Incorporation of a monolithic column into sequential injection system for drug-protein binding studies

A sequential injection analysis (SIA) manifold was incorporated with a monolithic strong anion-exchanger disk for on-line drug-protein interaction studies. The antibiotic ciprofloxacin (CF) was selected as a model drug compound. The separation principle was based on the strong retention of bovine serum albumin (BSA) on the monolithic strong anion-exchanger and the liberation/release of the free form of the drug. Elution of the retained BSA was easily achieved by delivering a different mobile phase via the SIA manifold. The type of functional group of the monolithic support, the breakthrough volume and the injected volumes of CF and BSA were studied and optimized. The influence of the variation of incubation time was studied in on-line binding assays. Scatchard plot was employed to obtain the number of binding sites and the equilibrium binding constants. For the off-line study of the CF-BSA binding, two binding classes were determined with constants of (3.16 ± 0.21) × 106 M−1 and (1.27 ± 0.48) × 104 M−1 and 6.1 ± 1.3 and 17.8 ± 3.9 binding sites per class, respectively. In non-equilibrium binding experiments the binding rate constant was k1 = 785 M−1 min−1. All measurements were monitored with fluorescence (λext = 300 nm, λem = 460 nm) and spectrophotometric detection (λ = 280 nm). To evaluate the accuracy of the developed method the obtained results were compared versus ultrafiltration experiments and were found in good agreement.