Stereoselective determination of free warfarin concentration in protein binding equilibrium using direct sample injection and an on-line liquid chromatographic system.

A new on-line high-performance liquid chromatography system was developed for the stereoselective determination of free drug concentration in drug-protein binding equilibrium. When a 40-300-microL portion of a sample solution containing 50-200 microM racemic warfarin (Wf) and 100-550 microM human serum albumin was directly injected into the internal-surface reversed-phase silica column, Wf gave a trapezoidal peak exhibiting a plateau region. The concentration in the plateau region was equal to the free Wf concentration in the initial sample solution. By the delivery of a portion (90 microL) of the eluent in the plateau region into the chiral separation column (Chiral AGP column) by column switching, the free concentrations of respective enantiomers of Wf were determined. The results agreed well with those obtained by the conventional ultrafiltration method. The precision was also confirmed by the within-run and day-to-day reproducibilities (coefficient of variation less than or equal to 3.05%, n = 5). The present method is simple and rapid and four sample solutions can be analyzed within 1 h without pretreatment.