Separation and quantification of two fluoroquinolones in serum by on-line high-performance immunoaffinity chromatography

To demonstrate that two structurally similar chemicals can be extracted fro m a complex matrix and then separated from each other on the basis of their relative affinities for an antibody, an automated column-sn;itching system was used, incorporating on-line, high-performance immunoaffinity chromatog raphy (HPIAC), A high-affinity monoclonal antibody (Mab Sara-95) against th e fluoroquinolone sarafloxacin was covalently cross-linked to a protein G c olumn and used to capture fluoroquinolones in fortified serum samples, Inte rference from matrix components adhering nonspecifically to the column was minimized by the insertion of a protein G cleanup column between the inject ion port and the Mab Sara-95 derivatized HPIAC column. Upon injection, seru m samples containing the fluoroquinolones passed through both columns. The cleanup column detained serum components, that otherwise would bind nonspec ifically to the HPIAC column, but allowed the fluoroquinolones to pass thro ugh unhindered to the HPIAC column. The fluoroquinolones were then eluted f rom the HPIAC column according to their relative affinities for the antibod y, and individual peaks were monitored using fluorescence detection. BS usi ng an on-line cleanup column in tandem with an HPIAC column, the fluoroquin olones could be separated from the serum matrix and then separated from eac h other on the basis of their affinity for Mab Sara-95 without the use of o rganic solvents or reversed-phase liquid chromatography (RPLC). This method demonstrates true immunoaffinity separation of structurally related compou nds in a complex matrix.