High-Performance Frontal Analysis/High-Performance Liquid Chromatographic System for the Enantioselective Determination of Unbound Fenoprofen Concentration in Protein Binding Equilibrium

Abstract

High-performance frontal analysis (HPFA) and high-perfon-ance liquid chromatography (HPLC) were incorporated in an on-line coupled column system for the enantioselective determination of unbound fenoprofen (FP) concentrations in the state of a protein binding equilibrium following direct sample injection. This system consists of a “restricted access” type column for HPFA and al-acid glycoprotein-immobilized silica column for chiral separation. When a 80-μL portion of a sample solution containing 50 or 90μg/mL FP and 300 or 550,μM human serum albumin (HSA) was directly injected into the HPFA column, FP gave a trapezoidal peak exhibiting a plateau region. The concentration in the plateau range was equal to the unbound FP concentration in the initial sample solution. By the delivery of a 90-μL portion of the eluent in this plateau region into the chiral separation column, the unbound concentrations of FP enantiomers were determined. The results agreed with those obtained by the conventional ultrafil-tration-HPLC method. The precision was also confirmed by the within-run and day-to-day reproducibilities (CV<9.95%, n=5). It was found that the binding between FP and HSA is stereoselective, and S-FP binds with HSA more strongly than R-FP.