Theoretical study of high-performance frontal analysis:  a chromatographic method for determination of drug-protein...

High-performance frontal analysis (HPFA), a chromatographic method to determine unbound drug concentration in drug-protein binding equilibrium, has been considered on the basis of a theoretical plate model, where a rapid equilibrium of drug-protein binding in the mobile phase in the interstices of packing materials and a chromatographic partition equilibrium of the drug were taken into account simultaneously. When a certain excess volume of drug-protein mixed solution is injected directly into a HPFA column packed with a restricted-access type phase that excludes protein but retains drug in the micropores, the drug is eluted as a zonal peak with a plateau region. The elution profile can be well simulated by the mass balance equation derived according to a relatively simple plate theory concept, which confirms that the drug concentration in the plateau range agrees with the unbound drug concentration in the sample solution. The model was applied to the theoretical and systematic investigation of the dependence of the HPFA profile on several chromatographic conditions and the properties of the sample solution, such as injection volume of sample solution, drug and protein concentrations in sample solution, capacity factor of the drug, theoretical plate number, and binding parameters. The smaller capacity factor and the higher column efficiency lead to the larger plateau volume. The lower drug concentration, the higher protein concentration, and the stronger binding constant, which give the lower unbound drug fraction, lead to the larger plateau volume and allow frontal analysis with a smaller sample size.