Determination of fluoroquinolones in serum using an on-line clean-up column coupled to high-performance immunoaffinity–reversed-phase liquid chromatography

A simple, rapid and reliable method for the simultaneous analysis of the fluoroquinolones ciprofloxacin, enrofloxacin, sarafloxacin, and difloxacin in bovine serum has been developed. Upon injection of serum samples, an on-line protein G-linked column was employed to automatically remove serum components that otherwise would interfere with analyses. A high-performance immunoaffinity chromatography (HPIAC) column containing covalently bound anti-sarafloxacin antibodies was then used to capture the fluoroquinolones while allowing the remainder of the serum components to elute to waste. After binding to the HPIAC column, the fluoroquinolones were eluted directly onto a reversed-phase (RP) column for final separation of the compounds prior to fluorescence detection at excitation and emission wavelengths of 280 and 444 nm, respectively. Due to use of a clean-up column in tandem with a highly selective HPIAC column, the only off-line sample preparation required was dilution (10-fold) in phosphate buffered saline (PBS) and passage of the samples through a 0.2-μm filter to remove particulate matter prior to injection. No significant interferences from the sample matrix were observed, indicating good selectivity with the HPIAC column. The method yielded high recoveries from fortified bovine serum that were >95% for all four fluoroquinolones with good reproducibility (C.V. values <7.0%). The on-line, automated method described here provides a simple, sensitive and specific assay for multiresidue detection of fluoroquinolones in serum.