Submitted to: Journal of Chromatography A
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/22/2008
Publication Date: 9/6/2008
Publication URL: http://handle.nal.usda.gov/10113/22679
Citation: Li, C., Wang, Z., Cao, X., Beier, R.C., Zhang, S., Ding, S., Li, X., Shen, J. 2008. Development of an immunoaffinity column method using broad-specificity monoclonal antibodies for simultaneous extraction and cleanup of quinolone and sulfonamide antibiotics in animal muscle tissues. Journal of Chromatography A. 1209:1-9. Interpretive Summary: Fluoroquinolones (FQs) and sulfonamides (SAs) are both synthetically derived antibiotics. These antibiotics are used for treatment in livestock and poultry. We have developed an immunoaffinity column separation method using antibodies that recognize a broad group of twelve FQ and six SA antibiotics. Antibodies are substances that are produced by the immune system in response to foreign substances which enter the body. Once the antibody to a foreign substance is isolated, it can be used in a method to bind or detect the presence of that foreign substance. This immunoaffinity column may be used to simultaneously extract and cleanup FQ and SA antibiotics in animal muscle tissue samples. Tests using these columns would allow the simultaneous analysis of 19 different antibiotics.
Technical Abstract: This paper describes a novel mixed-bed immunoaffinity column (IAC) method. The IAC was produced by coupling anti-fluoroquinolone and anti-sulfonamide broad-specificity monoclonal antibodies (Mabs) to Sepharose 4B for simultaneously isolating 13 fluoroquinolones (FQs) and 6 sulfonamides (SAs) from swine and chicken muscle tissues, followed by antibiotic determination using liquid chromatography-tandem mass spectrometry (LC-MS/MS). A new broad-specificity Mab (B1A4E8) toward sulfonamides was produced using sulfamethoxazole (SMX) as hapten that demonstrated cross-reactivities to 6 SAs in the range of 31–112%. The Mab was used to investigate a correlation between antibody binding and conformational and electronic properties of 6 SA analogs. IAC optimized conditions were found that allowed the IAC to be reused for selective binding of both SAs and FQs. The dual-broad-specificity IAC selectively retained a total of 13 FQs and 6 SAs and allowed for efficient removal of interfering sample matrix compounds. Recoveries of all 19 antibiotics from animal muscle ranged from 72.6–107.6%, with RSDs below 11.3% and 15.4% for intra-day and inter-day experiments, respectively. The limit of quantification (LOQ) ranged from 0.5–3.0 ng/g. The strategy used here for a mixed-bed IAC may be used to study other compounds and more than two classes of analytes simultaneously.