Chromatographic analysis of carbamazepine binding to human serum albumin: II. Comparison of the Schiff base and <i>N</i>-hydroxysuccinimide immobilization methods

Recent studies with carbamazepine on human serum albumin (HSA) columns have noted an appreciable degree of non-specifi c binding on supports prepared by the Schiff base immobilization method. This work examines an alternative immobilization method for HSA based on N-hydroxysuccinimide (NHS)-activated silica. This support was prepared by reacting HPLC-grade silica directly with disuccinimidyl carbonate. The resulting material was compared to an HSA support prepared by the Schiff base method in terms of its activity for carbamazepine and non-specifi c interactions with this drug. When examined by frontal analysis, both supports gave comparable association equilibrium constants for carbamazepine interactions with HSA ((0.53–0.55) × 104 M−1 at 37 °C). However, columns prepared by the Schiff base method gave greater non-specifi c binding. These columns, as well as control columns prepared using the carbonyldiimidazole (CDI) immobilization method, were also evaluated for their non-specifi c binding to a variety of other solutes known to interact with HSA. From these results it was concluded that the NHS method was an attractive alternative to the Schiff base technique in the preparation of immobilized HSA for HPLC-based binding studies for carbamazepine. However, it was also noted that nonspecifi c binding varies from one drug to the next in these immobilization methods, indicating that such properties should be evaluated on a case-by-case basis in the use and development of HSA columns for binding studies.