for the liquid chromatographic resolution of racemic compounds. The column showed strong retention of acidic solutes, a characteristic attributed to the structure of the stationary phase support gel. Although the efficiency of the column was lower than that of an ovomucoid-conjugated silica gel column, enantiospecific chlorprenaline determination in plasma was achieved with solute amounts from 1.0 ng to 0.1 microgram.