Carol K. Holtzapple

To demonstrate that two structurally similar chemicals can be extracted from a complex matrix and then separated from each other on the basis of their relative affinities for an antibody, an automated column-switching system was used, incorporating on-line, high-performance immunoaffinity chromatography (HPIAC). A high-affinity monoclonal antibody (Mab Sara-95) against the fluoroquinolone sarafloxacin was covalently cross-linked to a protein G column and used to capture fluoroquinolones in fortified serum samples. Interference from matrix components adhering nonspecifically to the column was minimized by the insertion of a protein G cleanup column between the injection port and the Mab Sara-95 derivatized HPIAC column. Upon injection, serum samples containing the fluoroquinolones passed through both columns. The cleanup 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 were then eluted from the HPIAC column according to their relative affinities for the antibody, and individual peaks were monitored using fluorescence detection. By using an on-line cleanup column in tandem with an HPIAC column, the fluoroquinolones could be separated from the serum matrix and then separated from each other on the basis of their affinity for Mab Sara-95 without the use of organic solvents or reversed-phase liquid chromatography (RPLC). This method demonstrates true immunoaffinity separation of structurally related compounds in a complex matrix.

Four fluoroquinolones were analyzed in fortified chicken liver using an automated, on-line immunoaffinity extraction method. The fluoroquinolones were extracted from the liver matrix using an immunoaffinity capture column containing anti-sarafloxacin antibodies covalently cross-linked to protein G. After interfering liver matrix components had been washed away, the captured fluoroquinolones were automatically eluted directly onto a reversed phase column. Liquid chromatographic analyses were performed by isocratic elution using 2% acetic acid/acetonitrile (85:15) as the mobile phase and an Inertsil phenyl column with fluorescence detection at excitation and emission wavelengths of 280 and 444 nm, respectively. No significant interferences from the sample matrix were observed, indicating good selectivity with the immunoaffinity column. Overall recoveries from fortified liver samples (20, 50, and 100 ng/g) ranged between 85.7 and 93.5% with standard deviations of <5%. The limit of quantification for each fluoroquinolone was 1 ng/mL. The limits of detection, based on a signal-to-noise ratio of 5:1, were 0.47, 0.32, 0.87, and 0.53 ng/mL for ciprofloxacin, enrofloxacin, sarafloxacin, and difloxacin, respectively. Keywords: Fluoroquinolone; immunoaffinity chromatography; on-line detection method; bovine tissues; food safety

An automated, on-line immunoaffinity extraction method was developed for the analysis of 4 fluoroquinolones in milk: ciprofloxacin, difloxacin, enrofloxacin, and sarafloxacin. This method involves analyte extraction using an immunoaffinity capture column containing anti-fluoroquinolone antibodies coupled on-line with reversed-phase column chromatography. Liquid chromatographic analyses were performed by isocratic elution using a mobile phase of 2% acetic acid-acetonitrile (85 + 15) and an Inertsil phenyl column with fluorescence detection at excitation and emission wavelengths of 278 and 444 nm, respectively. No significant interferences from the sample matrix were observed, indicating good selectivity with the immunoaffinity column. Recoveries from fortified raw milk samples (5–50 ppb of each fluoroquinolone) ranged from 72 to 90%, with standard deviations of ≤8%.