THEORETICAL-STUDY OF HIGH-PERFORMANCE FRONTAL ANALYSIS - A CHROMATOGRAPHIC METHOD FOR DETERMINATION OF DRUG-PROTEIN INTERACTION

High-performance frontal analysis (HPFA), a chromatographic method to determine unbound drug concentration in drag-protein binding equilibri um, has been considered on the basis of a theoretical plate model, whe re a rapid equilibrium of drug-protein binding in the mobile phase in the interstices of packing materials and a chromatographic partition e quilibrium of the drug were taken into account simultaneously. When a certain excess volume of drug-protein mixed solution is injected direc tly into a HPFA column packed with a restricted-access type phase that excludes protein but retains drug in the micropores, the drug is elut ed as a zonal peak with a plateau region. The elution profile can be w ell simulated by the mass balance equation derived according to a rela tively simple plate theory concept, which confirms that the drug conce ntration in the plateau range agrees with the unbound drug concentrati on in the sample solution, The model was applied to the theoretical an d systematic investigation of the dependence of the HPFA profile on se veral chromatographic conditions and the properties of the sample solu tion, such as injection volume of sample solution, drug and protein co ncentrations in sample solution, capacity factor of the drug, theoreti cal plate number, and binding parameters, The smaller capacity factor and the higher column efficiency lead to the larger plateau volume, Th e lower drug concentration, the higher protein concentration, and the stronger binding constant, which give the lower unbound drag fraction, lead to the larger plateau volume and allow frontal analysis with a s maller sample size.