Separation and Quantification of Two Fluoroquinolones in Serum by On-Line High-Performance Immunoaffinity Chromatography

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• 作者：Carol K. Holtzapple ; Elizabeth J. Pishko ; and Larry H. Stanker
• 刊名：Analytical Chemistry
• 出版年：2000
• 出版时间：September 1, 2000
• 年：2000
• 卷：72
• 期：17
• 页码：4148 - 4153
• 全文大小：71K
• 年卷期：v.72,no.17(September 1, 2000)
• ISSN：1520-6882

文摘

To demonstrate that two structurally similar chemicals canbe extracted from a complex matrix and then separatedfrom each other on the basis of their relative affinities foran antibody, an automated column-switching system wasused, incorporating on-line, high-performance immunoaffinity chromatography (HPIAC). A high-affinity monoclonal antibody (Mab Sara-95) against the fluoroquinolonesarafloxacin was covalently cross-linked to a protein Gcolumn and used to capture fluoroquinolones in fortifiedserum samples. Interference from matrix componentsadhering nonspecifically to the column was minimized bythe insertion of a protein G cleanup column between theinjection port and the Mab Sara-95 derivatized HPIACcolumn. Upon injection, serum samples containing thefluoroquinolones passed through both columns. Thecleanup column detained serum components, that otherwise would bind nonspecifically to the HPIAC column,but allowed the fluoroquinolones to pass through unhindered to the HPIAC column. The fluoroquinolones werethen eluted from the HPIAC column according to theirrelative affinities for the antibody, and individual peakswere monitored using fluorescence detection. By usingan on-line cleanup column in tandem with an HPIACcolumn, the fluoroquinolones could be separated from theserum matrix and then separated from each other on thebasis of their affinity for Mab Sara-95 without the use oforganic solvents or reversed-phase liquid chromatography(RPLC). This method demonstrates true immunoaffinityseparation of structurally related compounds in a complexmatrix.