Chemiluminescence detection coupled to high-performance frontal analysis for the determination of unbound concentrations of drugs in protein binding equilibrium

High-performance frontal analysis coupled with chemiluminescence detection (HPFA-CL) was developed for the determination of unbound oxacillin concentration in human serum albumin solution. The HPFA system consisted of an ISRP column and a mobile phase of 67 mM potassium phosphate buffer of pH 7.4 and ionic strength of 0.17. The luminol-H2O2-Co2+ system was used in the chemiluminescence detection. An enhancement of luminol chemiluminescence by oxacillin was investigated and employed for determining the concentration of oxacillin in the HPFA eluate. Sample solutions were directly injected onto the column; the drug was eluted as a zonal peak with a plateau region. The unbound drug concentrations were determined by using the height of the plateau. The results agreed with those obtained with conventional ultrafiltration-HPLC method. Good reproducibility was confirmed by the within run and between run RSD < or = 7.4%. HPFA-CL provided a selective method for determination of unbound drug concentration in protein binding equilibrium.